In vitro metjhods for testing immunotoxicity and skin Sensitization-AVRLinkedIN
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Applied in vitro toxicology course 8-13 January 2017, Belvaux - Luxembourg
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Transcript of In vitro metjhods for testing immunotoxicity and skin Sensitization-AVRLinkedIN
Applied in vitro toxicology course
8-13 January 2017 Belvaux - Luxembourg
VITO AREAS OF EXPERTISE
wwwvitobe vitovitobe
Energy Materials Chemistry
Health Land use
TOXICOLOGY SERVICES
3
bull In vitro toxicity amp Biocompatibility tests
bull Computational Models amp expert analysis
bull Ecotoxicity amp Biodegradation tests
3
4
5
6
SOME WORKING DEFINITIONS
raquo Immunotoxicity
Study of immune dysfunction resulting from exposure of an organism to
a xenobiotic
The immune dysfunction may take the form of immunosuppression or
alternatively hypersensitivity autoimmunity or any number of inflammatory-
based diseases or pathologies
raquo Immunosuppression
Act that reduces the activation or efficacy of the immune system
raquo Autoimmunity
System of immune responses of an organism against its own cells and tissues
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
VITO AREAS OF EXPERTISE
wwwvitobe vitovitobe
Energy Materials Chemistry
Health Land use
TOXICOLOGY SERVICES
3
bull In vitro toxicity amp Biocompatibility tests
bull Computational Models amp expert analysis
bull Ecotoxicity amp Biodegradation tests
3
4
5
6
SOME WORKING DEFINITIONS
raquo Immunotoxicity
Study of immune dysfunction resulting from exposure of an organism to
a xenobiotic
The immune dysfunction may take the form of immunosuppression or
alternatively hypersensitivity autoimmunity or any number of inflammatory-
based diseases or pathologies
raquo Immunosuppression
Act that reduces the activation or efficacy of the immune system
raquo Autoimmunity
System of immune responses of an organism against its own cells and tissues
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
TOXICOLOGY SERVICES
3
bull In vitro toxicity amp Biocompatibility tests
bull Computational Models amp expert analysis
bull Ecotoxicity amp Biodegradation tests
3
4
5
6
SOME WORKING DEFINITIONS
raquo Immunotoxicity
Study of immune dysfunction resulting from exposure of an organism to
a xenobiotic
The immune dysfunction may take the form of immunosuppression or
alternatively hypersensitivity autoimmunity or any number of inflammatory-
based diseases or pathologies
raquo Immunosuppression
Act that reduces the activation or efficacy of the immune system
raquo Autoimmunity
System of immune responses of an organism against its own cells and tissues
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
4
5
6
SOME WORKING DEFINITIONS
raquo Immunotoxicity
Study of immune dysfunction resulting from exposure of an organism to
a xenobiotic
The immune dysfunction may take the form of immunosuppression or
alternatively hypersensitivity autoimmunity or any number of inflammatory-
based diseases or pathologies
raquo Immunosuppression
Act that reduces the activation or efficacy of the immune system
raquo Autoimmunity
System of immune responses of an organism against its own cells and tissues
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
5
6
SOME WORKING DEFINITIONS
raquo Immunotoxicity
Study of immune dysfunction resulting from exposure of an organism to
a xenobiotic
The immune dysfunction may take the form of immunosuppression or
alternatively hypersensitivity autoimmunity or any number of inflammatory-
based diseases or pathologies
raquo Immunosuppression
Act that reduces the activation or efficacy of the immune system
raquo Autoimmunity
System of immune responses of an organism against its own cells and tissues
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
6
SOME WORKING DEFINITIONS
raquo Immunotoxicity
Study of immune dysfunction resulting from exposure of an organism to
a xenobiotic
The immune dysfunction may take the form of immunosuppression or
alternatively hypersensitivity autoimmunity or any number of inflammatory-
based diseases or pathologies
raquo Immunosuppression
Act that reduces the activation or efficacy of the immune system
raquo Autoimmunity
System of immune responses of an organism against its own cells and tissues
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
7
IMMUNOTOXICITY
raquo REACH does not require immunotoxicity as a standard information
requirement
raquo Gold standard animal tests
raquo lsquoInterpreting data from animal immunotoxicology studies for risk
assessment has proven challenging especially when the immunological
effects are minimal-to-moderate in naturersquo (Germolec 2004)
raquo Hypersensitivity reactions represent most frequently reported
immunotoxic effects of chemical exposure in industrialized countries
Hartung amp Corsini 2013
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
8
IMMUNOSUPPRESSION
raquo Before starting with in vitro tests bioavailability should be considered
raquo Mitogen stimulation assays
raquo Tiered approach
raquo Tier 1 myelotoxicity
raquo Tier 2 lymphotoxicity
raquo Overt cytotoxicity vs cell functionality
Key Targets In Vitro Opportunities
Bone marrow Humanmurine GM-CFU assay for myelotoxicity
NK cell activity
Cytokine production ie whole blood assay lsquofluorescent chip assayrsquo etc
T cells proliferation and cytokine production
B cells proliferation
Transcriptomic profiles
In vitro antibody production
Innate immunity
Acquired immunity
Corsini Altex 2011
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SKIN SENSITIZATION IN A REGULATORY ENVIRONMENT
raquo Europe protection through
legislation
raquo REACH = Regulation Evaluation and
Authorization and Restriction of
Chemicals
raquo Between 25000 and 50000
substance registrations expected
for the 2018 deadline
raquo Cosmetic Products Directive (76768EEC)
raquo Globally Harmonized System (GHS)
raquo 77 chemicals require skin sensitization testing
9
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
raquo Contact dermatitis
raquo Irritant contact dermatitis activation of the innate immune system pro-
inflammatory molecules
raquo Allergic contact dermatitis activation of antigen-specific acquired immunity Cell-
mediated Hypersensitivity reaction type IV delayed-type
raquo 2 temporally discrete stages
raquo Sensitization acquired ability to respond immunologically to allergen in heightened
fashion
raquo Elicitation subsequent encounter of allergen accelerated and more agressive
secondary immune response with clinical symptoms
SOME WORKING DEFINITIONS
A skin sensitiser is an agent that will lead to an allergic
response in susceptible individuals following skin contact As a
consequence of a secondary - usually organ-specific -
subsequent re-exposure adverse health effects on the skin
(allergic contact dermatitis) (ECHA Chapter R7a 2016)
bull +3700 substances are skin sensitizers
bull 20 adult populaon is allergic to 1 or more
10
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SKIN SENTIZATION FROM WORKING ON KEYBOARD + PATCH TEST
11
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SENSITIZATION IS A MULTIPLE STEP PROCESS
Induction Phase Elicitation Phase
Chemical Edema and Erythema
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Langerhans Cell (LC)
Required for Immune
response
Lymphocyte
Proliferation
Specific
Response
Exaggerated
Response on
reexposure
Cellular
Influx
12
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SKIN SENSITIZATION PROCESS
EVENTS
1 Haptenation attachment of allergen
to skin protein (key event 1)
2 Epidermal inflammation release of
pro-inflammatory signals by
epidermal keratinocytes (key event
2)
3 Dentritic cells (DC) activation and
maturation (key event 3)
4 DC migration movement of DC
bearing hapten-protein complex
from skin to draining local lymph
node
5 T-cell proliferation clonal
expansion of hapten-peptide
specific T-cells (key event 4)
13
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
ANIMAL TESTS FOR SKIN SENSITIZATION
bull Legislation on skin sensitization testing 18 million animals required
bull In-house pre-production screening and synthesis billions more
ECHA for Skin sensitization (2016)
Potency required so Cat 1A (strong) vs Cat 1B (moderate)
bull in vitro test method must always be the start
bull in vivo testing only if
bull Methods are not suitable for the substance
bull Results of the in vitro tests are not adequate for classification and risk assessment
14
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
LOCAL LYMPH NODE ASSAY (LLNA) (OECD TG 429)
15
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
REDUCED LOCAL LYMPH NODE ASSAY (OECD TG 429)
16
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SKIN SENSITIZATION IMMUNOLOGY PROCES
17
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITIZATION
18
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITIZATION
Modified version of flowchart OECD report The Adverse Outcome Pathway for Skin Sensitisation
Initiated by Covalent Binding to Proteins Part 1 Scientific Evidence Series on Testing and Assessment
No168 ENVJMMONO(2012)10PART1 19
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
20
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
21
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
22
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
VALIDATION AND ADOPTION STATUS OF IN CHEMICOIN VITRO METHODS
23
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DIRECT PEPTIDE REACTIVITY ESSAY (DPRA)
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DPRA DATA ANALYSIS
raquo Integrate peaks of calibration solutions controls and samples
raquo Generate linear calibration curve
raquo Calculate peptide concentrations
raquo Determine percent peptide depletion
raquo
25
y = 37400x + 11465 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Cysteine y = 34451x + 22929 Rsup2 = 1
0
5000
10000
15000
20000
25000
0 01 02 03 04 05 06
Are
a
conc
Lysine
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
VALIDATION OF DPRA
raquo Proficiency substances (cfr OECD 442C) score ge 810 for each peptide
raquo See DPRA validation study template (httpecvam-
dbalmjrceceuropaeuprotocolscfmid_met=1953)
26
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DPRA PREDICTION MODEL AND APPLICABILITY
27
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
The KeratinoSensTM assay
28
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
raquo ARE element Genetic switch
raquo Nrf2-protein Transcription factor lsquoPresses the buttonlsquo on ARE
raquo Keap1 Sensor protein activates Nrf2 in presence of reactive molecule
KeratinoSensTM assay mechanism
Nrf2
Keap1
SH SH SH
Luciferase gene ARE DNA SV40 SV40 promotor
for stable background
Reaction at sensor surface
Antioxidant response element
from AKR1C2 29
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
KeratinoSensTM READ OUT DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
viability
fold luciferase induction
Chemical surpasses
threshold positive rating
Luciferase gene induction
Cytotoxicityviability
hair dye component p-phenylendiamine (strong sensitizer)
Endpoints EC 15 + Imax
Sensitizer 1) I max gt15 fold basal LI + EC 15 lt 1000 microM or lt 200 microgml
(2 out of 3 rep)
+ 2) Lowest conc with 15 FI should have viability gt 70
I max Maximal fold induction of
luciferase gene over solvent control
IC50 and IC30 conc for 50 and 30
reduction in viability
EC 15 Extrapolated concentration
of a test compound needed for a
15 fold luciferase induction
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
KeratinoSensTM DOSE-RESPONSE CURVES AND RELATIONSHIP TO POTENCY-
EXAMPLE ARYL HALIDES
1
10
01 1 10 100
conc (micromolar)
fold
in
du
cti
on
4-Nitrobenzylbromide
DNCB
24-
Dichloronitrobenzene
24 dichloro-nitro benzene is a weak sensitizer
24 dinitrochlorobenzene (DNCB) and 4-nitrobenzylbromide are extreme sensitizers
There is a clear correlation to potency within structural classes 31
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
KeratinoSensTM DOSE-RESPONSE CURVES
raquo In each test chemicals are tested against 12 test concentrations
raquo Each experiment repeated two ndash three times
raquo Each repetition done in three replicates
viability
fold luciferase induction
Luciferase gene induction
Cytotoxicity
A ndash the hair dye component p-phenylendiamine (strong sensitizer)
B ndash the fragrance and flavor component cinnamic aldehyde moderate sensitizer
C ndash the preservative methyldibromoglutaronitrile a moderate sensitizer
32
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
The KeratinoSensTM assay PREDICTION MODEL AND APPLICABILITY
33
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
KeratinoSensTM APPLICABILITY DOMAIN
raquo KeratinoSens was mainly tested against substances
raquo Cosmetic companies want answers for plant extracts ndash but also
animal tests were never validated for these Complex mixtures
raquo Best option test single constituents
raquo Limitations
- Very high cLogP not soluble substances
- Some phenolic prohaptens (opportunities for S9 assay)
- Chemicals with exclusive amine-reactivity (detected with peptide
reactivity assay)
- Larger polymers ndash preliminary data show limitations for silicones
- Probably overprediction for some flavonoids polyphenolics from
plants
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
- Surfactants and cytotoxic chemicals
- Surfactants with the prediction model taking into account cytotoxicity correctly
predicted
- Most cytotoxic sensitizers induce below the cytotoxic level ndash thus not an issue
- Very membrane active compounds ndash few cases for which cytotoxicity starts before
induction
KeratinoSensTM Applicability domain
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DPRA and KeratinoSensTM SUMMARY
36
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
Human Cell Line Activation Test (h-CLAT)
37
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
h-CLAT TEST SET-UP
38
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
h-CLAT
39
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DPRA KeratinoSensTM and h-CLAT
Depending on the regulatory framework positive results may be used on
their own to classify a chemical to UN GHS Category 1
The TGrsquos cannot be used on their own to subcategorize skin sensitizers into
UN GHS subcategories 1A and 1B or to predict potency for safety
assessment decisions
Given the limited mechanistic coverage and inherent limitations of
available methods combinations of different non-animal methods (in
silico in chemico in vitro) are needed especially to support negative
conclusions
40
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
OECD TG soon
raquo LuSens me-toosimilar test method to KeratinoSensTM
raquo U-SENS U937 Myeloid cells ndashCD86 expression Flow cytometer
raquo Il-8 Luc IL-8 reporter cell line derived from THP-1 luminometer
raquo SENS-IS Reconstructed human Epidermis RhE - EpiSkin RT-PCR
raquo GARD (Genomic Assay Rapid Detection) ~MUTZ Myeloid cells NanoString nCounter
raquo KeratinoSensTM and h-CLAT animal free conditions raquo 10 human serum in place of 10 foetal calf serum + non-animal recombinant
trypsin (TrypZean) instead of recombinant trypsin
raquo 10 human serum in place of 10 foetal calf serum + Human Serum Albumin (HSA) used in place of Bovine Serum Albumin (BSA) + Flow cytometry antibodies BioRad HuCAL anti-human antibodies in place of mouse anti-human antibodies
41
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DIFFERENCES BETWEEN IATA
(Integrated Approaches to Testing and Assessment) FOR
SKIN CORROSIONIRRITATION AND FOR SKIN SENSITISATION
42
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
DATA INTEGRATION A CHALLENGE
43
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
44
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
IATA Integrated Approaches to Testing and Assessment
45
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
IATA Integrated Approaches to Testing and Assessment
46
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
Examples of published data integration strategies for skin sensitization
47
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
Two out of three 1
Bauch et al 2012 54 substances
(LLNA and Human information)
WoE Results of 2 out of 3 tests
determine the classification
48
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
Two out of three 2
54 and 145 substances
raquo Similar accuracy between both studies despite data set from 54 to 154
raquo for the 145 substances accuracy compared to human data could not be determined
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
TWO OUT OF THREE 3
50
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
Limitations of the two out of three
No toxicological test is perfect
ndash including the animal tests ndash
it is important to know their strengths and limitations
51
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
Quantitative Risk Assessment (QRA)
for skin Sensitization
52
Kimber I Gerberick GF Basketter DA Quantitative risk assessment for skin sensitization Success or
failure Regul Toxicol Pharmacol 2017 Feb83104-108
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
QUESTIONS amp CONTACT
VITO-ABS your 1 independent provider of toxicology and
ecotoxicology services
The ABS test facility is certified for GLP ISO9001(Quality)
ISO14001(Environment) and OHSAS18001 (Safety)
Thank you
Dr An Van Rompay ERT (European Registered Toxicologist) Project Manager amp Study director in Toxicology VITO ndash Toxicology Services Industriezone Vlasmeer 7 2400 Mol BELGIUM +32 (0)14 33 5246 or +32 (0) 496 56 5386 anvanrompayvitobe httpsehsvitobe
VitoToxtesting
httpwwwlinkedincominanvanrompay
53
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SOME USEFUL REFERENCES
bull ECHA report 2016 Practical Guide How to use alternatives to animal testing to fulfull your
information requirments for REACH registration
httpsechaeuropaeudocuments1016213655practical_guide_how_to_use_alternatives_
enpdf
bull United Nations Economic Commission (UNECE) (2013) Global Harmonized System of
Classification and Labelling of Chemicals (GHS) 5th Revised Edition p149
bull Frosch PJ et al (2006) Contact dermatitis 4th Edition BerlinHeidelberg Springer Verlag
bull EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report pp1-74
bull OECD Guideline for the Testing of Chemicals In Chemico Skin Sensitisation Direct Peptide
Reactivity Assay (DPRA) Test Guideline 442C adopted February 2015
bull Natsch A et al(2013) A dataset on 145 chemicals tested in alternative assays for skin
sensitisation undergoing prevalidation J Appl Toxicol 33(11) 1337-1352
bull Natsch N et al (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSens assay to predict skin sensitizers in vitro Results of a ring-study in five
laboratories Toxicol In Vitro 25(3) 733-744
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation ARE-Nrf2 Luciferase
Test Method Test Guideline 442D adopted February 2015
bull OECD Guideline for the Testing of Chemicals In Vitro Skin Sensitisation human Cell Line
Activation Test (h-CLAT) Test Guideline 442E adopted July 2016
bull OECD Series on Testing amp Assessment No 256 Guidance document on the terporting of
defined approaches and individual information sources to be used within integrated
approaches to testing testing and assessment (IATA) for Skin Sensitisation
bull Benigni R Bossa C Tcheremenskaia O A data-based exploration of the adverse outcome
pathway for skin sensitization points to the necessary requirements for its prediction with
alternative methods Regul Toxicol Pharmacol 2016 Jul7845-52
54 54
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
bull OECD Guidance document No 168 The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins Part 1 and part 2
bull ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7a Endpoint specific guidance
bull ECHA Report 2014 The Use of Altneratives to Testing on Animals for the REACH Regulation Second report under article 117(3) for the REACH Regulation
bull Basketter D Aleacutepeacutee N Casati S Crozier J Eigler D Griem P Hubesch B de Knecht J Landsiedel R Louekari K Manou I Maxwell G Mehling A Netzeva T Petry T Rossi LH Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU Regul Toxicol Pharmacol 2013 Dec67(3)531-5
bull Bauch C Kolle SN Ramirez T Eltze T Fabian E Mehling A Teubner W van Ravenzwaay B Landsiedel R Putting the parts together combining in vitro methods to test for skin sensitizing potentials Regul Toxicol Pharmacol 2012 Aug63(3)489-504
bull Jaworska J Dancik Y Kern P Gerberick F Natsch A Bayesian integrated testing strategy to assess skin sensitization potency from theory to practice J Appl Toxicol 2013 Nov33(11)1353-64
bull Jaworska JS Natsch A Ryan C Strickland J Ashikaga T Miyazawa M Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment a decision support system for quantitative weight of evidence and adaptive testing strategy Arch Toxicol 2015 Dec89(12)2355-83
bull Basketter DA White IR McFadden JP Kimber I Skin sensitization Implications for integration of clinical data into hazard identification and risk assessment Hum Exp Toxicol 2015 Dec34(12)1222-30
raquo Ezendam J Braakhuis HM Vandebriel RJ State of the art in non-animal approaches for skin sensitization testing from individual test methods towards testing strategies Arch Toxicol 2016 Dec90(12)2861-2883
SOME USEFUL REFERENCES
55
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
SOME USEFUL REFERENCES
bull Nukada Y Miyazawa M Kazutoshi S Sakaguchi H Nishiyama N Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals Toxicol In Vitro 2013 Mar27(2)609-18
bull Patlewicz G Kuseva C Kesova A Popova I Zhechev T Pavlov T Roberts DW Mekenyan O Towards AOP application--implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization Regul Toxicol Pharmacol 2014 Aug69(3)529-45
bull Urbisch D Mehling A Guth K Ramirez T Honarvar N Kolle S Landsiedel R Jaworska J Kern PS Gerberick F Natsch A Emter R Ashikaga T Miyazawa M Sakaguchi H Assessing skin sensitization hazard in mice and men using non-animal test methods Regul Toxicol Pharmacol 2015 Mar71(2)337-51
bull Urbisch D Honarvar N Kolle SN Mehling A Ramirez T Teubner W Landsiedel R Peptide reactivity associated with skin sensitization The QSAR Toolbox and TIMES compared to the DPRA Toxicol In Vitro 2016 Aug34194-203
bull Tsujita-Inoue K Atobe T Hirota M Ashikaga T Kouzuki H In silico risk assessment for skin sensitization using artificial neural network analysis J Toxicol Sci 2015 Apr40(2)193-209
bull Tsujita-Inoue K Hirota M Ashikaga T Atobe T Kouzuki H Aiba S Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays Toxicol In Vitro 2014 Jun28(4)626-39 doi 101016jtiv201401003
raquo van der Veen JW Rorije E Emter R Natsch A van Loveren H Ezendam J Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals Regul Toxicol Pharmacol 2014 Aug69(3)371-9
raquo Luechtefeld T Maertens A Russo DP Rovida C Zhu H Hartung T Analysis of publically available skin sensitization data from REACH registrations 2008-2014 ALTEX 201633(2)135-48
raquo OECD GUIDANCE DOCUMENT 256 ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION ENVJMMONO(2016)29
raquo hellip
56
