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Alternative Methods to Animal Testing – A Cosmetic Industry Perspective Conference on Alternative Approaches to Animal Testing EUROPE GOES ALTERNATIVE.

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Presentation on theme: "Alternative Methods to Animal Testing – A Cosmetic Industry Perspective Conference on Alternative Approaches to Animal Testing EUROPE GOES ALTERNATIVE."— Presentation transcript:

1 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 LOréal

2 LOREAL contribution to Validation Studies and International Programmes on Alternative Methods 1989LOréal 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) Ring study on the BCOP test 1995 ECVAM/COLIPA Validation study on Phototoxicity 1996 Colipa Validation study on eye irritation (2 methods) th 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 th FWP - Sensitiv

3 PCOP for liquid and water-soluble materials Prediction of MAS Statistical analysis on 41 substances predicted MAS = X DO 30 – 5.47 X DO 30 ² = overpredicted = underpredicted. Concordance : 90 % Kappa = 0.83 (p<0.01). R2 = % confidence intervals too wide

4 Reconstructed skin and LOréal «The living epidermis : the Episkin kit » «The : HTS human epidermis model» « The mini living kit epidermis kit epidermisEKIN » « The full reconstructed human skin kit » « EPISKIN » Industrial models « Epidermis mimicking an allergenic response » « Epidermis mimicking an allergenic response » « Tanned epidermis » 2001 The 1rst living reconstructed human epidermis (M. Pruniéras, M. Régnier) 1986 The full reconstructed human skin ( E. Bell, D. Asselineau) RESEARCH

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

6 Replacement of the skin irritancy test EpiSkinLimits* Sensitivity 85.0%> 60% Specificity 78.6%> 60% Accuracy 81.3%> 60% Positive Predictivity 73.9%nd Negative Predictivity 88.0%nd False Pos. 26.1%<40% False Neg. 12.0%<40% Viability endpoint -Predictive model * Recommended ECVAM limits ViabilityClass.EUOECD < 50 %IrritantR38Irritant > 50 %Non IrritantNo ClassNo label Performance Set of 48 chemicals -20- Irritants -28- Non Irritants EpiSkin Optimized Protocol (EOP) Irritants Non Irritants

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

8 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 Quantification

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

10 Naive T Lymph node Langerhans cells Sens T U937 cell line Read out system : 48h In vitro identification of contact sensitizers with human cell lines : one component of the battery ? U937 / CD86 test: LOréal internal validation 67 references tested including 52% sensitizers Accuracy with human clinic : 95% Kappa : 0.91

11 EPISKIN® 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 In vitro: no change in epidermis viability. Good tolerance In vivo(clinical data): no significant increase of irritation values. Well tolerated 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 C-16 carbons

12 - 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.

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

14 COLIPA-SCAAT ACTIVITIES The Steering Committee for Alternatives to Animal Testing (SCAAT) has provided a focal point for the cosmetics industrys 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

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

16 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 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. IDENTIFICATION OF SIGNAL TRANSDUCTION PATHWAYS INSERM Lyon, LVMH 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 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) LANGERHANS CELLS & DENDRITRIC CELL LINES -Changes in gene expression P&G – Syngenta -Examination of Markers Wella and Cosmital -Interlaboratory ring trials Shiseido, KAO, LOREAL, HENKEL, P& G, LVMH, Wella THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN SENSITISATION

17 THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN IRRITATION 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 CYTOKINES RESPONSES Henkel EPIDERMAL BIOAVAILABILITY Univ. Cincinnati, P&G FURTHER PROJECTS Under consideration and following the outcome of the ECVAM study CHANGES IN GENE EXPRESSION IN KERATINOCYTES Unilever

18 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. 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. 3D HUMAN CELL CULTURE MODELS INITIATED in January 2002 At the Bristol University (UK) Dr M. Berry M. Radburn-Smith AIM : identify new endpoints 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 DEVELOP NEW OR IMPROVED METHOD READY FOR PREVALIDATION

19 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. COLLABORATION WITH ECVAM Through its TF ASSESSMENT OF THE PREDICTIVE CAPACITY OF THE CURRENT IN VITRO METHODS : THE REASONS FOR FALSE POSITIVES (DR KIRKLAND) PROJECT UNDER CONSIDERATION TO IMPROVE RISK ASSESSMENT, USING BIOLOGICAL TARGETS RELEVANT FOR SKIN EXPOSURE, BASED ON INTERNAL WORK OF COMPANIES, ON 3D MODELS PROJECT UNDER DISCUSSION TO IDENTIFY MECHANISMS RESPONSIBLE FOR FALSE POSITIVES AND TO DEVELOP A STRATEGY BASED ON THIS NEW KNOWLEDGE DEVELOPMENT OF THE COLIPA STRATEGY

20 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)

21 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)

22 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


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