Development and Validation of Non-Animal Tests and Testing Strategies: the Identification of a Coordinated Response to the Challenge and the Opportunity Presented by the Sixth Amendment to the Cosmetics Directive (76/768/EEC)

The Report and Recommendations of ECVAM Workshop 71,2

Reprinted with minor amendments from ATLA 23, 398-409

Michael Balls3, Walter De Klerck4, Frank Baker5, Michel van Beek4, Claude Bouillon6, Leon Bruner5, Jorgen Carstensen7, Mark Chamberlain8, Martine Cottin9, Rodger Curren10, Jack Dupuis11, Frank Fairweather12, Uta Faure13, Julia Fentem3, Chris Fisher14, Corrado Galli15, Fritz Kemper16, Ada Knaap17, Gill Langley18, Gregorio Loprieno19, Nicola Loprieno20, Wolfgang Pape21, Georges Pechovitch22, Horst Spielmann23, Krys Ungar24, Ian White25 and Valérie Zuang3
3ECVAM, JRC Environment Institute, 21020 Ispra (Va), Italy; 4Consumer Policy Service, European Commission, 200 rue de la Loi, 1049 Brussels, Belgium; 5Procter & Gamble Health & Beauty Care Europe, Rusham Park, Whitehall Lane, Egham, Surrey TW20 9NW, UK; 6Direction Générale Scientifique, L'Oréal, 41 rue Martre, 92117 Clichy Cédex, France; 7Department of Toxicology and Applied Pharmacology, Novo Nordisk A/S, 2880 Bagsvaard, Denmark; 8Environmental Safety Laboratory, Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK; 9Laboratoire de Recherche Fondamentale, L'Oréal, 1 Avenue Eugéne Schueller, 93601 Aulnay-sous-Bois, France; 10Microbiological Associates Inc., 9900 Blackwell Road, Rockville, MD 20878, USA; 11COLIPA, 223 rue de la Loi, 1040 Brussels, Belgium; 12394 London Road, Langley, Berks. SL3 7HX, UK; 13DGXII/C/5, European Commission, 200 rue de la Loi, 1049 Brussels, Belgium; 1466 Higham Road, London N17 6NQ, UK; 15Istituto di Scienze Farmacologiche, Universita degli Studi di Milano, 9 via Balzaretti, 20093 Milan, Italy; 16Umweltprobenbank für Human-Organproben, Westf. Wilhelms Universität, Domagkstrasse 11, 48129 Münster, Germany; 17Hoofd Adviescentrum Toxicologie, Rijksinstitut voor Volksgezondheid en Milieuhygiene, 3720 BA Bilthoven, The Netherlands; 18Dr Hadwen Trust for Humane Research, 22 Bancroft, Hitchin, Herts. SG5 1JW, UK; 19TEC.SER. Sas, 59 via Vecchia Lucchese, 56123 Pisa, Italy; 20Department of Environmental Sciences, University of Pisa, 22 via S. Giuseppe, 56126 Pisa, Italy; 214232-Safety Assessment Center, Beiersdorf AG, 20245 Hamburg, Germany; 22DGXI/C/6, European Commission, 200 rue de la Loi, 1049 Brussels, Belgium; 23ZEBET, BgVV, Diedersdorfer Weg 1, 12277 Berlin, Germany; 24FRAME, Russell & Burch House, 96-98 North Sherwood Street, Nottingham NG1 4EE, UK; 25St Thomas' Hospital, London SE1 7EH, UK.

1ECVAM - The European Centre for the Validation of Alternative Methods. 2This document represents the agreed report of the participants as individual scientists.

Address for correspondence: Professor Michael Balls, ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (Va), Italy

Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy

Preface

This is the report of the seventh of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1).

This particular workshop was a joint initiative of ECVAM and the Consumer Policy Service (CPS), and the two Commission services cooperated closely in its organisation. The workshop was held in Angera, Italy on 11-13 April 1994, under the co-chairmanship of Michael Balls (ECVAM) and Walter De Klerck (CPS). A number of relevant developments have taken place since the workshop was held. Some of these have been noted in this report, in order to make it as up-to-date as possible.

Introduction

Article 1 of Council Directive 93/35/EEC (2), which amends Council Directive 76/768/EEC (3) for the sixth time, defines a cosmetic product as any substance or preparation intended to be placed in contact with the various external parts of the human body (epidermis, hair system, nails, lips, and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance and/or correcting body odours and/or protecting them or keeping them in good condition.

Article 2 of the Sixth Amendment requires that a cosmetic product put on the market within the Community must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use, taking account, in particular, of the product's presentation, its labelling, and any instructions for its use and disposal, as well as any other indication or information provided by the manufacturer or his authorized agent or by any other person responsible for placing the product on the Community market.

Article 4 states that, without prejudice to their general obligations deriving from Article 2, Member States shall prohibit the marketing of cosmetic products containing ingredients or combinations of ingredients tested on animals after 1 January 1998 in order to meet the requirements of this Directive. However:

If there has been insufficient progress in developing satisfactory methods to replace animal testing and in particular in those cases where alternative methods of testing, despite all reasonable endeavours, have not been scientifically validated as offering an equivalent level of protection for the consumer, taking into account OECD toxicity test guidelines, the Commission shall, by 1 January 1997, submit draft measures to postpone the date of implementation of this provision, for a sufficient period, and in any case for no less than two years, in accordance with the procedure laid down in Article 10. Before submitting such measures, the Commission will consult the Scientific Committee on Cosmetology.

The Commission shall present an annual report to the European Parliament and the Council on progress in the development, validation and legal acceptance of alternative methods to those involving experiments on animals. That report shall contain precise data on the number and type of experiments relating to cosmetic products carried out on animals. The Member States shall be obliged to collect that information in addition to collecting statistics as laid down by Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes. The Commission shall in particular ensure the development, validation and legal acceptance of experimental methods which do not use live animals.

At the joint ECVAM/CPS workshop, representatives of various services of the European Commission discussed the implications of the Sixth Amendment with representatives of the cosmetic industry, centres involved in organising validation studies, databases on alternative methods, and the animal welfare movement, and with academic and industrial in vitro toxicologists.

The main objectives of the workshop were:

  1. To assess the progress being made in the development of alternative methods.
  2. To identify any obstacles to the successful validation and acceptance of these alternative methods.
  3. To define the actions necessary for removing any such obstacles.
  4. To identify the most promising areas for replacement of the current animal tests.
  5. To assess the value of the existing databases on alternative methods.
  6. To compare the predictive values of in vivo and in vitro tests.
  7. To strengthen cooperation between those concerned in any way in the cosmetics testing issue.
  8. To suggest criteria for developing and validating alternative methods to be used in the safety evaluation of cosmetics.

Responsibilities Within the Commission

The CPS (now DGXXIV) is responsible for the administration of Council Directive 76/768/EEC, including the implementation of the Sixth Amendment and the production of the annual report for the European Parliament and the Council. The performance of these duties requires liaison with other services of the Commission and with the cosmetic industry, notably with COLIPA (the European Cosmetic, Toiletry, and Perfumery Association). The first annual report was published in December 1994 (4).

The CPS is advised by the Scientific Committee on Cosmetology (SCC), which consists of highly qualified scientists from the Member States of the European Union (EU). The SCC assists the CPS with all matters related to the safety evaluation of cosmetic ingredients (5). The SCC is consulted about every Commission proposal to adapt the Cosmetics Directive to technical progress. Furthermore, the Sixth Amendment requires that the European Commission consults the SCC before submitting draft measures to postpone the date of implementation of the ban on animal testing in the event that there is insufficient progress, by 1 January 1997, in developing satisfactory methods to replace animal testing.

Unit C/6 of DGXI, the Directorate-General for Environment, Nuclear Safety and Civil Protection, is responsible for the administration of Council Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes (6). Article 7(2) of this directive requires that an experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available. Article 26 of Directive 86/609/EEC requires that statistics on animal use should be submitted to the Commission by the Member States at regular intervals not exceeding three years. The first set of statistics was published in May 1994 (7), and it is proposed that publication will take place every two years in the future. Discussions are taking place between the CPS, DGXI/C/6 and the Member States concerning the annual publication of statistics on animals used, in compliance with Directive 76/768/EEC.

Article 23 of Directive 86/609/EEC states that the Commission and Member States should encourage research into the development and validation of alternative techniques which could provide the same level of information as that obtained in experiments using animals but which involve fewer animals or which entail less painful procedures, and shall take such other steps as they consider appropriate to encourage research in this field. This led to the announcement by the Commission in 1991 that a European Centre for the Validation of Alternative Methods (ECVAM) was to be established, as a relatively independent unit of the Environment Institute of the Commission's Joint Research Centre, at Ispra, in Italy (8). The duties of ECVAM were spelled out as follows:

  1. To coordinate the validation of alternative test methods at EU level. This will involve the specification of test protocols, the organisation of ring-test exercises, the choice of chemicals to be used in these tests, and the analysis and evaluation of the results, etc.
  2. To act as a focal point for the exchange of information on the development of alternative test methods.
  3. To set up, maintain, and manage a data base on alternative procedures, with associated user services.
  4. To promote dialogue among legislators, industrial companies, biomedical scientists, consumer organisations and animal welfare groups, with a view to the development, validation, and international recognition of alternative test methods.

ECVAM's principal aim is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine, or replace the use of laboratory animals. ECVAM's attention will not be focused solely on regulatory toxicity testing, but also, for example, on the use of alternatives in assessing the efficacy of medicines and the potency of vaccines. ECVAM has a Scientific Advisory Committee composed of individuals from the Member States, industry, the academic world, and animal welfare organisations.

Also in response to Article 23 of Directive 86/609/EEC, support for the development of non-animal tests is included in the BIOTECH and BIOMED 2 programmes of Directorate E - R & TD: Life Sciences and Technologies of DGXII, the Directorate-General for Science, Research, and Development. Directorate C - R & TD: Industrial and Materials Technologies of DGXII is also interested in the development of new methods for routine use by industrial companies, including in vitro toxicity tests.

The Scientific Committee on Cosmetology

The SCC was set up in 1978 to advise the Commission on all matters related to the safety of cosmetics (5). SCC guidelines for the safety evaluation of cosmetic ingredients were first published in 1982 and were last revised in 1990 (9). The SCC must be consulted on any problem of a scientific or a technical nature in the field of cosmetic products and, in particular, on substances used in the preparation of cosmetic products and on the composition and conditions of use of such products (5). Therefore, under the terms of Directive 76/768/EEC, and acting on the advice of the SCC, the Commission published lists of chemicals which may not be used in cosmetic products manufactured or marketed in the EU, and positive lists of ingredients which can be used as UV-filters, colouring agents, and preservatives. The main goal of the SCC/CPS is the safety evaluation of cosmetic ingredients for human use and application. In fulfilment of this task, the SCC has also to advise on which alternative methods are applicable in this safety evaluation. Moreover, the SCC and its responsible sub-committees will update the appropriate guidelines.

To date, the SCC has evaluated about 300 cosmetic ingredients for their safety. The Committee takes into account physico-chemical data, in vivo and in vitro test results, and likely human exposure route, pattern and level. For toxicological characterisations, data on the following are required: acute toxicity, dermal absorption, dermal irritation, mucous membrane irritation, skin sensitisation, sub-chronic toxicity, mutagenicity, phototoxicity, and human responses (if available). When considerable oral intake is possible, or when the dermal absorption data indicate the possibility of considerable skin penetration, further in vivo data may be required on toxicokinetics, teratogenicity, reproductive toxicity, additional genotoxicity, and carcinogenicity.

The SCC's approach assumes that the safety evaluation of products can usually be based on the toxicological potentials of their ingredients. However, in certain cases, the testing of finished products may be advisable, for example, where the formulations of the finished products incorporate solvents which are different from those used in the toxicity testing of the ingredients, since this may affect dermal absorption considerably. Other examples are where the potentiation of toxic effects might occur because of interactions between the ingredients, and when manufacturers claim that inclusion in the formulation reduces the potential hazard of one or more of its ingredients.

The SCC is keen to encourage the use of alternative methods as soon as their relevance and reliability have been established and has, therefore, established a subgroup on alternatives with the following remit:

The members of the SCC who participated in the workshop emphasised that, since the three categories for which positive lists of ingredients have been produced (i.e. UV-filters, colouring agents, and preservatives) constitute chemicals of many different classes, validation studies on the testing of chemicals likely to be used for these purposes should be given a high priority.

The Animal Welfare Viewpoint

The participants from animal welfare organisations emphasised that the Sixth Amendment to Directive 76/768/EEC meant that the ban on the use of ingredients tested in animals after 1 January 1998 would come into force, unless it could be shown that the necessary scientifically-validated alternatives were not available. Thus, significant progress must be achieved by 1 January 1997, or there would be great pressure for a complete ban on animal tests, regardless of the status of the alternatives to replace them. It was therefore essential that there be ways of quantitatively assessing the progress being made toward the elimination of the need for animal testing. This, in turn, meant that the precise data specified by the Sixth Amendment must be provided. These data should include the purpose of testing, the animal species used, the types of experiment conducted, and the categories of ingredients and products tested.

Moreover, although it could be argued that the Commission need only produce statistics on animal use directly in compliance with Directive 76/768/EEC, information should also be provided on the testing of cosmetic ingredients conducted within the EU in compliance with other Directives and/or in accordance with the regulatory or other requirements of other countries and regions. In addition, information should be provided on the regulation and licensing of the testing of cosmetic ingredients in animals in each of the Member States of the EU.

The conclusions reached at a meeting organised jointly by the International Fund for Animal Welfare and Eurogroup for Animal Welfare (10), held in Munich in March 1994, were discussed. The participants in that discussion had identified three categories of testing in terms of the prospects of meeting the 1998 deadline:

The Cosmetic Industry Perspective

The workshop was informed of a number of COLIPA initiatives, including:

Participants from the cosmetic companies represented at the workshop outlined their in-house strategies for developing non-animal tests and incorporating them into the decision-making process.

One approach is to compare the results obtained with a range of in vitro methods, as a means of evaluating their respective advantages and disadvantages when used alone or in combination. For example, in the case of alternatives to the Draize eye irritation test, the performances of physico-chemical, cell culture, organotypic, and eye-part methods have been evaluated by deriving Pearson and Spearman correlation coefficients and by multivariate analysis. It was found that different tests and combinations of tests can be useful for surfactant-based and finished products. Therefore, the selected alternative methods have been incorporated in a stepwise approach into the decision-making process for finished products, and are part of the evaluation of ingredients.

Another approach involves a sequential consideration of:

This approach is being used to distinguish between corrosive and non-corrosive materials and to predict skin sensitisation for new chemicals.

An example was given of a stepwise approach for making decisions with respect to new chemicals. It involves three steps. Step 1 is to study the physico-chemical properties of a molecule and ask whether it can be classified on this basis alone. If not, Step 2 involves the use of an in vitro test or the conduct of a limited animal study. If necessary, a full OECD/Annex V study is undertaken in Step 3. In the case of particularly harmful materials, decisions can sometimes be taken on the basis of the results of Step 1 alone, or those from Step 1 and Step 2. For example, some chemicals can be classified as eye irritants on the basis of their pH values alone, while others can be classified as irritants on the basis of results obtained in cytotoxicity tests, eye-part tests (for example, the isolated rabbit eye test) and/or organotypic tests (for example, the HET-CAM method).

The strategy used for the eye and skin irritation testing of chemicals by the regulatory authorities in Germany was outlined. The stepwise scheme is as follows:

  1. If the pH of a material is higher than 11 or less than 2, it is labelled as being corrosive (R34).
  2. If the pH is between 2 and 11, it is tested for skin corrosivity.
  3. If the result is positive, the material is labelled as being corrosive (R34 or R35).
  4. If the result is negative, SAR analysis is performed.
  5. If the SAR analysis clearly indicates that the material is likely to be a severe irritant, the material is labelled as such (R41).
  6. If the result is negative, the material is tested by in vitro methods (for example, the HET-CAM test).
  7. If the in vitro result is positive, the material is labelled as being a severe irritant (R41).
  8. If the result is negative, the material is tested on one animal.
  9. If the in vivo result is positive, the material is labelled as a severe irritant (R41).
  10. If the result is negative, the material is tested on two more animals; according to the result obtained, the material is labelled as being irritant (R36) or does not require labelling (no risk phrase).

Validation

The proper scientific validation of alternative methods is the key to their acceptance into the regulatory process to complement or replace animal tests. The key elements in a successful validation study are (12):

  1. A clear and unequivocal statement of what the validation study is designed to accomplish.
  2. A well-defined plan for the study.
  3. A sufficiently large set of test substances covering the relevant chemical classes, the different categories of cosmetic ingredients, and the range of toxic endpoints to be evaluated.
  4. In vivo data of high quality on all the test substances to be used.
  5. Evidence that the methods to be evaluated are scientifically-sound, relevant, reproducible, and that they have the potential to replace the animal test in question.
  6. An optimised protocol for each test, with any necessary standard operating procedures.
  7. A clear description of how each alternative method can be used to predict an in vivo endpoint.
  8. Agreed statistical procedures for testing whether the methods can predict the in vivo endpoints defined by their developers.
  9. Agreed criteria to be met in order to show that an alternative method could successfully and safely replace an animal test.
  10. Results which meet these criteria.

Major problems arise when sufficient in vivo data are not available, or when the results of animal tests are highly variable.

Toxicology Databases

For the safety evaluation of cosmetic ingredients required by Directive 76/768/EEC, information is needed on both in vivo and in vitro toxicity tests conducted in the past, before any decision is taken about the need for further in vivo or in vitro testing. Bawden (13) has suggested that a comprehensive information system can assist in reducing the number of animal experiments conducted by:

In the EU, the potential users of an information system focusing on cosmetic ingredients would include the following:

The data required are not readily available from established databases, such as MEDLINE and TOX-LINE, but the following sources are making, or potentially could make, valuable contributions to such an information system:

Conclusions and Recommendations

General

  1. The approach to replacing animal testing should be subdivided, and attention should be focused separately on types of toxic endpoints, degrees of toxicity, and types of test materials (especially finished products versus ingredients). The results of current validation studies can be expected to emphasise the need for such an approach.
  2. Effective safety evaluation should be recognised as the ultimate goal, and it should be recognised that animal tests, in vitro tests, and human volunteer studies, etc., are all merely means of providing information for use in that evaluation.
  3. It would be wrong to insist that non-animal tests or testing strategies must be significantly more relevant and more reliable than the animal test procedures they will replace; this could delay their acceptance, and the Sixth Amendment requires only that they provide an equivalent level of protection for the consumer.
  4. The validation process will be much easier if the non-animal tests which are being developed provide less variable results than the currently-accepted animal tests, and if they have a clear mechanistic relationship with the type of toxicity to be assessed.
  5. Consideration should be given to ways of incorporating non-animal test data into safety assessment dossiers. Both industry and regulators must share information regarding the use of alternative methods, if they are to be ultimately accepted as replacements for the currently used in vivo toxicity tests.
  6. The CPS and DGXI/C/6 should seek to obtain the most comprehensive annual statistical information possible on cosmetics testing on animals within the Member States. This should include information on the tests that were performed, the animals which were used, and the purpose of the testing.
  7. A wider survey should also be conducted, in order to provide information on what animal testing for cosmetics safety purposes, including tests on finished products, is required, both in each Member State and in other countries which represent major markets, for example, Japan and the USA.

Product testing

  1. There are no major barriers to the cessation of finished product testing on animals; however, there might be a slightly increased risk of certain types of adverse reactions occurring in some consumers. For example, problems might arise with respect to skin sensitisation.

Non-animal tests and testing strategies

  1. The importance of conducting validation studies of the highest possible standard cannot be overemphasised; excellence of study design is essential, as is the selection of appropriate methods and test materials.
  2. Attention should be focused on whether non-animal methods could be used to identify materials which were so unlikely to cause any adverse effects (for example, eye irritancy) that no animal testing would be necessary, rather than considering only whether such methods could be used to avoid the animal testing of materials likely to cause severe effects. The ultimate objective is the validation and acceptance by regulatory authorities of non-animal tests and testing strategies capable of providing information on all levels of toxic responses.
  3. For a variety of reasons and purposes, more effort should be invested in developing computer models and appropriate in vitro systems for assessing biokinetics (absorption, distribution, metabolism and excretion), not least to assist in determining the appropriate range of chemical concentrations to be used in in vitro toxicity tests.
  4. Similarly, the introduction of QSAR approaches into the safety assessment of cosmetic ingredients, either alone or in conjunction with other approaches, such as the use of in vitro tests, should be encouraged in every possible way.
  5. Since it has become clear that it will often not be possible to replace an animal test with a single in vitro test, attention should be focused on the principles of devising, optimising, and using tier-testing strategies.
  6. Many companies are already using in vitro methods for internal risk assessment purposes, using their own "islands" of knowledge; they should be encouraged to pool this information in order to provide larger islands, but it must be recognised that extrapolation from such in-house approaches to the wider sphere is not necessarily straightforward. Particular attention should, therefore, be paid to the accuracy of analysis of the original experimental data on which the conclusions drawn by the various industrial companies are based.

Eye irritation

  1. Organotypic models (for example, isolated eye, isolated cornea, and HET-CAM methods) are already used as screens for chemicals from certain classes, and as a means of identifying severe eye irritants.
  2. More research is needed on modelling recovery from ocular damage and on the effects of repeated chemical insult.
  3. The results of the European Commission/Home Office-sponsored international study on alternatives to the Draize eye irritancy test are eagerly awaited, as are the conclusions of the US Inter-agency Regulatory Alternatives Group (IRAG) workshop held in November 1993.
  4. The current COLIPA-sponsored study on alternative tests for cosmetic ingredients and products is crucial, as it may be the last major eye irritation study to be completed before the implementation of the proposed January 1998 ban on animal testing is considered (i.e. during 1996).
  5. A better mechanistic understanding of eye irritation, particularly in the human eye, would facilitate the development and validation of non-animal testing strategies directly relevant to human safety assessment.

Skin irritation

  1. There is debate within the cosmetic industry about whether in vitro tests for skin irritancy are needed, or whether it would be possible to screen new ingredients for genotoxicity, systemic toxicity, corrosivity, and sensitisation, and then proceed directly to human volunteer studies without undertaking animal or non-animal tests for dermal irritancy per se.
  2. Attention should, therefore, be paid, as a matter of urgency, to what information is essential before in vivo human skin testing can be considered to be ethically acceptable.
  3. A standard guideline for human skin testing for irritancy should be devised, agreed, and approved, also as a matter of urgency.
  4. Promising in vitro methods for detecting corrosive chemicals are currently being subjected to an international validation study (18); however, it should be noted that such chemicals are not normally used in cosmetic products.

Skin penetration

  1. Encouraging developments are taking place with regard to the development of in vitro and (Q)SAR systems for evaluating the possibility of percutaneous absorption of chemicals; this is a key step toward improving safety evaluation.
  2. When it can be shown satisfactorily that a chemical will not penetrate the skin, certain other toxicity tests, for example, for reproductive toxicity, should not be required.
  3. Human skin should be used in in vitro systems in preference to rat and pig skin, and effort should be put into solving current ethical, safety, and logistical problems connected with the supply of human skin for in vitro studies. However, studies on the usefulness of pig skin as a possible alternative to human skin for in vitro studies should be encouraged.
  4. Reconstructed human skin equivalents should be improved with respect to their barrier/absorption properties.

Skin sensitisation

  1. Skin sensitisation is another key problem. Effort should be put into finding reliable and relevant in vitro testing strategies for identifying the sensitising potential of chemicals. A better fundamental understanding of the immunobiology of the skin will be essential.
  2. The question of the significance of metabolic activation in the skin must also be addressed.
  3. It must be recognised that low levels of impurities in commercial-grade chemicals will remain a problem to be taken into account.

Photosensitisation

  1. Although there is pressure from some quarters for an OECD guideline for an animal test or tests for phototoxicity to be produced, at present there is no widely accepted animal procedure to be replaced by non-animal methods. However, there are a number of promising non-animal tests for photoirritancy (19), which are currently being evaluated in a European Commission/COLIPA international validation study.
  2. Photoallergy is no less of a problem than other types of (contact) allergy (sensitisation).

Genotoxicity

  1. Generally speaking, negative results obtained from in vitro genotoxicity tests, such as the Salmonella typhimurium bacterial point mutation test and the chromosomal aberration test in mammalian cells in vitro, are indicative of the non-genotoxicity of a given chemical, with some exceptions, for example, those chemicals which present one or more 'structural alerts' in their molecules.
  2. Positive results obtained in one or both of these types of in vitro genotoxicity tests are indicative of the mutagenic activity of the chemical. However, for an assessment of the mutagenic risk involved upon exposure to such a chemical, in vivo genotoxicity testing (for example, the micronucleus test) is necessary.
  3. At present, several attempts are being made to improve the ability of in vitro genotoxicity tests to identify chemicals able to produce genotoxic effects by mechanisms other than gene mutation, chromosomal aberration, and DNA damage (such as aneuploidy).

Carcinogenicity

  1. The specificities of in vitro tests for predicting carcinogenic potential, for example, the in vitro mammalian cell transformation assay, are being improved by incorporating specific endpoints for carcinogenicity, such as the activation of oncogenes and the inactivation of tumour suppressor genes.

Difficult areas

  1. For certain types of toxicity, it is unlikely that the replacement of animal tests will be possible in the short-to-medium term. These include acute systemic and sub-acute toxicity testing, teratogenicity and reproductive toxicity testing, and toxicokinetic studies. However, research in these areas is in progress and breakthroughs are possible.

Databases and information centres

  1. There is now a need to improve the collection, storage, analysis, and availability of the various types of information, so that a better service can be provided for interested parties.
  2. Criteria for selecting the data to be included in databases need to be defined and agreed.
  3. Some coordination of the ways of processing data and comparing various methodologies and in vivo and in vitro data needs to be encouraged.
  4. A network should be established, to provide the fastest possible supply of information to those needing to use it.
  5. Where questions of confidentiality of the source or exact details of the data may be involved, an independent organisation or organisations should be identified, to be responsible for the safe storage and appropriate use of such data, in order to encourage owners to make the data available.

References

  1. Anon. (1994). ECVAM News & Views. ATLA 22: 7-11.
  2. Anon. (1993). Council Directive 93/35/EEC of 14 June 1993 amending for the sixth time Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. Official Journal of the European Communities L151: 32-36.
  3. Anon. (1976). Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products. Official Journal of the European Communities L262: 169-172.
  4. Anon. (1994). Development, Validation and Legal Acceptance of Alternative Methods to Animal Experiments. Com(94), 606 final, 27 pp. Brussels: Commission of the European Communities.
  5. Anon. (1978). Commission Decision of 19 December 1977 (78/45/EEC) establishing a Scientific Committee on Cosmetology. Official Journal of the European Communities L13: 24-25.
  6. Anon. (1986). Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations, and administrative provisions of the Member States regarding the protection of animals used for experimental and other purposes. Official Journal of the European Communities L358: 1-29.
  7. Anon. (1994). First Report from the Commission to the Council and the European Parliament on the Statistics on the Number of Animals Used for Experimental and Other Scientific Purposes. Com(94), 195 final, 59 pp. Brussels: Commission of the European Communities.
  8. Anon. (1991). Establishment of a European Centre for the Validation of Alternative Methods (ECVAM). Communication from the Commission to the Council and the European Parliament, 6 pp. Brussels: Commission of the European Communities.
  9. Loprieno, N. (1992). Guidelines for safety evaluation of cosmetics ingredients in the EC countries. Food & Chemical Toxicology 30: 809-815.
  10. Anon. (1994). Non-Animal Alternatives in Cosmetics Testing. Proceedings of the meeting held on 25-27 March 1994 at Akademie für Tierschutz, Neubiberg, Germany organised by the International Fund for Animal Welfare and EUROGROUP for Animal Welfare, p. 6. London: IFAW.
  11. Spielmann, H., Balls, M., Brand, M., Döring, B., Holzhütter, H.G., Kalweit, S., Klecak, G., L'Eplattenier, H., Liebsch, M., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfanenbecker, U., Potthast, J., De Silva, O., Steiling, W. & Willshaw, A. (1994). EEC/COLIPA project on in vitro phototoxicity testing: first results obtained with a Balb/c 3T3 cell phototoxicity assay. Toxicology In Vitro 8: 793-796.
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