The Contributions of the European Cosmetics Industry to the Development of Alternatives to Animal Testing: Dialogue with ECVAM and Future Challenges

Transcription

1 ATLA 30, Supplement 2, , The Contributions of the European Cosmetics Industry to the Development of Alternatives to Animal Testing: Dialogue with ECVAM and Future Challenges Odile de Silva On behalf of the COLIPA Steering Committee on Alternatives to Animal Testing (SCAAT) L Oréal, 90 rue de Général Roguet, Clichy Cedex, France Summary COLIPA (the European Federation of the Cosmetics Industry) represents 24 international companies and 2000 small and medium-sized enterprises. Together with ECVAM, COLIPA has been involved in the development and validation of alternative methods since the beginning of the validation efforts. The work of the Steering Committee on Alternatives to Animal Testing (SCAAT) is based on collaboration between companies, but also with academia, trade associations, the Scientific Committee on Cosmetics and Non-Food Products (SCCNFP), European Commission Directorates General, and ECVAM. Some success has been achieved, but some validation efforts have failed. One lesson is that the search for alternatives requires a lot of humility. Key words: alternative methods, COLIPA, cosmetics testing, percutaneous absorption, phototoxicity, sensitisation, skin corrosion, skin tolerance, validation. Introduction COLIPA (the European Federation of the Cosmetics Industry) is committed to the highest safety standards for its products, which are used by millions of customers daily, and is equally committed to the elimination of animal testing, as soon as it is satisfied that this is scientifically possible without compromising human safety or environmental protection. COLIPA will encourage the use of validated alternative tests whenever there are no legal requirements to use conventional animal tests, and will seek to persuade regulatory bodies to accept alternatives whenever animal tests are specified by legislation. In 1992, COLIPA created SCAAT (the Steering Committee on Alternatives to Animal Testing) to coordinate the efforts of the cosmetics industry in the development and promotion of alternatives to animals in cosmetic safety evaluation. SCAAT s strategy is based on three principles. 1) The cosmetics industry will be responsible for developing alternative methods that will be valid for the materials used in the cosmetics industry. 2) The cosmetics industry will be the lead industry for developing safety tests of particular relevance to cosmetics, such as skin and eye irritation, skin penetration and skin allergy. 3) COLIPA is European, and the efforts of SCAAT will therefore be primarily directed toward Europe. SCAAT works with four task forces, which focus on the following domains: phototoxicity, percutaneous absorption, eye irritation, and skin tolerance (which involves skin allergy and irritation). Collaboration and Validation with ECVAM From the 1970s to the 1990s, the cosmetics industry s efforts were focused on the development of finished-product evaluation strategies without the use of animal tests, as well as research on the development of new alternative methods (1 3). For the past ten years, the collaboration with ECVAM has opened up the validation process and has made it into a reality: hard, challenging, and rewarding. During all the years that COLIPA has collaborated with ECVAM, we have worked together on many issues related to alternative methods. Eye irritation was the first, followed by photoirritation, the challenge of the 6th Amendment to the EU Cosmetics Directive, skin corrosion, skin irritation, percutaneous absorption, skin allergy, and skin tolerance in human volunteers. All these subjects have been discussed in ECVAM workshops, with many of the co-chairs belonging to the cosmetics industry. The first real experience of validation was with eye irritation. This was where we had learned what validation is about. Before SCAAT was created, a cosmetics industry task force had been convened, and started work in 1990 (4). In vitro methods and protocols looked promising and, in 1992, we all joined in the European Community/Home Office

2 190 O. de Silva (EC/HO) study with considerable enthusiasm. The COLIPA Eye Irritation Task Force even set up a validation study to build on the EC/HO study. However, no alternative method was shown to be sufficiently reliable and relevant. In 1995, learning from this lesson, ECVAM created the concept of prevalidation (5), and the COL- IPA Task Force proposed the concept of the prediction model (6). These two contributions have fundamentally transformed the validation process, and have helped it considerably. One of the conclusions was that insufficient knowledge was available on the mechanisms of chemically induced eye irritation to enable us to mimic in vitro some of the key steps of eye irritation. COLIPA therefore convened a workshop with ophthalmologists to identify research hypotheses (7) and, in 1998, ECVAM organised a workshop with all the stakeholders from academia and industry (8). The COLIPA Eye Irritation Task Force worked on a research proposal and submitted it to the European Commission under the 5th Framework Programme. However, two attempts to obtain support were unsuccessful, for non-scientific reasons, so the cosmetics industry started the research on its own. Phototoxicity is a success story. It is also about lessons learned and the importance of good collaboration. In 1991, the COLIPA Photoirritation Task Force brought together representatives of the cosmetics and pharmaceutical industries, of DG Environment, and of ZEBET. Together, they organised a first multicentre study on several promising in vitro alternatives (9). ECVAM organised a workshop on alternatives to phototoxicity in 1993 (10), and in 1995 the ECVAM/COLIPA formal validation study, which built on the results of the previous Phase I study, was conducted (11). The 3T3 neutral red uptake phototoxicity method was successfully validated, and this result was endorsed by the ECVAM Scientific Advisory Committee (ESAC) in 1997: it was the first time that a method had been successfully validated! To demonstrate that the method was applicable to cosmetics ingredients, the COLIPA Task Force, in collaboration with ECVAM and ZEBET, established that UV filters could be assessed by the same method. The Scientific Committee for Cosmetics and Non-Food Products (SCCNFP) then endorsed the method (12). It has now been introduced into Annex V of Directive 65/548/EEC, and a draft guideline was accepted by the OECD National Coordinators in May As has already been highlighted, the combination of good science and up-front collaboration is the key to a successful validation (13). For skin corrosion, the prevalidation and validation phases were much shorter, and benefited from the experience gained in the previous validation studies (14). The SCAAT companies collaborated actively with ECVAM, leading to the endorsement by ESAC of two replacement alternatives: the TER and the EPISKIN methods, followed by endorsement by SCCNFP (15), the publication of the methods in Annex V of the Dangerous Substances Directive, and acceptance of draft guidelines by OECD National Coordinators for the Test Guidelines Programme, in May Percutaneous absorption is a key endpoint in the safety assessment of cosmetics ingredients, as it helps to determine the margin of safety. In this field, cosmetic companies have been using in vitro methods for the past 30 years. In 1994, ECVAM organised a workshop (16), and COLIPA set up a specific task force which issued guidelines reflecting the protocols and practices of industry (17, 18). The SCCNFP accepted the in vitro methods, and defined an additional set of basic criteria for cosmetic ingredients (19). COLIPA and ECPA (the European Crop Protection Agency) worked together to contribute data to the OECD, with a view to preparing guidelines and a guidance document for in vitro and in vivo percutaneous absorption studies. Following many years of discussion with many experts, these have now been accepted by the OECD National Coordinators for the Test Guidelines Programme. In this particular field, no new study was initiated, but a retrospective analysis of all the protocols and available data was carried out. This new approach is complex, but it also shows that the validation concept is very flexible, as long as it is based on good science, and the process is fully transparent. In the field of skin tolerance, ECVAM organised a workshop on the potential use of non-invasive methods in the safety assessment of cosmetic products (20). This was followed by the creation of an ECVAM task force to discuss alternative methods for skin irritation testing. An ECVAM workshop was held (21), and a prevalidation study was initiated. Three methods were identified as promising, but not yet ready for validation (22): the human skin models, EpiDerm and EPISKIN, and the SIFT model. Further work is under way, which should lead to improved protocols (23). Recent results are promising, and could lead to a validation study in An important aspect of safety for the cosmetics industry is to correctly identify contact sensitisers. An ECVAM workshop was organised in 1995, which established the promising areas of research for nonanimal alternatives (24). One of these was the evaluation of interleukin-1β as a specific signal produced by human dendritic cells when in contact with allergens. ECVAM co-funded this project with COLIPA. It was concluded that this endpoint was not sensitive enough, although the results were promising, and that further work and other endpoints should be studied (25). Another important area identified by the ECVAM workshop was skin metabolism and

3 Contributions of the European cosmetics industry to alternatives 191 allergy: COLIPA funded a project to gather all the available knowledge on skin metabolism. This was made concrete in a text-book review of the detailed metabolic chemistry associated with sensitisation (26). The cosmetics industry was involved for 15 years in the development of a refinement and reduction alternative, the local lymph node assay (LLNA), which today is accepted as valid by the Interagency Co-ordinating Committee on the Validation of Alternative Methods (ICCVAM) and by ECVAM, is accepted by the SCCNFP, and will now be published as an OECD guideline (27). In 1994, ECVAM organised a workshop to support the cosmetics industry, which was aimed at responding to the challenge of the 6th Amendment to the Cosmetics Directive (28). All the participants tried to identify a coordinated response, which would lead to validated methods and strategies. The close collaboration which followed has led to success in some areas. However, in the face of the enormous task of replacing animal experiments for all toxicity endpoints, we more than ever need a coordinated and comprehensive response. ECVAM is the key, having shown the way, and we, with all the stakeholders, remain committed to finding safe alternative solutions. The Way Forward and the Future with ECVAM To make progress in research in, and development of, new alternative tests, and to propose new candidate tests for validation by ECVAM, COLIPA is funding a research programme, which started in the autumn of The areas covered by this research programme are eye irritation, skin irritation and skin allergy, and five projects are being funded. The two projects on eye irritation are building on work performed on the area and depth of injury, which was conducted during the previous two years by Procter & Gamble. This had been planned by the COLIPA Eye Irritation Task Force, as part of the larger programme originally submitted for funding under the EU 5th Framework Program, but which was not supported. The first project is being conducted by the University of Aachen, Germany (Dr Alice Nietgen, Professor Norbert Schrage and Professor Martin Reim). It is focused on the pharmacokinetics of the response of the in vitro perfused cornea to chemical irritants. It will show whether patterns of change in physiological functions, and signals of injury released from the cornea, can be used to predict a chemical s potential to damage the eye. The timecourse of morphological changes will be studied for up to 28 days. The physiological parameters being investigated are: barrier function; corneal thickness and opacity; changes in ph; redox potential and levels of K + ; and release of enzymes and inflammatory mediators. The second project is looking at injury and recovery in human corneal cells, in conventional monolayer cultures and in 3-dimensional models. It is being conducted at the University of Bristol, UK (Dr Monica Berry and Dr Marcus Radburn-Smith). It will identify endpoints related to the magnitude of injury and the quality of repair in human immortalised cells and 3-dimensional constructs, and will address the characterisation and selection of cell lines. The quality of the constructs will be studied, and opacity will be measured by confocal microscopy. Keratocyte activation and the release of enzymes and inflammatory mediators will be assessed, as well as recovery. Four chemicals will be tested in both projects in the first year. It is hoped that new endpoints will be found which will help to improve existing models and/or aid the design of new methods and prediction models. Promising methods and prediction models will then be transferred to ECVAM for prevalidation and validation. In the field of skin irritation, ECVAM is planning to follow the ongoing prevalidation study on human skin models and the SIFT with a formal validation study. If successful, the results will permit chemicals to be classified as irritants or non-irritants according to current hazard phrases. The SCAAT project will complement the ECVAM approach. It will evaluate the detailed mechanisms of the less-severe skin irritation response, and will try to identify novel markers of irritation by using genomic and proteomic analyses of early changes in human skin models, in response to chemical irritants, with a view to submitting methods to ECVAM for prevalidation. Two industry laboratories are involved in this work, namely those of Unilever and Henkel. They will review the current state of the art on mechanisms and models, define the major cellular chemically induced mechanisms, and determine in vitro recovery after a simple application of irritants. They will also study the irritation response at the genomic level and confirm the response by the polymerase chain reaction, as well as the selection of endpoints at the protein/functional level. This will be performed with various classes of irritants. Finally, we have started two research projects on skin allergy. Some progress has been achieved recently in the evaluation of the response of in vitro-generated dendritic cells obtained from human myeloid cell lines (U937, TMP-1). Previous work on primary dendritic cells has shown the pivotal role of these cells, but with poor reproducibility and therefore some lack of sensitivity (29). The first three-year project, which will be conducted by Wella, will build on recent results

4 192 O. de Silva obtained by other teams at L Oréal, Kao and Shiseido), will try to define an optimised cell line culture system for the identification of contact allergens, with the intention of developing a method and a prediction model ready for prevalidation by ECVAM. The first year of the project will be used to evaluate and compare culture protocols for obtaining dendritic cell-like characteristics with human monocytic cell lines (U-937, TMP1 and KG- 1). The modulation of markers at the protein and mrna levels will be investigated. A large number of reference sensitisers and irritants will be tested during the second and third years. Possible signal transduction pathways and co-culture with keratinocytes will also be explored. This first project will be developed in a close relationship with the second one, which will examine the modulation of gene expression. During the first year, the project will identify candidate genes which are regulated following the exposure of dendritic cells to sensitisers, as well as new genes in dendritic cells and T-cells during primary immune responses. During the second year, it is planned to determine whether the expression profiles of selected genes are regulated selectively by contact allergens. The dynamic range of the response of these specific genes and therefore the level of sensitivity of the response will also be explored, with a view to developing an in vitro model for skin sensitisation potential. We support the European Council s Common Position on the 7th Amendment to the Cosmetics Directive. We strongly believe, like ECVAM, that a step-by-step replacement of animal tests, as soon as alternative methods have been validated or endorsed as valid by ECVAM or other agencies, is the most efficient approach to replace laboratory animal test procedures. The Commission s White Paper on chemicals has brought the issue of animal testing for chemical safety more sharply into focus. There is a strong need to replace animal tests by scientifically validated alternative methods: the SCAAT research programme is addressing some of these tests, but the task is huge. That is why we call for a coordinated and comprehensive European strategy to guide research and development on alternatives to animal testing, involving all the stakeholders industry, competent authorities and public and private research institutions. References 1. Lisansky, S., Macmillan, R. & Dupuis, J., eds (1996). Alternatives to Animal Testing. Proceedings of an International Scientific Conference Organised by the European Cosmetics Industry, Brussels, Belgium 1995, 377pp. Newbury, UK: CPL Press. 2. COLIPA (1997). Guidelines for the Safety Assessment of Cosmetic Products. Brussels, Belgium: COL- IPA. 3. Clark, D., Lisansky, S. & Macmillan, R., eds (1999). Alternatives to Animal Testing II. Proceedings of the Second International Scientific Conference Organised by the European Cosmetics Industry, Brussels, Belgium, 294pp. Newbury, UK: CPL Press. 4. Bagley, D.M., Bruner, L.H., de Silva, O., Cottin, M., O Brien, K.A.F., Uttley, M. & Walker, A.P. (1992). An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo. Toxicology in Vitro 6, Curren, R.D., Southee, J.A., Spielmann, H., Liebsch, M., Fentem, J.H. & Balls, M. (1995). The role of prevalidation in the development, validation and acceptance of alternatives methods. ATLA 23, Brantom, P.G., Bruner, L.H., Chamberlain, M., de Silva, O., Dupuis, J., Earl, L.K., Lovell, D.P., Pape, W.J.W., Uttley, M., Bagley, D.M., Baker, F.W., Bracher, M., Courtellemont, P., Declercq, L., Freeman, S., Steiling, W., Walker, A.P., Carr, G.J., Dami, M., Thomas, G., Harbell, J., Jones, P.A., Pfannenbecker, U., Southee, J.A., Tcheng, M., Argembeaux, H., Casteli, D., Clothier, R., Esdaile, D.J., Itagaki, H., Junk, K., Kasai, Y., Kojima, H., Kristen, U., Larnicol, M., Lewis, R.W., Marenus, K., Moreno, O., Peterson, A., Rasmussen, E.S., Robles, C. & Stern, M. (1997). A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test. Toxicology in Vitro 11, Bruner, L.H., de Silva, O., Earl, L.K., Pape, W. & Spielmann, H. (1998). Report on the COLIPA workshop on mechanisms of eye irritation. ATLA 26, Balls, M., Berg, N., Bruner, L.H., Curren, R.D., de Silva, O., Earl, L.K., Esdaile, D.J., Fentem, J.H., Liebsch, M., Ohno, Y., Prinsen, M.K., Spielmann, H. & Worth, A.P. (1999). The report and recommendations of ECVAM workshop 34. ATLA 27, Spielmann, H., Balls, M., Brand, M., Döring, B., Holzhütter, H-G., Kalweit, S., Klecak, G., Lépannetier, H., Liebsch, M., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfannenbecker, U. Potthast, J., de Silva, O., Steiling, W. & Willshaw, A. (1994). EC/COLIPA project on in vitro phototoxicity testing: first results obtained with the Balb/c 3T3 cell phototoxicity assay. Toxicology in Vitro 8, Spielmann, H., Lovell, W.W., Hölzle, E., Johnson, B.E., Maurer, J., Miranda, M., Pape, W.J.W., Sapora, O. & Sladowski, D. (1994). In vitro phototoxicity testing. The report and recommendations of ECVAM workshop 2. ATLA 22, Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G., de Silva, O., Holzhütter, H.G., Clothier, R., Desolle, P., Gerberick, G.F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J.M., Csato, M., Sladowski, D., Steiling, W. & Brantom, P. (1998). The international EU/COL- IPA in vitro phototoxicity validation study: results of Phase II (blind trial); part I: the 3T3 NRU phototoxicity test. Toxicology in Vitro 12, SCCNFP (1998). Opinion on In Vitro Methods to Assess Phototoxicity in the Safety Evaluation of Cosmetic Ingredients or Mixtures of Ingredients. SCCNFP/0069/98. Brussels, Belgium: European Commission, Health & Consumer Protection Directorate General. 13. de Silva, O. & Loprieno, N. (2000). Collaboration as the way to the acceptance of alternative methods. In

5 Contributions of the European cosmetics industry to alternatives 193 Progress in the Reduction, Refinement and Replacement of Animal Experimentation (ed M. Balls, A-M. van Zeller & M.E. Halder), pp Amsterdam, The Netherlands: Elsevier Science. 14. Fentem, J.H., Archer, G.F.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhütter, H.G. & Liebsch, M. (1998). The ECVAM international validation study on in vitro tests for skin corrosivity. II. Results and evaluation by the Management Team. Toxicology in Vitro 12, SCCNFP (1998). Opinion on In Vitro Methods to Assess Skin Corrosivity in the Safety Evaluation of Cosmetic Ingredients or Mixtures of Ingredients. SCCNFP/0070/98. Brussels, Belgium: European Commission, Health & Consumer Protection Directorate General. 16. Howes, D., Guy, R., Hadgraft, J., Heylings, J., Hoeck, U., Kemper, F., Maibach, H., Marty, J-P., Merk, H., Parra, J., Rekkas, D., Rondelli, I., Schaefer, H., Täuber, U. & Verbiese, N. (1996). Methods for assessing percutaneous absorption. The report and recommendations of ECVAM workshop 13. ATLA 24, COLIPA (1995). Cosmetic Ingredients: Guidelines for Percutaneous Absorption/Penetration. 20pp. Brussels, Belgium: COLIPA. 18. Diembeck, W., Beck, H., Benech-Fieffer, F., Courtellemont, P., Dupuis, J., Lovell, W., Paye, M., Spengler, J. & Steiling, W. (1999). Test Guidelines for in vitro assessment of cosmetic ingredients. Food and Chemical Toxicology 37, SCCNFP (1999). Opinion on In Vitro Methods to Assess Percutaneous Absorption of Cosmetic Ingredients. SCCNFP/0088/98. Brussels, Belgium: European Commission, Health & Consumer Protection Directorate General. 20. Rogiers, V., Balls, M., Basketter, D.A., Berardesca, E., Edwards, C., Elsner, P., Ennen, J., Lévêque, J.L., Loden, M., Masson, P., Parra, P., Paye, M., Pierard, G., Rodrigues, L., Schaefer, H., Salter, D. & Zuang, V. (1999). The potential use of non-invasive methods in safety assessment of cosmetics. The report and recommendations of ECVAM workshop 38. ATLA 27, Van de Sandt, H., Roguet, R., Cohen, C., Esdaile, D., Ponec, M., Corsini, E., Barker, C., Fusenig, N., Liebsch, M., Benford, D., de Brugerolle de Fraissinett, A. & Fartasch, M. (1999). The use of human keratinocytes and human skin models for predicting skin irritation. ATLA 27, Fentem, J.H., Briggs, D., Chesné, C., Elliott, G.R., Harbell, J.W., Keyling, J.R., Portes, P., Roguet, R., Van de Sandt, H. & Botham, P. (2001). A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, Zuang, V., Balls, M., Botham, P., Coquette, A., Corsini, E., Curren, R.D., Elliott, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., Van de Sandt, H., Wiemann, C. & Worth, A.P. (2002). Follow up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ECVAM Skin Irritation Task Force Report 2. ATLA 30, de Silva, O., Basketter, D.A., Barratt, M.D., Corsini, E., Cronin, M.T.D., Das, P.K., Degwert, J., Enk, A., Garrigue, J.L., Hauser, C., Kimber, I., Lepoittevin, J.P., Peguet, J. & Ponec, M. (1996). Alternative methods for skin sensitisation testing. The report and recommendations of ECVAM workshop 19. ATLA 24, Pichowski, J.S., Cumberbatch, M., Dearman, R.J., Basketter, D.A. & Kimber, I. (2000). Investigation of induced changes in Interleukin 1β mrna expression by cultured human dentritic cells as an in vitro approach to skin sensitisation testing. Toxicology in Vitro 14, Smith, C. & Hotchkiss, S. (2001). Allergic Contact Dermatitis: Chemical and Metabolic Mechanisms, 344pp. London, UK: Taylor and Francis. 27. Basketter, D., Gerberick, F. & Kimber, I. (2000). Validation in practice the reality for skin sensitisation. In Progress in the Reduction, Refinement and Replacement of Animal Experimentation (ed. M. Balls, A-M. van Zeller & M.E. Halder), pp Amsterdam, The Netherlands: Elsevier. 28. Balls, M., De Klerck, W., Baker, F., Van Beck, M., Bouillon, C., Bruner, L., Carstensen, J., Chamberlain, M., Cottin, M., Dupuis, J., Faure, U., Fentem, J., Fisher, J., Galli, C., Kemper, F., Knaap, A., Langley, G., Loprieno, G., Loprieno, N., Pape, W., Pechovitch, G., Spielmann, H., Ungar, K., White, I. & Zuang, V. (1995). Development and validation of non-animal tests and testing strategies: the identification of a coordinated response to the challenge and the opportunity to the Cosmetics Directive (76/768/EEC). The report and recommendations of ECVAM workshop 7. ATLA 23, Rousset, F., Verda, D., Arrighi, J.F., Péguet- Navarro, J. & Cottin, M. (2000). A strategy for the prediction of skin sensitisation: an alternative to animal testing. In Progress in the Reduction, Refinement and Replacement of Animal Experimentation (ed. M. Balls, A-M. van Zeller & M.E. Halder), pp Amsterdam, The Netherlands: Elsevier.