1. Ankara, 12 Novembre 2007 – Alternatives 2007 1 ECVAM Key Area Topical Toxicity Summary of ongoing activities Chantra Eskes European Centre for the Validation of Alternative Methods - ECVAM Institute for Health and Consumer Protection European Commission Joint Research Centre http://ecvam.jrc.cec.eu.int

2. Ankara, 12 Novembre 2007 – Alternatives 2007 2 Cosmetics industry EU: 2000 companies, 60 billion € turnover EU: 5000 new products per year 25% turnover due to products released within last 6 months 2003: EU 7th Amendment to Cosmetics Directive  Critical need for alternatives

3. Ankara, 12 Novembre 2007 – Alternatives 2007 3 200420052006200720092008 2004 Ingredients as soon as alternatives are a) validated by ECVAM and b) adopted in EU legislation Ban on animal testing Cosmetic finished products 7th Amendment to EU Cosmetics Directive Marketing ban Cosmetic products and their ingredients as soon as alternatives are a) validated by ECVAM, b) adopted in EU legislation c) and with due regard to OECD process Complete ban on animal testing Irrespective of availability of alternatives Most human health endpoints Except  2013 ■ repeated dose tox. ■ reproductive tox. ■ toxicokinetics

4. Ankara, 12 Novembre 2007 – Alternatives 2007 4 Base-set testing (~ 20’000 substances) In vitro testing required for eye & skin irritation billions of Euro, millions of animals, decades of testing EU Chemical legislation REACH (Registration, Evaluation, Authorisation of Chemicals) 30,000 chemicals > 1 t per year to be assessed Major saving impact of in silico and in vitro tests

5. Ankara, 12 Novembre 2007 – Alternatives 2007 5  Support to policies - Cosmetics - REACH  Evaluation of advanced in vitro alternatives - Phototoxicity, Skin Corrosion - Skin Irritation - Eye Irritation  Collaboration Task Forces, ICCVAM, COLIPA, Industry Topical Toxicity Key Area Activities

6. Ankara, 12 Novembre 2007 – Alternatives 2007 6 Time estimation for phasing-out animal testing 100 scientists & stakeholder representatives 6 DG services 4 Industry associations 3 Animal Welfare NGOs OECD 11 areas of human health effects of concern Inventory & status of validation of most promising alternative methods Recommendations to achieve validation in a timely manner Eskes & Zuang (2005)ATLA 33S1

7. Ankara, 12 Novembre 2007 – Alternatives 2007 7 Eye Irritation – exp. ’ 09 Photogenotoxicity – exp. ’ 08 Skin Irritation – exp. ’ 08 hazard identification Skin Absorption / Penetration Acute Phototoxicity Skin Corrosion Acute Toxicity Genotoxicity / Mutagenicity Reproductive & Developmental Toxicity Carcinogenicity Photo-allergy (-sensitisation) Skin Sensitisation Subacute & Subchronic Toxicity Toxicokinetics / Metabolism EC Timetables for phasing-out animal testing * * substantial reduction of animal use could be achieved earlier 2009 / 2009 2009 / 2013 Testing / Marketing ban

8. Ankara, 12 Novembre 2007 – Alternatives 2007 8 ECVAM Pre- validation Validation ESAC Peer Review Research Animal model In vitro model OECD Pharmacopoeia ECB DGs Regulation chemicals cosmetics drugs ecology work safety biomaterials Optimised test

9. Ankara, 12 Novembre 2007 – Alternatives 2007 9 Skin Corrosion & Acute Phototoxicity 1998 and 2000: ECVAM validated replacement methods TER, EPISKIN TM, EpiDerm TM 3T3NRU 2000 EU acceptance (B.40, B.41) 2004 OECD adoption Skin Corrosion TG 430 & 431 Acute Phototoxicity TG 432 Similar tests submitted to ECVAM for skin corrosion  Catch-up validation SkinEthic: validated in 2006 Cellsystems EST-100: peer review Straticell

10. Ankara, 12 Novembre 2007 – Alternatives 2007 10 1999-2001Prevalidation study 2002Optimisation studies 2003-2006ECVAM Validation Study Goal To replace the Draize skin irritation test performed in albino rabbits Study design Three models: EpiDerm TM, EPISKIN TM, Mouse, Skin Integrity Function Test SIFT Phase 1. Protocol optimisation & training Phase 2. Testing 58 chemicals in 3 labs/test Skin irritation

11. Ankara, 12 Novembre 2007 – Alternatives 2007 11 Skin irritation  Test Definition Harmonised exposure: 15 min exposure + 42 hr post-incubation Endpoints:- Cell viability - MTT - Release of IL1  in media (when cell viability > 50%) Prediction model: MTT cell viability  50%  skin irritant (R38) MTT cell viability > 50%  non irritant Additional: MTT > 50% + IL1  > 60 pg/ml  skin irritant (R38)  Reproducibility EPISKIN (MTT)EpiDerm (MTT) Within-laboratory (a)93.9 %96.0 % Between-laboratory (b)89.5 %88.5 % Proportion of identical MTT classifications (a) over 3 independent experiments, (b) based on median classifications

12. Ankara, 12 Novembre 2007 – Alternatives 2007 12 EPISKIN (MTT)EPISKIN (MTT+IL1-α)EpiDerm (MTT)* SENSITIVITY74.7 %90.7 %57.3 % SPECIFICITY80.8 %78.8 %83.8 % Concordance78.2 %83.0 %72.4 % Replacement of the Draize test. (EU Annex V B.4. OECD TG 404) ESAC Only specificity acceptable. No replacement recommendation, but useful in testing strategy. ESAC Skin irritation * Addition of IL1-α to the EpiDerm protocol gave no improvement to the outcome  Predictive Capacity

13. Ankara, 12 Novembre 2007 – Alternatives 2007 13

14. Ankara, 12 Novembre 2007 – Alternatives 2007 14 6 Major international studies took place in the 90’s EC/HO, COLIPA, CTFA, IRAG, BFA/BMBF, MHW/JCIA No single assay capable of replacing the Draize rabbit eye test - In vitro tests only partially model in vivo response - Animal data of limited quality (low reproducibility across laboratories) - Insufficiently developed protocols and prediction models - Limitations in statistical analyses specific and limited purposes The usefulness of alternative methods however well established within industry and EU regulatory agencies for specific and limited purposes Eye Irritation Background

15. Ankara, 12 Novembre 2007 – Alternatives 2007 15  Evaluation of advanced in vitro alternatives - Organotypic assays - Cytotoxicity / cell function based assays - Human Reconstituted Tissue models  Identification of testing strategies  Critical evaluation of Draize rabbit eye test  Mechanistic developments ECVAM activities on Eye Irritation

16. Ankara, 12 Novembre 2007 – Alternatives 2007 16 Bovine Corneal Opacity and Permeability test (BCOP) Isolated Chicken Eye (ICE) Isolated Rabbit Eye (IRE) Hen`s egg test on the Chorio-Allantoic Membrane assay (HET-CAM) Underwent several multi-laboratory studies Underwent several multi-laboratory studies Routinely used within industries & contract laboratories for specific purposes Routinely used within industries & contract laboratories for specific purposes 2002 Regulatory Acceptance by EU Competent National Authorities: 2002 Regulatory Acceptance by EU Competent National Authorities: Positive outcome accepted for C&L of severe irritants (R41), although not validated  Retrospective validation Organotypic assays

17. Ankara, 12 Novembre 2007 – Alternatives 2007 17 2003-2006ICCVAM Retrospective evaluation with ECVAM coll. Expert Panel Review ICCVAM Recommendations Current status for detecting ocular corrosives / severe irritants April ’07ESAC Statement - BCOP & ICE: endorsed ICCVAM recommendations - HET-CAM & ICE: further analyses requested Follow-up activities - OECD & EU Test Guidelines under preparation - Further improvements and analyses as recommended by ESAC - Evaluation for mild ranges of irritancy Organotypic assays

18. Ankara, 12 Novembre 2007 – Alternatives 2007 18 Neutral Red Release (NRR) Red Blood Cell Test (RBC) Fluorescein Leakage (FL) Cytosensor Microphysiometer (CM) Underwent several multi-laboratory studies Underwent several multi-laboratory studies Routinely used within industries & contract laboratories Routinely used within industries & contract laboratories Specific purposes, e.g., non irritants versus irritants, surfactants Specific purposes, e.g., non irritants versus irritants, surfactants  ECVAM Retrospective weigh-of-evidence evaluation Cytotoxicity- / Cell function- based assays

19. Ankara, 12 Novembre 2007 – Alternatives 2007 19 General management I.Test Definition II.Within Laboratory Variability III.Transferability IV.Between Laboratory Variability V.Predictive Capacity VI.Applicability Domain VII.Performance Standards Modular ApproachWeight-of-evidence Principles Background Review Documents Retrospective evaluation – initiated fall 2005 ECVAM WS (May ’04)

20. Ankara, 12 Novembre 2007 – Alternatives 2007 20 Retrospective weight-of-evidence evaluation Sponsor & oversight Study design Data & evidence collection Weigh-of- evidence assessment Peer review ECVAM Core Validation Management Group Data Compilation Group & QC Audit Group Core Validation Management Group ESAC Weight-of-evidence principles - Balls et al. 2006, ATLA 34, 1-19

21. Ankara, 12 Novembre 2007 – Alternatives 2007 21 Summary Data Collection NRR, RBC, FL, CM Data Gaps NRR data on French Official protocol / Predisafe Raw data & protocol of RBC from Japanese studies Major protocols NRR Predisafe, Invittox 54, IIVS protocol RBC Invittox 37 & 99, Japanese protocols FL Invittox 120, 71, 86 & 82 CM IIVS protocol, Invittox 97 & 102 Within Lab. Variability 62 - 130 test substances from 4 to 14 studies Between Lab. Variability 55 -112 tested substances 2 to 9 laboratories, 1 to 4 major studies Predictive Capacity 86 - 175 tested substances from 3 to 4 studies

22. Ankara, 12 Novembre 2007 – Alternatives 2007 22 Outcome Submission to ECVAM ECVAM TF recommendations Protocol Optimisation Human Reconstituted Tissue Models 200520062007 SkinEthic HCE Corporate pre-validation 4 laboratories 20 chemicals Protocol Validation WS Prediction Model Enlarge Appl. Domain & Validation WS ECVAM Workshop to plan Validation Study EpiOcular OCL-200 Corporate validation Surfactant ingredients 4 laboratories 54 test substances Protocol optimisation & COLIPA feasibility study Protocol optimisation & feasibility study

23. Ankara, 12 Novembre 2007 – Alternatives 2007 23 Test Strategies

24. Ankara, 12 Novembre 2007 – Alternatives 2007 24 Test Strategies Proposed Testing Strategy ECVAM Expert Meeting, Feb 2005 30 Partners: COLIPA, Industry, CROs, Regulators, Academia, Welfare

25. Ankara, 12 Novembre 2007 – Alternatives 2007 25 Regulatory acceptance Topical Toxicity - Summary of activities Phototoxicity Skin Corrosion Eye Irritation Skin Irritation * Validation ESAC statement Prevalidation Follow-up regulatory acceptance * Partial replacement for severe irritants Skin Penetration Status in 2003

26. Ankara, 12 Novembre 2007 – Alternatives 2007 26 Ongoing * Regulatory acceptance Topical Toxicity - Summary of activities Phototoxicity Skin Corrosion Eye Irritation Skin Irritation Validation ESAC statement Prevalidation Follow-up regulatory acceptance Skin Penetration * Ongoing * Partial replacement for severe irritants Ongoing * Progress in 2007

27. Ankara, 12 Novembre 2007 – Alternatives 2007 27 Valérie Zuang Claudius Griesinger Acknowledgements

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