Alternative Methods to Animal Testing – A Cosmetic Industry Perspective Conference on Alternative Approaches to Animal Testing “EUROPE GOES ALTERNATIVE” Brussels, 7 November 2005 Odile de Silva L’Oréal

L’OREAL contribution to Validation Studies and International Programmes on Alternative Methods 1989 L’Oréal ends tests on cosmetic finished products 1992 EC/HO: Validation study on eye irritation (3 methods) 1993 MEIC Programme and acute toxicity IRAG: Alternatives to eye irritation (8 methods). 1994 Ring study on the BCOP test 1995 ECVAM/COLIPA Validation study on Phototoxicity 1996 Colipa Validation study on eye irritation (2 methods) 1997 4th FWP – Langerhans cells in reconstructed skin - Human skin models 1999 Colipa Guidelines on in vitro percutaneous absorption 2000 ECVAM pre validation study on skin irritation 2001 BIOMED II 2002 Dendritic cells and the Colipa Research Programme 2004 ECVAM validation study on skin irritation 2005 6th FWP - Sensitiv

PCOP for liquid and water-soluble materials Prediction of MAS Statistical analysis on 41 substances predicted MAS = 8.08 + 26.16 X DO30 – 5.47 X DO30² Concordance : 90 % Kappa = 0.83 (p<0.01). R2 = 0.84 95% confidence intervals too wide  = overpredicted = underpredicted.

«The living epidermis : « The full reconstructed Reconstructed skin and L’Oréal RESEARCH « Tanned epidermis » « Epidermis mimicking an allergenic response » The full reconstructed human skin ( E. Bell, D. Asselineau) The 1rst living reconstructed human epidermis (M. Pruniéras, M. Régnier) 1983 1986 1994 1997 2001 2002 2005 «The living epidermis : the Episkin kit  » «The : HTS  human epidermis model» « The mini living kit epidermis EKIN » « The full reconstructed human skin kit » « EPISKIN » Industrial models

Replacement of the skin irritancy test Stratum corneum Stratum granulosum Stratum spinosum Basal layers Normal Human Epidermis Reconstructed Human Epidermis EpiSkin The EpiSkin Model -12 tissues / kit -QC on all components -Tissue histology, viability, SDS IC.50 -ISO 9001 norms

Replacement of the skin irritancy test Viability endpoint -Predictive model Viability Class. EU OECD < 50 % Irritant R38 > 50 % Non Irritant No Class No label Set of 48 chemicals -20- Irritants -28- Non Irritants EpiSkin Optimized Protocol (EOP) Performance EpiSkin Limits* Sensitivity 85.0% > 60% Specificity 78.6% Accuracy 81.3% Positive Predictivity 73.9% nd Negative Predictivity 88.0% False Pos. 26.1% <40% False Neg. 12.0% Irritants Non Irritants * Recommended ECVAM limits

Episkin® : ranking compounds according their transcutaneous diffusion potential Stratum corneum Viable epidermis 4h Collagen Matrix Used in wells Low penetration Medium High penetration %Dose RF =100 Q applied Analysis performed with LCMSMS Group 1 Group 2 RF : receptor fluid

Comet assay on Episkin : Detection of photogenotoxic compounds UV-A 15 min + Lomefloxacin in a formulation (topical application) UV-A 15 min + Lomefloxacin in the medium UV-A 15 min Quantification

Epidermis and Langerhans cells Physiology of Langerhans cells Histology Langerin positive cells “Epidermal sheet” Sensitisation Photoimmunosuppression and UV Control Irritant Sensitiser Control SSR SSR + UV filters M. Régnier et al., J.Invest.Dermatol., 1997 –V. Facy et al. , J. Invest. Dermatol. , 2004

In vitro identification of contact sensitizers with human cell lines : one component of the battery ? Naive T Lymph node Langerhans cells Sens T U937 cell line Read out system : 48h U937 / CD86 test: L’Oréal internal validation 67 references tested including 52% sensitizers Accuracy with human clinic : 95% Kappa : 0.91

The use of the Reconstructed Human Epidermis Model EPISKIN® as a predictive in vitro irritation model for cosmetic ingredients. Comparison of 2 cationic surfactants differing only by their carbon chain length (C-22 and C-16).Similarity between human clinical irritation scores and in vitro results C-16 carbons C-22 carbons In vitro: strong decrease of epidermis viability (50% reduction). Poor tolerance, dose-effect. In vivo(clinical data): Significant increase of irritation values since 0.125%. Irritant, with dose-effect In vitro: no change in epidermis viability. Good tolerance In vivo(clinical data): no significant increase of irritation values. Well tolerated

The COLIPA research and development programme (since 1992) To develop novel approaches in the cosmetics fields of expertise for product safety assessment that do not involve any new animal testing.

THE COSMETIC CONTEXT RISK ASSESSMENT and NOT HAZARD Article 2 of the Cosmetics Directive NOT ONLY TOLERANCE BUT SYSTEMIC AND SUB-CHRONIC TOXICITY

COLIPA-SCAAT ACTIVITIES The Steering Committee for Alternatives to Animal Testing (SCAAT) has provided a focal point for the cosmetics industry’s efforts in the EU to develop alternative approaches for over 10 years The COLIPA R&D programme is directed towards identifying novel cellular and molecular endpoints for incorporation into new / improved alternative methods It is the intention that these alternative methods and strategies will be developed and evaluated to the stage that they are ready for prevalidation

Not applicable 2009 2013 Done Genotoxicity Eye irritation Photomutagenicity Subacute Subchronic Skin irritation Skin sensitisation Photosensitisation Acute toxicity Toxicokinetics Phototoxicity 2013 Teratogenicity Percutaneous absorption Reprotoxicity Finished Products Skin corrosion Carcinogenicity Done

THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN SENSITISATION COLLABORATION With ECVAM in their respective COLIPA & ECVAM TFs & ECVAM Workshops With other industry sectors : chemical & pharmaceutical companies With academia : through COLIPA & EU sponsored projects PENETRATION AND REACTION WITH ENDOGENOUS PROTEINS Toxicokinetic model Univ. Cincinnati and P&G Peptide reactivity assay Univ of Strasbourg and P&G Covalent binding assay Wella, Cosmital IDENTIFICATION OF SIGNAL TRANSDUCTION PATHWAYS INSERM Lyon, LVMH RESEARCH PROGRAMME INITIATED in 2001 OBJECTIVE : Understanding key mechanisms in order to build a battery of methods able to replace the current in vivo test for hazard evaluation and risk assessment purposes. LANGERHANS CELLS & DENDRITRIC CELL LINES Changes in gene expression P&G – Syngenta Examination of Markers Wella and Cosmital Interlaboratory ring trials Shiseido, KAO, L’OREAL, HENKEL, P& G, LVMH, Wella SENS-IT-IV IP – 6th FWP Novel in vitro approaches for skin & lung sensitisation COLIPA, ECVAM, Academia, Novozymes, Pharmaceutical, & Chemical companies, ECOPA, IVTIP. (30 partners)

THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN IRRITATION EPIDERMAL BIOAVAILABILITY Univ. Cincinnati, P&G CHANGES IN CYTOKINES RESPONSES Henkel RESEARCH PROGRAMME INITIATED in 2001 OBJECTIVE : To identify new markers of Skin Irritation in order to address risk assessment and not only hazard, the latter being investigated in the current ECVAM validation study CHANGES IN GENE EXPRESSION IN KERATINOCYTES Unilever FURTHER PROJECTS Under consideration and following the outcome of the ECVAM study

IN VITRO DYNAMIC CORNEAL CULTURE ASSAY 3D HUMAN CELL CULTURE MODELS THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES IN EYE IRRITATION COLLABORATION With academia through Colipa projects With ECVAM :  Participation in respective COLIPA & ECVAM TFs & in ECVAM Workshops With ICCVAM / NIEHS & ECVAM :  Contribution to expert reviews & workshop on Mechanisms. IN VITRO DYNAMIC CORNEAL CULTURE ASSAY INITIATED in January 2002 At the Aachen University, Germany Pr N. Schrage M. Frenz AIM : identify new signals & end points 3D HUMAN CELL CULTURE MODELS INITIATED in January 2002 At the Bristol University (UK) Dr M. Berry M. Radburn-Smith AIM : identify new endpoints RESEARCH PROGRAMME INITIATED in 2001 OBJECTIVE : Understanding mechanisms of eye irritation with a focus on injury and recovery of the CORNEA following an expert workshop and internal company research DEVELOPMENT OF GENE EXPRESSION FINGERPRINTS TO IDENTIFY DAMAGE TO THE CORNEA INITIATED in summer 2005 at Cardiff University (UK) Prof. Mike Boulton. DEVELOP NEW OR IMPROVED METHOD READY FOR PREVALIDATION

COLLABORATION WITH ECVAM THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES IN GENOTOXICITY OBJECTIVES : To develop new approaches in order to optimize the predictive capacity of current in vitro methods , or develop new methods able to predict genotoxic potential without the use of animal tests. Develop a battery in order to perform risk assessment and not only hazard. ASSESSMENT OF THE PREDICTIVE CAPACITY OF THE CURRENT IN VITRO METHODS : THE REASONS FOR FALSE POSITIVES (DR KIRKLAND) COLLABORATION WITH ECVAM Through its TF PROJECT UNDER DISCUSSION TO IDENTIFY MECHANISMS RESPONSIBLE FOR FALSE POSITIVES AND TO DEVELOP A STRATEGY BASED ON THIS NEW KNOWLEDGE PROJECT UNDER CONSIDERATION TO IMPROVE RISK ASSESSMENT, USING BIOLOGICAL TARGETS RELEVANT FOR SKIN EXPOSURE, BASED ON INTERNAL WORK OF COMPANIES, ON 3D MODELS DEVELOPMENT OF THE COLIPA STRATEGY

OTHER ENDPOINTS R&D NEEDS COLIPA current focus/priorities cover the fields where we have expertise : eye and skin irritation, skin sensitisation, and genotoxicity. There is a clear need to develop alternative approaches that cover all toxicological endpoints The cosmetic industry scientists have no specific expertise for developing systemic and chronic toxicity alternative approaches. Other partners : national government labs, ECVAM, and other industry sectors are very active in some of these areas (e.g. reproductive toxicity, acute toxicity)

THE CHALLENGES Good science : the cutting edge To attract the best scientists from academia To start now integrated projects for systemic and sub-chronic toxicity To combine all these data : Systems Biology New and pragmatic thinking Thresholds of Toxicological Concern (TTC)

FACING UP TO THE CHALLENGES The cosmetics industry has taken a leading role in its areas of expertise The cosmetics industry is ready to work in partnership with other stakeholders for the remaining challenges and to respond in a positive way to the EU political agenda in relation to developing alternative approaches to animal testing for assessing safety For the cosmetics industry , the SAFETY of its products is, and must always remain, the number one priority