La rivoluzione REACH
Transcript of La rivoluzione REACH
Costanza Rovida
REACH Mastery - Como
La rivoluzione REACH
23 maggio 2014
Costanza [email protected]
Center for Alternatives to Animal Testing – Europe (CAAT-Europe)University of Konstanz
La rivoluzione REACH
23 maggio 2014
Article 1
Aim and scope
1. The purpose of this Regulation is to ensure a high
level of protection of human health and the
environment, including the promotion of alternative
methods for assessment of hazards of
substances, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation
Article 1
Aim and scope
1. The purpose of this Regulation is to ensure a high
level of protection of human health and the
environment, including the promotion of alternative
methods for assessment of hazards of
substances, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation
List of requiredendpoints,
according toREACH
RegulationPhysico-chemical
information
List of requiredendpoints,
according to REACH Regulation
Toxicologicalinformation
List of requiredendpoints,
according toREACH
Regulation:Ecotoxicological
information
IUCLID fields to be completed
Guidelines on Information Requirements
TOTAL of 34 FILES !!! (How many thousands of pages?)
Guidelines on Information Requirements
ECHA Practical Guides
1. How to report in vitro data2. How to report Robust Study Summary3. How to report Weight of Evidence4. How to report waiving5. How to report (Q)SARs6. How to report read across and categories
http://echa.europa.eu/doc/press/na_10_16_practical_guides_20100409.pdf
Article 1
Aim and scope
1. The purpose of this Regulation is to ensure a high
level of protection of human health and the
environment, including the promotion of alternative
methods for assessment of hazards of
substances, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation
Articolo 13
REACH Regulation
ANNEXES VII-XBefore new tests are carried out to determine the properties listed in thisAnnex, all available in vitro data, in vivo data, historical human data, datafrom valid (Q)SARs and data from structurally related substances (read-across approach) shall be assessed first
ANNEX XIGeneral rules for adaptation of the standard testing regime set out in annexes VII to X
ANNEXES VII-X – Column 2Specific rules for adaptation from column 1
Annex XITESTING DOES NOT APPEAR SCIENTIFICALLY NECESSARY• Use of existing data• Historical human data
PRO and CONS
Studies are often very poor describedNo analytical identification of the substanceNo identification of the impurities……………..………………
Annex XI - Weight of EvidenceThere may be sufficient Weight of Evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion.
• newly developed test methods
• not yet included in the test methods referred to in
Article 13(3)
• different studies with the same reliability, no key study,
but consistent to assess the end point
Annex XI - (Q)SARQUALITATIVE OR QUANTITATIVE
STRUCTURE-ACTIVITY RELATIONSHIP ((Q)SAR)
PRO and CONS
Applicability domainFit the purpose“Poor” training data setsDifficulties in reporting……………..………………
Annex XI - Grouping and Read AcrossGROUPING OF SUBSTANCES AND READ-ACROSS APPROACH
Two approaches:
GROUPING IN CATEGORIES (HPV-SIDS approach)chemical similaritiesfunctional groupsphysical chemical propertiescommon environmental fatecommon metabolites
READ ACROSS FROM SIMILAR SUBSTANCESa specific end-point can be assessed with a result on a similar substancedifferent end point can be assessed from different substances
Pavia – 7 maggio 2011
Annex XI
ALTERNATIVE METHODS (ECVAM activities)
http://tsar.jrc.ec.europa.eu/
Reduction of animals: Sensitisation
Acute oral toxicity
Acute aquatic toxicity
Developing screening metods that can support the reliability of a category
or a read across
Pavia – 7 maggio 2011
Annex XI
GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH
Two approaches:
GROUPING IN CATEGORIES (HPV-SIDS approach)
chemical similarities
functional groups
physical chemical properties
common environmental fate
common metabolites
READ ACROSS FROM SIMILAR SUBSTANCES
a specific end-point can be assessed with a result on a similar
substance
different end point can be assessed from different substances
Pavia – 7 maggio 2011
Annex XI
SUBSTANCE-TAILORED EXPOSURE-DRIVEN TESTING
Testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and
Annex X may be omitted, based on the exposure scenario(s) developed in
the Chemical Safety Report
PERFORM a Chemical Safety Assessment complete with Exposure
Scenarios even if the substance is NOT DANGEROUS
CONS: Companies are often not cooperative even if they save money
In vitro methodsIn vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and direct food additives that may or may not taste good.
In vitro assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal movement to reduce the use of animals in research, and a desire to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects.
www.answers.com
I metodi alternativi: che cosa sono
Principio delle 3R:Reduction = RiduzioneRefinement = MiglioramentoReplacement = Sostituzione
Russell, W. M. S. and Burch, R. L. 1959. The principles of humane experimental technique
Direttiva EU 63/2010
Sostituisce la vecchia Direttiva EEC 86/609 con una maggiore attenzione al benessere animale e un esplicito riferimento alle 3RMaggiore controllo e rigore sugli esperimenti
Element Dir 86/609/EEC Dir 2010/63/EU
Ambito di applicazione Vertebrati. Vertebrati, cefalopdi, Feti
Compliance with the Three Rs Yes Yes
Project evaluation No Yes
Project authorisation (Yes/No) Yes
Housing and care standards guidelines bindingstandards
Main differences between the current & the new Dir
Element Dir 86/609/EEC Dir 2010/63/EU
Project follow-up (Yes) Yes
- Retrospective assessment No Yes
Non-technical summaries No Yes
Severity classification No Yes
Ispezioni periodiche Yes Obbligatorie
Main differences between the current & the new Dir
Shell Fish Biotoxins
Mouse bio-assay
Shell Fish Biotoxins
Metodo in vitro
Confronto metodo in vitro e in vivo
0
20
40
60
80
100
120
140
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157
µg
ST
X e
q/1
00
g
correlative sample number
In vitro Assay Mouse Bioassay
17.2% 52.6% 80.0%
In vitro methodsIn vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and direct food additives that may or may not taste good.
In vitro assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal movement to reduce the use of animals in research, and a desire to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects.
www.answers.com
IN VITRO METHODS
Validated methods:Skin corrosion/irritationSkin absorptionEye corrosion/irritation (partially)Screening for genotoxicity and mutagenicity (partially)Acute Toxicity (partially)
Pre-validation methods:SensitisationCarcinogenic potentialReproductive toxicity (embryotoxicity)
In vitro methods: Skin sensitisation
LOCAL LYMPH NODE ASSAYDays 1, 2 & 3Apply Chemical
Day 6 - Inject H-Thymidine3
5 hours LaterRemove Lymph Nodes
Make CellSuspension
Determine H-ThymidineIncorporation by LiquidScintillation Counting
3
In vitro approach to Skin sensitisation
SKIN Sensitisation
DPRA (Direct Peptide Reactivity Assay):
OECD 406 (Guinea pig): 2,000€OECD 429 (LLNA, traditional test): 3,500€OECD 442b (LLNA, BrdU-ELISA): 2,700€
Pavia – 7 maggio 2011
Strategy: sensitisation
MO
Test with OECD TG 428
Skin Absorption ≥ 0.1% Skin Absorption < 0.1%
MX2
LLNA positive
(Strong)
Negative
No classification as skin sensitiser
Positive
Classified as skin sensitiser H317Read Across
Metal salt Metal oxide
Pavia – 7 maggio 2011
Strategy: sensitisation
IN VITRO STUDIES : An example on how to assess doubtful sensitization
results on a basic dye supporting also exposure pattern.
No study available on both Oxalate and Chloride forms of the dye
Demonstration that the two salts are equivalent is missing
There is concern about the skin sensitisation potential
Negative result in an old study (1984)
Positive in a even older human patch test (1966)
• A new study performed on both salts is required
• A new study to confirm the sensitisation property is required
Pavia – 7 maggio 2011
ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: U937 method, which passed the pre-validation
and has just started the official validation at
ECVAM (European Centre for Validation of
Alternative Methods)
RATIONALE
Skin Absorption: Only adsorbed substances can cause skin sensitisation
The results can be also exploited as a comparison between
the two salts
Sensitisation: a POSITIVE results is accepted (Annex XI:positive results
from a suitable in vitro test can be accepted)
a NEGATIVE result is also accepted: (peer review literature)
Strategy: sensitisation
Pavia – 7 maggio 2011
COSTS:
OECD TG 428, in GLP:
2,700 €/per substance
4,320 € for the two salts when tested simultaneously
U937
Performed for free at the University of Milan, as the validating committee
needs new substances to test
The final result will be confidential and will not be published
(LLNA in vivo method):
7,200 €/per substance
10,800 € for both salts when tested simultaneously
Strategy: sensitisationoss
Pavia – 7 maggio 2011
Strategy: full dossier
In vitro studies to assess the category of nickel compounds and verify the
representative substance to take the existing data in Read Across
BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL
COMPOUNDS: INHALATION TOXICITY
Sample ID Code(s)2 CAS No.
Ni
Content
(%)3
Interstitial
Bioaccessibility
(% Ni/g sample)4
Lysosomal
Bioaccessibility
(% Ni/g sample)4
Acute Toxicity
(inhalation LC50,
mg/L)5
Ni Sulfate Hexahydrate N58-72 10101-97-0 22 12.80 21.35 2.48
Ni Oxide Green N9/N46 (N126) 1313-99-1 77 (81) 0.10 0.82 >5.08
Ni Sub-Sulfide N129 (N18) 12035-72-2 61 (70) 3.60 26.20 1.14
Ni Hydroxy Carbonate N128 (N109) 12122-15-5 49 (49) 1.65 47.206 >2.09 (F); 0.25 (M)7
Ni Chloride Hexahydrate N98 7791-20-0 25 8.10 25.056 NC
Ni Acetate Tetrahydrate N103 6018-89-9 24 10.90 24.856 NC
Ni Sulfamate
TetrahydrateN104 13770-89-3 18 8.60 18.306 NC
Ni Hydroxide N106 12054-48-7 54 0.02 55.80 NC
Ni Oxide Black N105 1313-99-1 75 0.56 24.50 NC
Ni Sulfide N97 16812-54-7 59 1.08 25.95 NC
Pavia – 7 maggio 2011
Strategy: full dossier
BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL
COMPOUNDS: ORAL SYSTEMIC TOXICITY
Sample ID Code1 CAS No.
Ni Content
(%)2
Bioaccessibility (%
Ni content
released)3
In Vivo Acute
Toxicity (oral LD50,
mg/kg/bw)4
Ni Sulfate Hexahydrate N58-72 10101-97-0 23 90.55% 362
Ni Oxide Green N9/N46 1313-99-1 77 0.33% >11000
Ni Oxide Green N112 1313-99-1 81 <LOD5 >11000
Ni Sub-Sulfide N18 12035-72-2 70 22.65% >11000
Ni Chloride Hexahydrate N98 7791 -20-0 25 89.85% 500
Ni Acetate Tetrahydrate N 103 6018-89-9 24 88.50% 550
Ni Hydroxy Carbonate N109 121 22-1 5-5 49 84.30% 2000
Ni Sulfamate
TetrahydrateN 104 13770-89-3 18 83.40% 1098
Ni Hydroxide N106 12054-48-7 54 26.30% 5000
Ni Oxide Black N105 131 3-99-1 75 29.60% 9990
Ni Sulfide N97 1681 2-54-7 59 9.75% NC6
Ni Fluoride Tetrahydrate7 N111 13940-83-5 32 82.35% 310
Pavia – 7 maggio 2011
European projects
OSIRIS
Optimised Strategies for Risk Assessment of Industrial Chemicals through
Integration of Non-Test and Test Information
http://www.osiris-reach.eu/
Pavia – 7 maggio 2011
Improvements
With the first dossiers many data have been generated, that can be used to
improve also the reliability of predictions models in the next years
More in vitro metods will be developed / validated
Multidisciplinary approach within the working groups
Will ECHA be ready?
An example for a Strategy
IN VITRO STUDIES : An example on how to assess doubtful sensitization results on a basic dye supporting also exposure pattern.
No study available on both Oxalate and Chloride forms of the dye
Demonstration that the two salts are equivalent is missing
There is concern about the skin sensitisation potential
Negative result in an old study (1984)Positive in a even older human patch test (1966)
• A new study performed on both salts is required• A new study to confirm the sensitisation property is required
ITS proposal:
1. Skin absorption assessment: OECD TG 4282. In vitro sensitisation: THP-1 method, which passed the pre-validation
and has just started the official validation at ECVAM (European Centre for Validation of Alternative Methods)
RATIONALE
Skin Absorption: Only adsorbed substances can cause skin sensitisationThe results can be also exploited as a comparison between the two salts
Sensitisation: a POSITIVE results is accepted (Annex XI:positive results from a suitable in vitro test can be accepted)a NEGATIVE result is also accepted: (peer review literature)
COSTS:
OECD TG 428, in GLP:2,700 €/per substance4,320 € for the two salts when tested simultaneously THP-1 Performed for free at the University of Milan, as the validating committee needs new substances to test
(LLNA in vivo method):7,200 €/per substance10,800 € for both salts when tested simultaneously
In vitro methods: Strategy
INTELLIGENT = INTEGRATED
Existingdata
Weight ofEvidence
In vitro dataGOOD SENSE!
Exposureassessment
Readacross
Categories
In silicodata
Validation modular approach
Test definition
Within-lab. variability
Transferability
Between-lab.variability
Predictive capacity
Applicability domain
Minimum performance standards
Reprodu-cibility
Relevance
“Standardised”
“Suitable/Adequate”
“Validated”
“Equivalent”
Hartung et al. ATLA 2004, 32:467-472
OECD Member Countries
Countries/Economies Engaged in Working
Relationships with the OECDOECD
428 Skin Absorption: In Vitro Method
430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)
431 In Vitro Skin Corrosion: Human Skin Model Test
432 In Vitro 3T3 NRU Phototoxicity Test
435 In Vitro Membrane Barrier Test Method for Skin Corrosion
437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
471 Bacterial Reverse Mutation Test
472 Genetic Toxicology: Escherichia coli, Reverse Assay
473 In Vitro Mammalian Chromosome Aberration Test
474 Mammalian Erythrocyte Micronucleus Test
476 In Vitro Mammalian Cell Gene Mutation Test
477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells
480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
483 Mammalian Spermatagonial Chromosome Aberration Test
487 In Vitro Mammalian Cell Micronucleus Test
OECD Methods
428 Skin Absorption: In Vitro Method
430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)
431 In Vitro Skin Corrosion: Human Skin Model Test
432 In Vitro 3T3 NRU Phototoxicity Test
435 In Vitro Membrane Barrier Test Method for Skin Corrosion
437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
471 Bacterial Reverse Mutation Test
472 Genetic Toxicology: Escherichia coli, Reverse Assay
473 In Vitro Mammalian Chromosome Aberration Test
474 Mammalian Erythrocyte Micronucleus Test
476 In Vitro Mammalian Cell Gene Mutation Test
477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells
480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
483 Mammalian Spermatagonial Chromosome Aberration Test
487 In Vitro Mammalian Cell Micronucleus Test
OECD Methods
OECD 437 BCOP Test
H318 Causes serious eye damage
Un esempio di una strategia
IN VITRO STUDIES : Un esempio di come una strategia in vitro è statautilizzata per la valutazione di un risultato dubbio sulla sensitization e per avere maggiori dati sull'esposizione.
Nessuno studio disponibile su entrambi i Sali in commercio del colorante (Ossalato e cloruro)
Mancanza di una dimostrazione che i due Sali fossero equivalenti
Dubbio che potesse essere allergenicoRisultato negativo in uno studio molto vecchio (1984)Risultato positivo in un human patch test ancora più vecchio (1966)
• Necessario uno studio da fare contemporaneamente sui due sali• Necessario uno studio per decidere sulla skin sensitisation
ITS proposal:
1. Skin absorption assessment: OECD TG 4282. In vitro sensitisation: THP-1 method, che ha già passato la fase di pre-
validation and ed è in corso di validazione ufficiale a ECVAM (European Centre for Validation of Alternative Methods)
RATIONALE
Skin Absorption: Solo le sostanze che vengono assorbite possono provocareallergie. Il risultato può essere utilizzato come base per ilconfronto tra i due sali
Sensitisation: un risultato POSITIVO è accettato (Annex XI: positive results from a suitable in vitro test can be accepted)un risultato NEGATIVO è accettato lo stesso (peer review literature)
Non si conosconoSono più difficili da applicareSpesso sono più costosiNon si è sicuri che vengano accettatiSi è sempre fatto in un altro modo
Altex 27, 3/10
… Approccio avanzato, ma ancora con impostazione classica…
The reproductive cycle
Courtesy of Michael Schwarz
Use of bovine gametes for reproductive toxicity testing
bovine ovaries
2 cm
bovine oocytes
Endpoint: % Metaphase II
Chemicalexposure (24h) during IVM
In vitro maturation (IVM) of bovine oocytes
Endpoint: % 2Pronuclei
In vitro fertilisation (IVF) of bovine oocytes
Chemicalexposure (20h) during IVF
IVM, 24 h
Coltura di cellule
embrionali staminali
dall’embrione alle cellule staminaliconcepimento blastocisti (5 giorni)
Developmental Toxicity
Acute Toxicty
Stem Cell Toxicity Approach
performanceconfidence
in vivo relevance
validationpredictive
QIVIVE Quantitative in vitro in vivo extrapolation
Target cells: permeability, transport, metabolic competenceExtracellular space: proteins, lipids, pH, plastic, other cellsExposure (concentration x time)
Modelli PBPK
Report del National Research
Council of the National Academies
Pubblicato nel 2007
(Google: "Toxicity Testing in the 21st Century")
Biologic
Inputs
Normal
Biologic
Function
Morbidity
and
Mortality
Cell
Injury
Adaptive Stress
Responses
Early Cellular
Changes
Exposure
Tissue Dose
Biologic Interaction
Perturbation
Low DoseHigher Dose
Higher yet
(Courtesy of Mel Andersen)
A New Paradigm: Activation of Toxicity Pathways
Progetto ToxCast
Toxicity PathwaysA cellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect.
Set Chemicals Assays Endpoints Completion Available
ToxCast Phase I 293 ~600 ~1100 2011 Now
ToxCast Phase II 767 ~600 ~1100 03/2013 10/2013
ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014
E1K (endocrine) 880 ~50 ~120 03/2013 10/2013
Tox21 8,193 ~25 ~50 Ongoing Ongoing
Effect of Concentration Response on Polypharmacology
72
Pentachlorophenol
“Non sopravvive chi è più forte o
più intelligente, ma chi
reagisce più velocemente ai
cambiamenti”
Charles Darwin