Defining the role of ECVAM in the development, validation and acceptance of alternative tests and testing strategies

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Toxic.in YirroVol. 9, No. 6, pp. 863-869, 1995 Copyright 0 1995Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/95 $9.50 + 0.00

Defining the Role of ECVAM in the Development, Validation and Acceptance of Alternative Tests and Testing Strategies M. BALLS European Centre for the Validation of Alternative Methods (ECVAM), Environment Institute, Joint Research Centre, 21020 Ispra (VA), Italy Abstract--The background to the establishment of the European Centre for the Validation of Alternative Methods (ECVAM) is reviewed, and the main events at the opening of the Centre and an ECVAM symposium on practical aspects of validation are summarized. Finally, recommendations made to ECVAM for consideration in developing the Centre’s strategy are listed.

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The possibility of establishing a European Centre for the Validation of Alternative Methods (ECVAM) was conceived shortly after the publication of Directive 86/609/EEC on The Approximation of Laws, Regulations and Administrative Provisions of the Member States Regarding the Protection of Animals Used for Experimental and other Scientific Purposes (O#icial Journal of the European Communities, 1986), since the Directive required that the Commission, as well as the Member States, ‘should encourage research into the development and validation of alternative techniques’. However, it was not until the Commission sent a Communication to the Council and the European Parliament in October 1991 (Commission of the European Communities, 1991) that the specific functions of ECVAM became public knowledge. The Communication stated that the primary task of the Centre would be to promote alternative methods to experiments on animals, primarily through the co-ordination of the validation of alternative test methods at the European Community level, including the specification of test protocols, the organization of ring-test exercises, the choice of the chemicals to be used, and the analysis and evaluation of the results obtained. In addition, the Centre would act as a focal point for the exchange of information on alternative test methods; would set up, maintain and manage a data base on alternative procedures; and would encourage dialogue among legislators, industrialists, biomedical scientists, consumer organizations and animal welfare groups, with a view to

Abbreviattons: ECVAM = European Centre for the Validation of Alternative Methods; ESAC = ECVAM Scientific Advisory Committee; EU =European Union; OECD = Organisation for Economic Cooperation and Development.

securing the international acceptance of alternative test methods. The Communication went on to say that funds had been set aside to provide for the appointment of competent staff for the Centre, to cover the costs of scientific and technical support, and to provide equipment and running costs for a purpose-designed new building and for external contract research. In addition, the Communication established an ECVAM Scientific Advisory Committee (ESAC), consisting of 12 individuals selected from lists of nominees submitted by the Member States and six individuals selected from lists of nominees submitted by COLIPA (Comite des Associations Europeenes de 1’Industrie de la Parfumerie, des Produits Cosmetiques et de la Toilette; the Federation of European Cosmetic Industry Associations), ECETOC (the European Centre for Ecotoxicology and Toxicology of Chemicals), EFPIA (the European Federation of Pharmaceutical Industry Associations), ERGATT (the European Research Group for Alternatives in Toxicity Testing), and the Eurogroup for Animal Welfare. When the new ECVAM building was officially opened on 17 October 1994, we took advantage of the presence in Ispra of members of the ESAC and other guests with experience in the development, validation and application of alternative methods, by organizing a symposium on 18 October. The symposium had two overlapping themes-practical aspects of validation, and identifying areas in which alternative methods were needed and/or were approaching readiness for validation. The speakers had been asked to give advice to ECVAM as it set out to try to live up to the expectations of all those who had supported its establishment. In this paper, I comment briefly on what was said by some of the speakers at the ECVAM Opening Ceremony itself, then on the contributions of the

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speakers at the ECVAM Symposium, before summarizing the advice received and its implications for ECVAM as we plan our strategy for the period of the Fourth Framework Programme for Research and Technological Development (1995-1998). The ECVAM Opening Ceremony

The Opening Ceremony was chaired by Jean-Pierre Contzen, Director-General of the Joint Research Centre. The first speaker, HRH Prince Laurent of Belgium, Founder of the Group for Reflection on the Quality of Life and Environment, who later formally opened the building, said that: As every day passes, we are increasingly confronted by the imperative need to recognize that the quality of our own lives is not something which can be isolated from what goes on around us, either close to home or elsewhere in the world, or even in space. This alone, even in the absence of any altruism, should force us to show concern, not only for ourselves and for those close to us, but also for other communities, other species and for the environment in general. The quality of the lives we currently enjoy in Europe is undoubtedly the result of industrial development and expansion in the past, and of skilful medical research involving laboratory animals, conducted in the past, which has provided much insight into the workings of the human body, understanding of a variety of diseases, and cures for many conditions which once severely curtailed the length of human life and greatly reduced its quality. One of the challenges before us is to see that these benefits are spread more widely to other parts of the human world. Nevertheless, we are also increasingly forced to recognize that the cost of our current economic prosperity and longevity has been high, both with respect to our use of finite natural resources and pollution of the air, soil and water on which all the living creatures in the world depend. Similarly, we have increasingly come to question the ethical acceptability of causing suffering to sentient animals as substitutes for ourselves, so that we can benefit, while they bear the cost. The escape from the moral dilemmas presented by laboratory animal experimentation was offered to us by William Russell and Rex Burch in The Principles of Humane

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Technique,

published in 1959. They called on all concerned in any way to reduce laboratory animal use to an unavoidable minimum, to refine the procedures used so that suffering was minimized, and to strive to replace laboratory animal use altogether, wherever possible, by developing and exploiting other, non-sentient experimental systems. Individuals and organizations in many parts of the world have taken up this challenge, and the philosophical and practical contributions made in

Europe have been particularly high. This led to a Council of Europe Convention and to Directive 86/609/EEC, both in 1986, and to changes in the laws of many individual countries. The adoption of the Three Rs principles of Russell and Burch is now almost universal. However, they are coming to be taken for granted, and only if our efforts truly lead to reduction, refinement and replacement will anything of lasting value have been achieved. Tom Garvey, Deputy Director-General of DGXI (Environment, Nuclear Safety and Civil Protection), which is responsible for the application of Directive 86/609/EEC, said that the establishment of ECVAM reflects the commitment of the European Union (EU) to the Three Rs. He emphasized that ECVAM should be regarded as complementary to, and not as a substitute for, work carried out in this field by commercial companies and academic researchers in the Member States. He noted the EU Target for an overall 50% reduction in animal experimentation by the year 2000 (Oficial Journal of the European Communities, 1993) and said that ECVAM should concentrate its efforts on areas in which the largest number of animals are used, especially where death is the inevitable end of the experiment. He also welcomed the support already being given by ECVAM in relation to other aspects of Directive 86/609/EEC concerning laboratory animal welfare and the collection and analysis of statistics on animal use in the Member States. David Wilkins, Director of the Eurogroup for Animal Welfare, read an address by Hanja MaijWeggen MEP, President of the European Parliament’s Intergroup on the Welfare and Conservation of Animals. This referred to the formation of Intergroup in 1983 and to its demands for a Directive on animal experimentation, which were accepted by the Parliament in 1984. Mrs Maij-Weggen’s address went on to say that, while the establishment of ECVAM was very much welcomed by Intergroup: Alternative, non-animal, tests are being developed and work on them has been going on for many years. However, many people, including myself, feel that progress has been too slow. The work of the Centre should provide the impetus for speedier progress in the future. But I must, on behalf of the Intergroup, sound a warning note. Setting up ECVAM is one step-a major step forward-but making it work efficiently and effectively is another, equally important, step. David Clark, a member of the ESAC, emphasized the need for ECVAM to remain neutral, for the identification of achievable goals, for an international outlook, for scientific excellence, and for outstanding leadership. He noted the key role played by OECD (Organisation for Economic Cooperation and Development) guidelines on animal testing, and reminded

Defining the role of ECVAM the audience that not all OECD member countries were as passionate about animal welfare as we are in some European countries. There was therefore a need for realism-ECVAM must not try to do too much too quickly, since it takes time for scientific endeavour to bear fruit. Alan Goldberg, Director of the Center for Alternatives to Animal Testing, Johns Hopkins University, Baltimore, Maryland, USA, praised the Commission for its initiative in establishing ECVAM, and emphasized the great importance of international cooperation in the widest sense, so that approaches to validation could be harmonized. The ECVAM Symposium The two sessions of the ECVAM Symposium, devoted to ‘Validation in Practice’ and ‘Progressing Toward Validation’, were chaired by Bas Blaauboer and by Jose Castell, respectively, and two round-table discussions, on ‘Lessons Learned from Practical Experience’ and ‘Alternatives and Cosmetics Testing’, were chaired by Philip Botham and Nicola Loprieno, respectively. The ‘Validation in Practice’ session was opened by Marcel Roberfroid (Catholic University of Louvain, Brussels, Belgium), who emphasized the difference between the science of toxicology and the art of hazard prediction and risk assessment (Roberfroid, 1995). He pointed out that risk assessment tends to be based on rather poor models, since so little is known about mechanisms of toxicity. This is where the alternative methods could be especially valuable, leading to new tests as the basis for a more scientific approach in place of the current dependence on formal regulations. He said that the three main purposes of testing must be recognized-for classification and labelling, for general risk assessment (e.g. consumer safety, safety at work) and for specific risk assessment (e.g. medicines). There was a paradox, in that alternatives exist, but while they can never be fully validated, they cannot be ignored. Therefore, flexibility in the application of regulations is essential and the results obtained in non-animal tests must be incorporated into the regulatory process. However, he warned that the empirical validation by means of retrospective in vitro/in vivo comparisons would lead us nowhere. John Frazier (ManTech Environmental Technology Inc., Dayton, Ohio, USA) concentrated his attention on the need for a multidisciplinary approach to test development and validation (Frazier, 1995). He then gave an example of how a cell culturist, a molecular toxicologist, a measurement technologist, a theoretical toxicologist (modeller), a chemist/ structure-activity expert, a kineticist and a statistician could all work together to develop a new test. He then turned to three difficult issues in validationthe selection of chemicals to be tested, the definition of the gold standard against which to evaluate a new

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test, and the way in which test performance should be evaluated, as a means of determining the relevance of the test for a specific purpose. He said that, while most of us have a fairly good idea of how to evaluate reliability, the evaluation of relevance remains a stumbling-block to progress. Herman Koeter (Environmental Health and Safety Division, OECD, Paris) pointed out that, while it had long been the policy of the OECD to encourage the development of non-animal test systems, and although the procedures for updating test guidelines had been modified to incorporate scientifically validated alternative methods, progress in the validation of such methods had so far been very disappointing (Koeter, 1995). He proposed that the relevance of the endpoints measured for human and/or environmental hazard characterization and risk assessment should be the foundation of any new test. Nevertheless, he favoured continuing to perform a limited number of animal tests to confirm either negative results or positive results obtained in in vitro tests. He suggested that validation studies should be more focused, and indicated that the National Co-ordinators of the OECD Test Guidelines Programme were qualified to review proposals for validation projects and their outcome. However, since other organizations would also be involved, there was a need to build consensus on criteria for the validation process itself. A great deal of experience was on display in the round-table discussion on ‘Validation-Lessons Learned from Practical Experience’ (Fentem et al., 1995) for which the panelists were Julia Fentem (ECVAM), Menk Prinsen (TNO, Zeist, The Netherlands), Horst Spielmann (ZEBET, BgW, Berlin, Germany) and Erik Walum (Pharmacia AB, Stockholm, Sweden), with Dr Botham (Zeneca, Macclesfield, UK) as Chairman. The panellists agreed with Dr Koeter’s regret that progress towards validating alternative methods has so far been painfully slow. We now recognize that our concept of validation in 1990 (Balls et al., 1990a) was somewhat theoretical, and an ECVAM workshop, held at the beginning of 1994, sought a much more practical approach (Balls et al., 1995). Nevertheless, while we ought to be able to overcome problems caused by poor design, poor management, inadequate test protocols, and even imprecise definition of the goals of particular validation studies, the solutions to other difficulties seem more intractable. These include the availability of sufficient animal data of sufficiently high quality on a sufficient range of materials to provide a basis for evaluating the performance of the new methods, and more importantly, the unresolved question of whether, even if freely available, in sufficient variety and of the highest scientific quality, animal data could ever be an appropriate basis for judging the performance and the relevance of non-animal test procedures. Perhaps the situation could be summed up like this: the stronger the mechanistic basis of the non-animal

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in relation to harmful effects in man which we understand, the less will be the need for reliance on data from animal tests which themselves have not been subjected to any rigorous validation process, but have become acceptable merely because they exist and because they have been performed so frequently. The question is, how long will it take us to provide the scientific basis for the greatly improved applied art of hazard prediction and risk assessment which Roberfroid urges us to insist upon? Meanwhile, can the OECD and all other agencies involved in the current practice of regulatory toxicology reassure us-indeed, prove to us-that their standards for the acceptance of new animal test procedures are not dramatically less stringent than those which will be applied to non-animal tests? The panelists discussed criteria for the selection of tests, the selection of laboratories, the selection of test chemicals, and the analysis and interpretation of data. It is becoming clear that a new way forward for the validation process must be devised, with many small studies rather than few large ones, and with a greater emphasis on a prevalidation phase to provide optimized protocols and clear evidence of interlaboratory transferability. The question of what should be validated and why was addressed in the Symposium session on ‘Progressing Toward Validation’. The general public could be forgiven for believing that toxicity testing is more or less confined to the LD,, test for industrial chemicals and the Draize eye irritation test for cosmetic products, such is the publicity given to those particular issues. However, in the knowledge that ECVAM would also be expected to contribute to the development, validation and acceptance of alternative methods for many other kinds of chemicals and products, speakers had been invited to address the development and testing of medicines and vaccines and the role of in vitro tests in ecotoxicology. Bernward Garthoff (Bayer AG, Leverkusen, Germany) dealt with the role of alternative methods in drug research and development (Garthoff, 1995). He distinguished between the discovery phase, in which in oitro methods are now the rule and animal studies the exception, and the development phase, which involves toxicity testing and safety evaluation according to standardized procedures. He placed particular emphasis on the great potential of transgenie animal models as a means of reducing the numbers of animals used in the preclinical phase of testing, because they can be constructed to provide for greater relevance and therefore a better-targeted approach. He nevertheless recognized the ethical concerns raised by the increased use of transgenic animals (Balls, 1994). Garthoff argued that progress in the development phase could best be achieved, at least in the short term, by the harmonization of testing requirements and guidelines and by question-

ing the value of certain animal test procedures, especially the need for a second rodent species in carcinogenicity testing. Vaccine development, quality control and testing is an area in which large numbers of animals are used at present, but where many initiatives are already under way in terms of reduction, refinement and replacement. A particularly successful ECVAM Workshop on this topic took place in 1994 (Hendriksen ef al., 1994). The principal organizer of the Workshop, Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) used the occasion of the ECVAM Symposium to discuss a specific example of the replacement of an animal test by an in vitro method. The determination of the tetanus antitoxin titre in a serum sample used to be based on a toxin neutralization (TN) test in mice, in which the test criterion used was the identification of the serum dilution containing non-neutralized tetanus toxin which caused the death of, or characteristic signs of tetanus in, half the mice in the test group within 96 hr. An in vitro method, the toxin binding inhibition (ToBI) test, a modified ELISA method, was developed at RIVM and has now been accepted in The Netherlands as a replacement for the TN test (Hendriksen, 1995). ECVAM will be working with RIVM, The Netherlands Centre for Alternatives (Utrecht), FRAME (Nottingham, UK), the Paul Ehrlich Institute (Langen, Germany) and the European Pharmacopoeia (Strasbourg, France), as well as with other appropriate agencies, to maximize the achievement of reduction, refinement and replacement in the vaccines field at the fastest possible rate. A totally different challenge was presented by Boris Isomaa (Abe Akademic University, Turku, Finlandtithe need for in vitro tests in ecotoxicity testing (Isomaa and Lilius, 1995). He pointed out that ecotoxicology is concerned with the ecosystem as the primary target, with the fate of individual species being of secondary concern. He warned that ecosystems should not be seen as superorganisms, and regretted that the single-species approach, based on Daphnia and fish tests, was still the backbone of aquatic environmental safety testing. He favoured the multispecies approach, with an emphasis on the mesocosm (many species) rather than the microcosm (few species), and suggested that in vitro methods would be very valuable, not merely as a way of getting many species involved, but also for providing links to other areas of toxicology. Isomaa went on to say that the need for integrated studies is self-evident in ecotoxicology, and saw something like the ECITTS (ERGATT/CFN Integrated Toxicity Testing Scheme; Walum et al., 1992) as a sensible example of the best way forward. Jean-Paul Mot-in (INSERM U295, University of Rouen, France) used the kidney as a case study of the potential value of in vitro strategies in organ toxicity assessment (Morin et al., 1995). This example is

Defining the role of ECVAM representative of the kind of prenormative research funded by the European Commission’s DirectorateGeneral XII (Science, Research and Development), in this case via the BRIDGE Programme, which can progress to pre-validation and validation under the auspices of ECVAM. Morin outlined the different kinds of organ, tissue and cell preparations currently used in acute nephrotoxicity studies, but argued that only the cell culture systems are likely to be suitable for the chronic toxicity tests that are so badly needed. He called for more emphasis to be placed on the identification of systems and endpoints for distinguishing between general cell toxicity and specific organ toxicity. He concluded that the criteria for the acceptance of alternative methods must not be too demanding, and saw studies of high scientific quality based on sound experience as the key factor. The round table on ‘Alternatives in Cosmetics Bruner Testing’ involved four panellists-Leon (Procter & Gamble Company, Egham, UK), Mark Chamberlain (Unilever Research, Sharnbrook, UK), Martine Cottin (L’OrCal, Aulnay-sous-Bois, France) and Shinobu Kato (Shisheido Cutaneous Biology Research Center, Charlestown, MA, USA), with Professor Loprieno (University of Pisa, Italy) as Chairman (Loprieno et al., 1995). This was a particularly interesting and informative discussion, since it showed how four of the world’s largest cosmetics companies have adopted different strategies in incorporating in vitro methods into their own in-house safety testing programmes. There is an important message here, reminiscent of what was pointed out by Gerhard Zbinden several years ago (Zbinden, 1988): there is more than one valid way of achieving the same objective. Thus, flexibility coupled with strong scientific justification should be the key to the acceptance of alternative methods into regulatory toxicology. Indeed, we should avoid replacing the existing animal test guidelines with equally rigid specifications of particular protocols, cell preparations, etc., and any well-designed and scientifically defensible strategy incorporating any properly validated and independently recommended testing strategies should be acceptable (Balls, 1992), as was recommended at an EC-sponsored workshop on regulatory acceptance, held in 1990 (Balls et al., 1990b). One problem was identified, however. The in-house validation of alternative testing strategies tends to be based on data which are held to be ‘commercially confidential’. This limits the scope for the truly independent assessment and full publication of data, which many consider to be essential to the validation process. Another interesting point, which arose in the general discussion, was the usefulness of benchmark chemicals and formulations, as a means of calibrating test systems and also providing a comparative means of expressing the results obtained for other test materials.

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Defining the role of ECVAM In addition to presenting their opinions, based on their own wide and varied experience, the speakers in the ECVAM Symposium very much lived up to what was expected of them-they provided a wealth of advice and recommendations for ECVAM, which I will try to summarize. 1. ECVAM has an important role, not only in the development, validation and acceptance of alternative tests, but also in the use of nonanimal methods in the fundamental science of pharmacotoxicology. 2. The rapid increase in the number of partially developed test methods seen in recent years, often encouraged by animal welfare considerations, presents a problem for the validation process. ECVAM should contribute to the elaboration of guidelines for the validation process itself, and how it relates to test development and the acceptance of methods shown to be relevant and reliable for specified purposes. 3. ECVAM should establish short-term and longterm goals, and should seek to ensure that the same issues are not discussed time and time again in the years to come, without any real progress being made. 4. The identification of priority areas must take into account the state of the science in particular fields, but also the numbers of animals currently used and the degrees of suffering caused to them. 5. ECVAM should ensure that alternative tests involve simple, distinct and well-defined endpoints, with a sufficient understanding of their mechanistic basis. The application of the results obtained to predicting the likelihood of a complex series of events in vivo should be seen as a separate consideration. The first stage concerns the meaning of the test in terms of events at the molecular and cellular levels. The second is concerned with the relevance of the information it produces to the hazard prediction and risk assessment process. 6. A multidisciplinary approach will be essential, and ECVAM should seek not only to promote discussion among the various interested parties but also to encourage them to collaborate in practical projects. A natural consequence of the multidisciplinary approach will be the development of integrated testing strategies incorporating quantitative structure-activity relationship (QSAR), biokinetic and cell/tissue models. An ECVAM workshop on this topic will have been held by the time this article is published. 7. ECVAM must encourage all concerned to face up to the serious difficulties resulting from the lack of availability of sufficient well-characterized test materials relevant to particular events and effects in vivo, backed by in vivo data, human or animal, of sufficiently high quality.

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8. In carrying out its duties for the Commission in relation to Directive 86/609/EEC in general, ECVAM should seek to encourage a re-evaluation of the scientific merit of existing test guidelines and regulatory procedures, including bioassays for carcinogenicity. with this need for a more realistic 9. Connected attitude towards the usefulness of currently accepted animal procedures, ECVAM should encourage the belief that a new process of hazard prediction and risk assessment will be necessary to replace that which is dependent on data from laboratory animals, and should take action accordingly. should seek to build consensus by 10. ECVAM bringing together scientists from the wide variety of disciplines involved, and by working with the appropriate governmental, industry. academic and other organizations, in the EU and its Member States, and more widely. 11. An essential part of this consensus will be concerned with establishing criteria for validation, for the independent assessment of the outcome of validation studies, and for the acceptance of alternative tests and testing strategies into regulatory practice. This will require active cooperation between ECVAM and, for example, the OECD and with ICCVAM (the Inter-agency Coordinating Committee for the Validation of Alternative Methods) in the USA. 12. Since the number of individuals involved in all these activities is still relatively small, ECVAM should see education and training as one of its most important activities.

Concluding remarks Perhaps the biggest mistake ECVAM could make would be to try to do too much, even though it is clear that there is a great deal to be done and a great deal is expected of the European Commission’s new Centre. We must therefore establish a list of priorities in consultation with our Scientific Advisory Committee, our colleagues in other services of the Commission, and our wide network of partners all over the world. The necessary discussions are already under way and we hope that the publication of the proceedings of the ECVAM Symposium will serve others as well as ourselves. Meanwhile, it is already clear that we must: Ensure that we are well informed and equipped to inform others; Identify areas where non-animal tests are most needed; Concentrate on the organization and funding of pre-validation studies; Organize large validation studies, alone or with other organizations, but only as and when necessary;

5. Define criteria for the acceptance of validated alternatives and ensure that they are fairly applied; 6. Work with others in any ways that will truly serve the goals of ECVAM and the European Commission. Finally, we need not be hesitant in demanding that scientifically valid and feasible non-animal methods and testing strategies should be incorporated into regulatory testing guidelines. Directive 86/609/EEC specifically requires that ‘an experiment [on a laboratory animal] should not be performed, if another scientifically satisfactory method, not entailing the use of an animal, is reasonably and practicably available’. The law is on our side. REFERENCES

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Cottin M. De Silva 0. and Kato S. (1995) Alternatives in cosmetics testing. Toxicology in Vitro 9, 827-838. Morin J.-P., Leclere C., Marouillat S. and Monteil C. (1995) Some milestones in in vitro organ toxicity assessment. The kidney as a case study. Toxicology in Vitro 9, 795-814. Robcrfroid M. (1995) Toxicology: a science and an art. Toxicology

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Russell W. M. S. and Burch R. L. (1959) The Principles of Humane Experimental Technique. Methuen, London. Walum E., Balls M., Bianchi V., Blaauboer B., Bolcsfoldi G., Guillouzo A., Moore G. A., Odland L., Reinhardt C. and Spielmann H. (1992) ECITTS: an integrated approach to the application of in vitro test systems to the hazard assessment of chemicals. ATLA 20, 406-428. Zbinden G. (1988) A look behind regulatory guidelines. In National and International Drug Safety Guidelines Edited by S. Adler and G. Zbinden. pp. 7-19. MTC Verlag, Zollikon.