Post on 26-Mar-2019
Potency classification of skin
sensitizers: development and
use of in vitro methods…
SCCS WG on methodology
Luxemburg, July 1st 2015
Prof. Jean-Pierre Lepoittevin
University of Strasbourg, France
Research funding sources…
Cosmetics Europe
Research Institute for Fragrance
Material (RIFM)
L’Oréal
Centre National de la Recherche
Scientifique (CNRS)
Université de Strasbourg
Chemical
Hazard
Potency
Risk assessment…
Step 1
Step 2
Development of alternative
methods to animal testing… in
vit
ro m
od
els
Structure activity relationships
Phenotypical modifications
Transcriptome
hapten-protein
Interactions
Production of soluble factors
in silico
models
in chemico
models
in vivo models
T
Maturation
Hapten
Epidermis
Dendritic cells
Draining lymphnode
TC activation
Migration
Intracellular signaling
Keratinocytes
Intracellular signaling
Adverse Outcome Pathway (AOP)
chemical
structures
& properties
metabolism
penetration
electrophilic
substance
molecular
initiating
events
covalent
interaction
with proteins
cellular
response
Dendritic cells
Keratinocytes
Organ
response
Organism
response
Inflammation
upon challenge
Skin (epidermis) Lymph node
• MHC
presentation
• Activation of
T-cells
• Proliferation
of activated
T-cells
Toxicity pathway
Mode of action
Adverse outcome
Adverse Outcome Pathway (AOP)
chemical
structures
& properties
metabolism
penetration
electrophilic
substance
In vitro skin
absorption
(Q)SARs
In silico
toxicokinetics
molecular
initiating
events
covalent
interaction
with proteins
• Peptide
depletion
• Adduct
formation
• Relative
reactivity
rate
cellular
response
Dendritic cells
Keratinocytes
Activation
(Keap-1/Nrf2-ARE
Gene expression
Pro-inflammatory
mediators
Co-stimulatory
adhesion molecules
Organ
response
Organism
response
Inflammation
upon challenge
Skin (epidermis) Lymph node
• MHC
presentation
• Activation of
T-cells
• Proliferation
of activated
T-cells
In vitro T-cell
priming /
proliferation
Adverse Outcome Pathway (AOP)
chemical
structures
& properties
metabolism
penetration
electrophilic
substance
In vitro skin
absorption
(Q)SARs
In silico
toxicokinetics
molecular
initiating
events
covalent
interaction
with proteins
• Peptide
depletion
• Adduct
formation
• Relative
reactivity
rate
cellular
response
Dendritic cells
Keratinocytes
Activation
(Keap-1/Nrf2-ARE
Gene expression
Pro-inflammatory
mediators
Co-stimulatory
adhesion molecules
Organ
response
Organism
response
Inflammation
upon challenge
Skin (epidermis) Lymph node
• MHC
presentation
• Activation of
T-cells
• Proliferation
of activated
T-cells
In vitro T-cell
priming /
proliferation
KE1
KE2
KE3 KE4
KE1: hapten-protein interactions in
vit
ro m
od
els
Structure activity relationships
Phenotypical modifications
Transcriptome
hapten-protein
Interactions
Production of soluble factors
in silico
models
in chemico
models
in vivo models
T
Maturation
Hapten
Epidermis
Dendritic cells
Draining lymphnode
TC activation
Migration
Intracellular signaling
Keratinocytes
Intracellular signaling
Development of alternative
methods to animal testing…
KE1: Direct Peptide
Reactivity Assay (DPRA)
Mechanistic basis: address haptenization of
proteins…
Test system: synthetic heptapeptides containing
either Cys or Lys residues…
End-point: Cys and Lys peptide % depletion
Protocol: Test chemical in acetonitrile; Cysteine
peptide in phosphate buffer, pH 7.5; Lysine peptide
in ammonium acetate, pH 10.2; Test chemical
reacted with peptide (10:1 or 50:1) for 24 hours…
Controls: cinnamic aldehyde (positive)…
KE1: Direct Peptide
Reactivity Assay (DPRA)
2 synthetic peptides:
Pep-Cys
Pep-Lys
Gerberick F et al. Toxicol Sci, 2004, 81, 332-343
Test chemical in acetonitrile
Cysteine peptide in phosphate buffer, pH 7.5
Lysine peptide in ammonium acetate, pH 10.2
Test chemical reacted with peptide (10:1 or 50:1) for 24 hours
Peptide loss monitored by HPLC at 220 nm
Un-reacted Peptide
Test Chemical
Reaction Mixture
KE1: Direct Peptide
Reactivity Assay (DPRA)
KE1:Direct Peptide
Reactivity Assay (DPRA)
Prediction model: mean percent cysteine and
lysine peptide depletion value of 6.38 is used as
threshold to discriminate between negative and
positive predictions…
Applicability: not applicable for metals, oxidizers,
highly hydrophobic substances, pre- prohaptens…
Potential for relation reactivity
vs sensitization potential
Molecule
Molecule
Minimal
Reactivity
High reactivity
Pep-Cys
Molecule
Low reactivity
Moderate reactivity
Gerberick GF et al. Toxicol Sci 2007, 97, 417-427
Pep-Lys Pep-Lys
> 22.6 % ≤ 22.6 %
> 42.5 % ≤ 42.5 % ≤ 6.4 % > 6.4 %
Non
sensitiser
Weak
sensitiser
Moderate
sensitisers
Strong
sensitisers
Development of alternative
methods to animal testing… in
vitro
m
od
els
Structure activity relationships
Phenotypical modifications
Transcriptome
hapten-protein
Interactions
Production of soluble factors
in silico
models
in chemico
models
in vivo models
T
Maturation
Hapten
Epidermis
Dendritic cells
Draining lymphnode
TC activation
Migration
Intracellular signaling
Keratinocytes
Intracellular signaling
Development of alternative
methods to animal testing…
KE2: KeratinoSens™…
Mechanistic basis: address response of
keratinocytes by measuring activation of the
electrophile response (Nrf2-Keap1-ARE pathway)…
Test system: human keratinocyte-derived cell line
with a luciferase gene under the control of an ARE
element…
End-point: luciferase gene fold induction
Protocol: cell exposed for 48h to 12 concentrations
of test chemical; luciferase fold induction
quantified by luminescence analysis…
Controls: cinnamic aldehyde (positive)…
Natsch A. Toxicol Sci 2010, 113, 284-292
ARE
Proteasomal
degradation
Nrf2
Nrf2
Nrf2
Keap1
SH SH
P
sMaf
Gene transcription
Antioxidant genes
Detoxication
Cell Survival
Stress inducers
NUCLEUS
Target gene functions
P P
Nrf2
HMOX
NQO1
NRF-2
Keap1
S S
KE2: KeratinoSens™…
Prediction model: luciferase activity 1.5 fold higher
at a concentration with >70% cell viability in at
least 2 of 3 independent repetitions…
Applicability: not applicable for chemicals only
reacting with lysine, non soluble substances
(W/DMSO), prohaptens requiring P450 activation…
Natsch A. Toxicol Sci 2010, 113, 284-292
KE3: Human Cell Line
Activation Test (h-CLAT)…
Mechanistic basis: address response of dendritic
cells (DC) by measuring modulation of co-
stimulatory and adhesion molecules…
Test system: human monocytic leukemia cell line
(THP-1)…
End-point: relative fluorescence intensity (RFI) of
CD86 and CD54 and cytotoxicity…
Protocol: Cells exposed for 24h to 8 concentrations
of test chemical; RFI of CD86 and CD54 compared
to vehicle controls quantified by flow cytometry…
Controls: DNCB (positive)…
Ashikaga, T. et al. Toxicol in Vitro 2006, 20, 767-773
KE3: Human Cell Line
Activation Test (h-CLAT)…
Prediction model: A chemical is rated positive if
the RFI of CD86 is ≥ 150% and/or if the RFI of CD54
is ≥ 200% at any tested dose (≥ 50% of cell
viability) in at least 2 independent repetitions…
Applicability: not applicable for chemicals with
logKow
greater than 3.5; prohaptens requiring
metabolic activation…
Ashikaga, T. et al. Toxicol in Vitro 2006, 20, 767-773
Compared predictive capacity ?
DPRA KeratinoSens™ h-CLAT
Data set size N = 82 N = 145 N = 53
Compared to LLNA LLNA Human
Accuracy 89.0% 77.0% 83.0%
Sensitivity 88.0% 79.0% 85.0%
Specificity 90.0% 72.0% 76.9%
Major pitfalls…
Solubility / Toxicity
Complex mixtures
Major pitfalls…
Activation
(Pre- and prohaptens)
Detoxication
Potency prediction?
Potential to
access…
Not validated…
Integrated Approaches to
Testing and Assessment (IATA)
How to combine the results of in
silico, in chemico and in vitro tests
Integrated Approaches to
Testing and Assessment (IATA)
Nukada et al. Toxicol in Vitro 2013, 27, 609-618
Bauch et al. Regul Toxicol Pharmacol 2012, 63, 489-504
Van der Veen et al. Regul Toxicol Pharmacol 2014, 69, 371-379
Conclusion…
Alternative methods developed have a
potential to approach potency…
However they have been validated only
for danger prediction…
IATA’s are still under development for
danger prediction and need to be
validated…
All categories of sensitizers are not yet
covered…