WellBeing International WBI Studies Repository

1995

The Three Rs: The Way Forward

Michael Balls European Centre for the Validation of Alternative Methods

Alan M. Goldberg Johns Hopkins University

Julia H. Fentem European Centre for the Validation of Alternative Methods

Caren L. Broadhead Fund for the Replacement of Animals in Medical Experiments (FRAME)

Rex L. Burch

See next page for additional authors

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Part of the Bioethics and Medical Ethics Commons, Laboratory and Basic Science Research Commons, and the Politics and Social Change Commons

Recommended Citation Balls, M., Goldberg, A. M., Fentem, J. H., Broadhead, C. L., Burch, R. L., Festing, M. F., ... & Van Zutphen, B. F. (1995). The three Rs: the way forward: the report and recommendations of ECVAM Workshop 11. Alternatives to laboratory animals: ATLA, 23(6), 838.

This material is brought to you for free and open access by WellBeing International. It has been accepted for inclusion by an authorized administrator of the WBI Studies Repository. For more information, please contact [email protected]. Authors Michael Balls, Alan M. Goldberg, Julia H. Fentem, Caren L. Broadhead, Rex L. Burch, Michael F.W. Festing, John M. Frazier, Coenraad F.M. Hendriksen, Margaret Jennings, Margot D.O. van der Kamp, David B. Morton, Andrew N. Rowan, Claire Russell, William M.S. Russell, Horst Spielmann, Martin Stephens, William S. Stokes, Donald W. Straughan, James D. Yager, Joanne Zurlo, and Bert F.M. van Zutphen

This article is available at WBI Studies Repository: https://www.wellbeingintlstudiesrepository.org/acwp_arte/58 ATLA 23, 838-866, 1995 838

The Three Rs: The Way Forward , The Report and Recommendations of ECVAM Workshop 11 ,2

Michael Balls; Alan M. Goldberg; Julia H. Fentem," Caren L, Broadhead,5 Rex L. Burch," Michael F.W. Festin!\'7 John M. Frazier,8 Coenraad F.M. Hendriksen; Margaret Jennin~s, 0 Margot D.O. van der Kamp,l1 David B. Morton,'2 Andrew N. Rowan, 3 Claire Russell," William

M.S. Russell, I. Horst SpielmannJ 15 Martin L. Stephens,'6 William S. Stokes, 17 Donald W. Straughan, 0 James D. Yager, 18 Joanne Zurlo 4 and Bert F.M. van ZutphenI.

"ECVAM, JRC Environment Institute, 21020 Ispra (Va), Italy; 'CAAT, School of Hygiene and Public Health,/ohns Hopkins University, 111 Market PlacelSuite 840, Baltimore, MD 21202-6709, USA; FRAME, Russell & Burch House, 96-98 North Sherwood Street, Nottingham NGJ 4EE, UK; 6Town Hall, Sheringham, NOifolk NR26 8QY, UK; 7MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LEI 9HN, UK; AToxic Hazani!..s Research Unit, ManTech Environmental Technology Inc., Dayton, OR 45431-0009, [tSA; RIVM, Antonie van Leeuwenhoeklaan 9, 3721 MA Billhoven, The Netherlands; °Research f}:nimals Department, RSPCA, The Causeway, Horsham, West Suss~x RH12 IHG, UK; NCA, Yalelaan 17, De Uithof, 3584 CL Utrecht, The Netherlands; I Department of Biomedical ~fiences and Ethics, University of Birmingham Medical School, Birmingham BI52TJ, UK; Tufts Center for Animals and Public ff.licy, School for VeterinalJ' Medicine, 200 Westboro Road, North Grafton, MA 01536, US1i Department of Sociology, University of Reading, Whiteknights.Reading RG6 2AA, UK; I ZEBET, BgW, Diedersdolfer Weg 1, 12277 Berlin, Germany; I Hu"!?ne Society of the United States, 2100 L Street NW, Washi"i/lton, DC 20037, USA; NIEHS, NTP, NIH, Research Triangle Park, NC 27709, USA; 1 Division of Toxicological Sciences, School of Hygiene and Publif Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD 21205, USA; Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands

Preface tance to the biosciences and which reduce, refine or replace the use of laboratory ani­ This is the report of the eleventh of a series mals. One of the first priorities set by of workshops organised by the European Eev AM was the implementation of proce­ Centre for the Validation of Alternative dures which would enable it to become well­ Methods (ECVAM), which was established in informed about the state-of-the-art of 1991 by the European Commission. non-animal test development and validation. ECVAM's main goal, as defined in 1993 by and the potential for the possible incorpora­ its Scientific Advisory Committee, is to pro­ tion of replacement alternative tests into mote the scientific and regulatory acceptance regulatory procedures. It was decided that of alternative methods which are of impor- this would be best achieved by the organisa-

Address for correspondence and reprints: Professor Michael Balls, ECVAM, TP ,')80, JRC Environment Institute, 21020 Ispra (Va), Italy

I ECVAM _ The Ellropean Centre for the Validation of Alternative Methods. 2This document represents the agreed report of the participants as individual scientists. 839 M. Balls et ai. tion of ECVAM workshops on specific topics, project was managed by a committee under at which small groups of invited experts the chairmanship of Sir Peter Medawar, would review the current status of various with William Lane-Petter, Secretary of the types of in vitro tests and their potential Research Defence Society, among its mem­ uses, and make recommendations about the bers. It was international from its outset, best ways forward (1). since Christine Stevens, of the Animal The workshop on The Three Rs - The Welfare Institute (AWl) in the USA, pro­ Way Forward, held in Sheringham, Norfolk, vided financial support and made frequent UK, on 30 May to 3 June 1995, under the co­ visits to UFAW while the study was being chairmanship of Michael Balls (ECVAMI and conducted. Alan M. Goldberg (Johns Hopkins Center for W.M.S. Russell and R.L. Burch were Alternatives to Animal Testing [CAAT], appointed to carry out the work. This led to Baltimore, MD, USAI, had a wider aim. The their book, The Principles of Humane principal objectives of this workshop were to Experimental Techniq'le (2), which provided discuss the current status of the Three Rs, a wealth of information and many remark­ and to make recommendations aimed at able ideas and insights, most of them as rel­ achieving greater acceptance of the concept evant today as they were more than 35 years of humane experimental technique and, in ago. The book has recently been reprinted the interests of both scientific excellence and (31, and copies can be obtained from UFAW.' the highest standards of animal welfare, the It was in this book that Russell and Burch more active implementation of reduction presented the concept of the Three Rs. They alternatives, refinement alternatives and defined replacement as "any scientific replacement alternatives. method employing non-sentient material The invited participants were individuals which may in the history of animal experi­ actively and professionally committed to the mentation replace methods which use con­ Three Rs, and we were privileged to have scious living vertebrates", reduction as a William Russell and Rex Burch, who devel­ means of lowering "the number of animals oped the Three Rs approach in the 1950s, as used to obtain information of a given amount participants in the workshop. and precision", and refinement as any devel­ The opening ceremony was held in opment leading to a "decrease in the inci­ Sheringham Town Hall, where Rex Burch dence or severity of inhumane procedures has practised as a microbiologist since the applied to those animals which have to be early 1970s. Since this was the first time that used". Russell and Burch had attended a scientific Nobody can recall precisely when the conference together for nearly forty years, Three Rs concept arose (4), but it was some­ the proceedingfi were recorded on videotape time between 1955 and 1957. UFAW held a (the VHS tape can be borrowed from symposium on Humane Technique in the ECVAM and a JVC version is available from LaboratolY (51 in 1957, and it was then that CAATI. The rest of the workshop was held at the concept of the Three Rs was first dis­ the Links Country Park Hotel, West Runton. cussed in public. More about the origins of the Three Rs concept can be found in a talk given by Charles Hume in Washington in Introduction October 1959 (61, when he said of The Principles of Humane Experimental The origins of the Three Rs concept Technique: What are now known as the Three Rs of "This deserves to become a classic for all Russell and Burch, replacement, reduction time, and we have great hopes that it will and refinement, have their origins in a pro­ inaugurate a new field of systematic study. posal made in 1954 by Charles Hume, We hope that others will follow up the lead it founder of the Universities Federation for has given, and that a generalised study of Animal Welfare tUFAWI, that UFAW should humane technique, as a systematic compo· undertake a scientific study of humane tech­ nent of the methodology of research, will nique in laboratory animal experiments. The come to be considered essential to the train-

18 Hamilton Close, Softth Mimms, Poilen; Bar, Herts, EN6.'JQD, UK. ECVAM Workshop 11: Three Rs 840 ing of a biologist." under which experiments on animals were allowed and regulated. This led in turn to the What is perhaps the central message (the formation of the Committee for the ,Reform "humanity criterion") of The Principles of of Animal Experimentation (CRAE), which Humane Experimental Techiqlle (2) is had as its principal goal the reform of the spelled out on page 157 of the book: 1876 Act (9). The concept of alternatives was "If we are to use a criterion for choosing also taking hold in the USA. as a result oflhe experiments to perform, the criterion of efforts of the AWl (who distributed Russell humanity is the best we could possibly and Burch's book), United Action for invent." Animals, and the Humane Society of the United States. "The greatest scientific experiments have Meanwhile, David Smyth, a distinguished always been the most humane and the most physiologist, was conducting a survey on the aesthetically attractive, conveying that sense Three Rs for the Research Defence Society, of beauty and elegance which is the essence which led to another important landmark, of science at its most successful." the publication of his book on Alternatives to Animal Experiments (10). Smyth provided a The evolution of the Three Rs concept Three Rs definition of alternatives, which Despite its originality and scholarship, and has since been widely accepted: the involvement of many distinguished sci· "All procedures which can completely entists in the discussions leading up to its replace the need for animal experiments, publication, Russell and Burch's book had reduce the numbers of animals required, or little obvious impact on thinking or practice diminish the amount of pain or distress suf­ in the early years after its publication. In fered by animals in meeting the essential fact, its authors did not meet each other needs of man and other animals." again for about 30 years, when they were "rediscovered" by a new generation of A number of particularly important reformers. changes began to take place at the beginning However, in 1969, one particularly signifi· of the 1980s. In the USA, animal activist cant development did take place - the foun­ Henry Spira launched a campaign to abolish dation by Dorothy Hegarty of the Fund for the Draize eye irritancy test, with the world­ the Replacement of Animals in Medical wide support of a coalition of 400 animal Experiments (FRAME), specifically to organisations. In Europe, discussions began advance Russell and Burch's vision that which were later to lead to the Council of humanitarian and scientific benefits would Europe Convention for the Protection of result from the systematic and rational Vertebl:afe Animals Used for Experimental application of the Three Rs approach. and Other Scientific Plllposes (111 and FRAME saw reduction and refinement as' COllllcil Directive 8616091EEC of 24 achievable in the short term, but decided to November 1986 on the Approximation of focus its own activities primarily on replace­ Laws, Regulations and Administrative ment as the ultimate, long-term goal (7). Provisions of the Member States Regarding FRAME was to succeed in establishing itself the Protection of Animals Used for in the middle ground between the antivivi­ Experimental and Other ScientiJ;c Purposes sectionists and the defenders of animal· 1121. based research, with a positive message, Meanwhile, the British Home Secretary, based not on confrontation, but on support Merlyn Rees, said that he would consider lis­ of the Three Rs concept. tening to proposals for the reform of the In the 1970s, there were a number of other 1876 Act, but only if animal welfare organi. significant events. For example, there was a sations would agree on a policy among them­ substantial increase in laboratory animal use selves. An alliance was therefore formed in the early part of the decade, which led to between CRAE, FRAME and the British great public concern in Great Britain, and an Veterinary Association (BVA; 9). A set of Animal Welfare Year campaign, involving BV NCRAE/FRAME proposals were submit­ many animal welfare organisations, was ted in 1983 113), which greatly influenced organised to mark the centenary of the British Government thinking, as revealed in Cruelty to Animals Act 1876 (8), the law two White Papers (14, 151. In what was a 841 M. Balls et ai. very dramatic development at the time, the 7.4. All experiments shall be designed to 1985 White Paper contained a commitment avoid distress and unnecessary pain and suf­ to the Three Rs concept, in these words: fering to the experimental animals. "Animal experiments that are unnecessary, Similar words are used in the Council of use unnecessarily large numbers of animals, Europe Convention (11), or are unnecessarily painful, are indefensi­ In Germany, when the national legislation ble." on animal protection was changed in 1987 to meet the requirements of Directive Members of the BV NCRAE/FRAME 86/609/EEC, a clause was inserted which alliance were invited to act as advisers to the British Government during the preparation requires the Federal Government to present a report (the Tierschzttzbericht) to the and passage through Parliament of the Animals (Scientific Procedures) Act 1986 Bundestag every two years, to document the progress made with respect to the implemen­ 061, which was supported by both the tation of animal protection measures. This Conservative and Labour parties and replaced the 1876 Act. The 1986 Act set up a puts pressure on the relevant government project and personal licensing system, as well institutions to take the necessary steps to as an independent Animal Procedures implement current legislative requirements Committee (APC), which can give advice to for animal protection. According to the the Government, whether or not it is German animal protection act (the wanted. The Act contains two particularly Tierschutzgesetz), nobody is allowed to cause pain, suffering or harm to an animal without important clauses (161: good reason. The legislation closely resem­ 5141. In determining whether and on what bles Direclive 86/609/EEC, with an addi­ terms to grant a project licence the Secretary tional provision which prohibits animal of State shall weigh the likely adverse effects experimentation for developing tobacco on the animals concerned against the benefit products, washing detergents and decorative likely to accrue as a result of the programme cosmetics. of work to be specified in the licence. In The Netherlands, the Act on Animal Experimentation was adopted in 1977. Items 515 J. The Secretary of State shall not grant a included in this Act which are of importance project licence unless he is satisfied that the with regard to the implementation of the applicant has given adequate consideration to the feasibility of achieving the purpose of Three Rs are: the programme to be specified in the licence a) Mandatory registration of animal use. by means not involving the use of protected bl Prohibition of the use of an animal for a animals. purpose that could be achieved equally by Directive 86/609/EEC 1121 spelled out its using in vitro methods or other non-ani~ Three Rs basis in Article 7, as follows: mal procedures. c) The requirement that persons involved in 7.2. An experiment shall not be performed if animal experimentation are shown to be another scientifically satisfactory method of competent. Education and training in the obtaining the result sought, not entailing the field of laboratory animal science, includ~ use of an animal, is reasonably and practica~ ing ethics and alternatives, is mandatory blyavailable. for scientists and animal technicians. 7.3. When an experiment has to be per­ d) The requirement that institutions where formed, the choice of species shall be care­ animal experiments are conducted must fully considered and, where necessary, be licensed. explained to the authority. In a choice e) The requirement that a certificated ani­ between experiments, those which use the mal welfare officer be appointed in asso­ minimum number of animals, involve ani­ ciation with the licence for the institute. mals with the lowest degree of neurophysio­ D Mandatory use of anaesthetics and anal­ logical sensitivity, cause the least pain, gesics when appreciable pain is antici­ suffering, distress or lasting harm and which pated. Their use may only be omitted are most likely to provide satisfactory results when this would jeopardise the purpose shall be selected. of the experiment. ECVAM Workshop 11: Three Rs 842

At present, the 1977 Act is being revised; the During the early 1980s, the campaigns amended Act will include provisions relating against the Draize and LD50 tests, and to Animal Experimentation Committees simultaneous attempts to pass legislation in (AECs; see section on Scientific and Ethical the USA to promote the use of alternatives, Justification). focused industrial and congressional atten­ Following the adoption of the Act on tion on alternative methods. Public pressure Animal Experimentation, several initiatives led to the revision and strengthening of the were undertaken. A Department of US Animal Welfal'e Act and the Public Laboratory Animal Science was established Health Service Policy on the Humane Care at Utrecht University in 1983; in this and Use of Labol'ato;y Animals (191, both of Department, research and education pro­ which incorporated the requirement that grammes have been developed which are consideration be given to the Three Rs specifically directed toward further imple­ before any research involving the use of ani­ mentation of the Three Rs, and courses on mals was started. The concept of alternatives laboratory animal science are routinely held was also promoted via legislation relating to for scientists. In 1987, the Dutch the role and activities of the National Alternatives to Animal Experiments Institutes of Health (NIH), Platform was established, through which In 1986, a report by the US Congress government, industry and animal welfare Office of Technology Assessment on organisations cooperate in order to stimulate Alternatives to Animal Use in Research, the development and use of alternative Testing a.nd Educa.tion (20) provided evi­ methods. The main task of the Platform is to dence of the broad scope and potential of the advise the Government on the funding of Three Rs concept of alternatives and, in the research projects concerned with the devel­ same year, the Health Research Extension opment of alternatives. The Netherlands Act gave legislative force to the revised Centre Alternatives to Animal Use (NCA) Public Health Service Policy on animal was established in Utrecht in 1994, as a research. In Europe, the European Research national information centre on alternatives. Group for Alternatives in Toxicity Testing The main objective of the NCA is to stimu­ (ERGATT) was also established in 1986. late the development, validation, acceptance A set of International Guiding Principles and use of alternative methods; that is, the for Biomedical Research Involving Animals NCA supports the Platform in seeking to were published in 1985 (21); the basic princi­ realise its goal. ples are outlined in Table 1. Thus, by the end In the USA, the Johns Hopkins Center for of the 1980s, new laws were in place in vari· Alternatives to Animal Testing (CAAT) was ous parts of the world, which not only recog­ founded in 1981, with the support of a grant nised Russell and Burch's concept, but from the Cosmetic, Toiletry and Fragrance placed legal and moral obligations on all con­ Association, to address the major issues fac­ cerned to seek to replace, reduce andlor ing the development of alternatives (17). refine laboratory animal experimentation CAAT's initial focus was on establishing a wherever possible. Full implementation of small grants programme, through which it these laws and the development of replace· has funded the development of new in vitro ment alternatives became the next chal· systems, and of assays that could ultimately lenges. be used for product safety testing, by funda­ In 1993, the US NIH Revitalization Act mental research scientists. Over the years, included statements drafted and supported CAA T has become a visible advocate of the by animal protection organisations and by Three Rs, and it fulfils a unique role in the several large corporations which promote the USA in liaising with scientists from acade­ concept of alternatives. In particular, the Act mia, industry and governmental organisa­ authorised the establishment of an Applied tions. In this capacity, CAAT has organised Toxicology Program within the National regular scientific symposia and has played a Institute of Environmental Health Sciences major role in bringing together diverse (NIEHS), which eventually developed into the groups to formulate a framework for the val­ Interagency Coordinating Committee for the idation of alternative methods for product Validation of Alternative Methods (ICCVAM). safety testing (18). At the same time, a group of scientists 843 M. Balls et al.

Table I: International Guiding Principles for Biomedical Research Involving Animals

1. The advancement of biological knowledge and the development of improved means for the protection of the health and well-being both of man and of animals require recourse to experimentation on intact live animals of a wide variety of species.

II. Methods such as mathematical models, computer simulation and in vitro biological sys­ tems should be used wherever appropriate.

III. Animal experiments should be undertaken only after due consideration of their rele­ vance for human or animal health and the advancement of biological knowledge.

IV. The animals selected for an experiment should be of an appropriate species and quality, and the minimum number required, to obtain scientifically valid results.

V. Investigators and other personnel should never fail to treat animals as sentient, and should regard their proper care and use and the avoidance or minimisation of discom­ fort, distress, or pain as ethical imperatives.

VI. Investigators should assume that procedures that would cause pain in human beings cause pain in other vertebrate species although more needs to be known about the per­ ception of pain in animals.

VII. Procedures with animals that may cause more than momentary or minimal pain or dis­ tress should be performed with appropriate sedation, analgesia or anaesthesia in accor­ dance with accepted veterinary practice. Surgical or other painful procedures should not be performed on unanaesthetised animals paralysed by chemical agents.

VIII. Where waivers are required in relation to the provisions of article VII, the decisions should not rest solely with the investigators directly concerned but should be made, with due regard to the provisions of articles IV, V and VI, by a suitably constituted review body. Such waivers should not be made solely for the purpose of teaching or demon­ stration.

IX. At the end of, or when appropriate during, an experiment, animals that would otherwise suffer severe or chronic pain, distress, discomfort, or disablement that cannot be relieved should be painlessly killed.

X. The best possible living conditions should be maintained for animals kept for biomed­ ~ ical purposes. Normally the care of animals should be under the supervision of veteri­ narians having experience in laboratory animal science. In any case, veterinary care should be available as required.

XI. It is the responsibility of the director of an institute or department using animals to ensure that investigators and personnel have appropriate qualifications or experience for conducting procedures on animals. Adequate opportunities shall be provided for in­ service training, including the proper and humane concern for the animals under their care.

Taken from Howard-Jones, (21). ECVAM Workshop 11: Three Rs 844 from key regulatory agencies in the USA considerable room for progress and improve­ established the Interagency Regulatory ment. Alternatives Group (IRAG; an ad hoc com­ mittee), to discuss the implementation of alternatives in the regulatory sector. Scientific and Ethical Justification In 1989, Alan Goldberg and Bert van Zutphen decided to initiate a series of world All proposed use of laboratory animals congresses devoted to alternatives and ani­ should be subject to review, to determine mal use in the life sciences (covering the whether such use appears to be scientifically Three Rs in research, testing and education), and ethically justifiable. In some circum­ which were to be held every three years. The stances, the "alternative" may simply be not first World Congress was held in Baltimore, to undertake the animal procedure at alL USA, in November 1993, and was attended Where the necessity of conducting certain by 725 people (representing academia, indus­ animal procedures cannot be justified suffi­ try, and government and animal protection ciently on scientific or ethical grounds, the organisations) from 24 countries (22). The project proposal should be rejected. second World Congress is to be held in Guidelines have been prepared to assist October 1996 in Utrecht, in The review committees in assessing whether Netherlands, while a third World Congress, alternatives have been adequately consid­ to be held in Italy in 1999, is already being ered (26). planned. In their consideration of the ethics of It is clear that significant changes have using animals in biomedical research, a occurred in the planning and conduct of bio­ Working Party of the Institute of Medical medical research projects. In Great Britain, Ethics (UK) concluded that "a research pro­ where reasonably accurate statistics on ani­ ject involving animal subjects should take mal use are available, the data indicate that place only when it can be shown: the use of animals increased by an average of a) that the aim of the project is worthwhile; 6% per year between 1937 and 1971, to a b) that the design of the project is such that total of over 5.5 million (23). From 1972 to there is the strong possibility that it will 1978, the number of animals used remained achieve the aim; relatively stable, and then animal use began c) that the aim could not be achieved using to decrease by an average of 5% per year morally more-acceptable and scientifi­ from 1979 onwards. The increase in animal cally no less-acceptable alternative sub­ use was driven largely by the search for new jects and procedures; and drugs and the expansion of the pharmaceuti­ dl that the likely benefits of the project are cal industry. Since the mid-1970s, the use of substantial enough in relation to the suf­ animals in commercial, government and uni­ fering likely to be caused to the animals versity/hospital laboratories in Britain has used (that is, the likely benefits of the dropped by 65%, 56% and 26%, respectively. research should be 'weighed' against the In The Netherlands, total animal use has 'costs' to the animals involved)"(27). declined by 50% since 1978 (24). While the data for the USA are less reliable, it has been It is these four main points, which include argued that animal use has also decreased the need to consider the potential for using significantly in American laboratories, alternative methods, which should be despite funding for biomedical research hav­ addressed to the satisfaction of the reviewers ing increased during this period (25). (who generally include biomedical scientists, In the mid-1990s, the question we face is veterinarians, ethicists, and community rep­ whether there will be a revolution in think­ resentatives with an interest in animal pro­ ing and practice, which is what is needed if tection). the expectations of Hume, Russell, Burch, The UK system involves licensing specific Lane-Petter, Medawar, Stevens, and all the persons both with respect to the projects to others involved in the original UFAW pro­ be undertaken and for their personal use of ject, are to be met, and the principles of laboratory animals (28). It provides a com­ humane experimental technique are to be prehensive and vigorous system of controls brought fully and effectively into operation. when taken together with the formal certifi· Much has been achieved, but there is still cation (designation) of heads of establish- 845 M. Balls et al. ments where experimental animals are used sion of the 1977 Act on Animal and the common species are bred. The Experimentation, the chairperson and at Animals (Scientific Procedures) Act 1986 also least two other members of such a committee provides for the appointment of inspectors in must be independent (that is, they must not the Home Office (the government departM have a working relationship with the institu­ ment equivalent to the ministry of internal tions for which the protocol is reviewed). affairs in other countries). Home Office Furthermore, the AEC must also include Inspectors check all designated establish­ experts on ethics and on alternative meth­ ments to ensure compliance with, or to ods, and the composition of the committee report on non-compliance with, the 1986 Act, must be approved by the National or with the terms and conditions of licences Committee on Animal Experimentation. The or certificates issued under the Act. main tasks of an AEC are to evaluate Uniquely in Britain, individual Home whether the expected benefit of the proposed Office Inspectors statutorily review projects experiment outweighs the likely suffering of and protocols, and advise the Minister (in the animals concerned. and to ascertain that practice, his officials) on the costs versus the feasibility for implementing the Three Rs benefits, with the aim of ensuring that only has been adequately taken into account properly justified work is licensed; these are when preparing the protocol. AECs are also functions performed by ethics review com­ required to evaluate the competence of the mittees in other countries. Where appropri­ persons involved in the design and perfor­ ate, views on proposed research projects are mance of the experiments. Rejection of a pro­ sought from other Inspectors and, occasion­ posed research protocol by the AEC can only ally, from external assessors or the APC. be overruled by the National Committee on Some special categories of work are manda­ Animal Experimentation. torily referred to the APC, for example, work In the USA, under both the Animal with primates and on cosmetics, and applica­ Welfare Act amendments of 1985 (regula­ tions to use animals for microsurgical train­ tions approved in 1989) and the Public ing. Home Office Inspectors only make Health Service revised policy, Institutional recommendations to government officials Animal Care and Use Committees ([ACUCs) but, in practice, their advice is usually are required to review and approve all ani­ accepted. Inspectors have almost no formal mal research proposals before the research is enforcement or executive powers with regard allowed to proCeed. The IACUCs are to licensees, but can order the immediate expected to ensure that approved animal humane killing of animals they consider to research protocols are worthwhile, that they be suffering excessively. Experimenters use the minimum number of animals neces­ rarely disregard the views of Home Office sary, that animal pain and distress are min­ Inspectors on the extent to which practical imised, and that, in any procedures likely to outcomes match the detailed protocols cause animal pain and distress (whether or authorised by project licences. not anaesthetics or analgesics are used). In Germany, the Department of principal investigators document that they Agriculture, which is responsible for animal have established "that alternatives were ade­ protection, adopts the general philosophy quately considered"(29). that, even if it is more expensive to use a While the manner in which IACUCs pur­ non-animal method than to conduct an ani­ sue their duties varies, the inspectors enforc­ mal procedure, a lower cost is not sufficient ing the Act have paid particular attention to justification for using animals. However, in the requirement that investigators docu­ 1994, the highest constitutional court in ment the lack of alternatives, and to the sec­ Germany ruled that an animal experiment tion of the regulations which reads: which is scientifically justifiable cannot be prohibited for ethical reasons, interpreting "Research facilities will be held responsible if this to be in compliance with both the it is subsequently determined that an alter­ national legislation and Directive native procedure was available to accomplish 86/609/EEC. the objectives of the proposed experiment ... In The Netherlands, the performance of or if it is subsequently determined that an animal experiments is not permitted unless experiment is unnecessarily duplicative and the protocol has been reviewed and approved that a good-faith review of available sources by an AEC. According to the proposed revi- would have indicated as much." ECVA11 Workshop 11: Three Rs 846

As a result, the consideration of alternatives for each assay and the degree of discomfort in the USA, while not necessarily embraced caused by current assay procedures. enthusiastically, is becoming routine during Where uniformity is important, it can also the preparation of research proposals. be promoted by controlling the environment in which the animals are reared and used. As Chance discovered many years ago (reviewed Selection of Appropriate Animals in Russell & Burch 1211, this does not mean keeping the environment uniform and con­ In whatever country the research is to be stant in all respects, but rather keeping it conducted, in designing a project the investi­ uniform, constant and appropriate in certain gator should first consider whether the aims key respects. Reduction here generally coin­ of the project could be realised by using in cides with the concept of refinement. For vitro techniques or less sentient animal example, as described by Fox (33), "handling species, such as insects or nematodes. The weanling female rats for three days prior to replacement of one animal species with experiments using the Steelman-Pohley another, particularly if the species which is method of follicle-stimulating hormone assay then used is non-vertebrate, could also be reduced the variability of their response", so considered to be an alternative method (30). that "about twice as many non-handled rats Therefore, the model selected should be the would be required in an assay to obtain the lowest phylogenetic species, and also the same degree of precision as with handled least sentient species, which will allow the rats". scientific objectives to be realised. If the use of living vertebrates is considered to be essential, the aim should be to use the mini­ Reduction Alternatives mum possible number of animals (see the section on Reduction Alternatives), and to The term reduction alternatives describes use strategies which will ensure that the ani­ methods for obtaining comparable levels of mals which must be used are subject to the information from the use of fewer animals in minimum discomfort (see the section on scientific procedures, or for obtaining more Refinement Alternatives). information from a given number of animals, A variety of strains of certain species are so that, in the long run, fewer animals are available. For example, over 400 inbred needed to complete a given research project strains of mice and over 200 inbred strains of or test. rats have been developed. These provide a The greater the number of animals used in wide range of phenotypes which are of poten­ an experiment, the greater will be the overall tial value in many areas of research. The costs, in terms of animal suffering (27l. choice of strain for a particular project Thus, the number of animals used should be should largely be governed by a knowledge of the minimum which is consistent with the its characteristics, and by the need to control aims of the experiment. However, past expe­ phenotypic variability. In most cases, iso­ rience in the area of regulatory toxicity test­ genic (inbred or Fl hybrid) animals are more ing, for example, suggests that laboratory suitable than outbred stocks, because of animal welfare considerations and common their high phenotypic uniformity, long-term sense do not always prevail (for example, in stability, identifiability, and detailed back­ LD50 testing; 341. Saving of time or personal ground information on their characteristics convenience, or other non-scientific reasons, (31). are not sufficient justification for using more However, in biological assays against stan­ animals than the minimum necessary to dards (where phenotypic uniformity is espe­ obtain meaningful results. Proper statistical cially important), although Fl hybrids are design, prior to undertaking the study, and usually more uniform than inhreds, there appropriate analysis of the resulting data, are no a priori grounds for choosing the most may make it possible to obtain .results of suitable strain or cross for a particular assay, comparable precision by using fewer ani­ and further studies of the type described by mals. Hendriksen et al. (32) should be undertaken, The precision of an experiment depends preferably as part of ongoing studies to min­ mainly on the sample size and the "error" imise the number of animals which are used variance and not on the body weight of the 847 M. Balls et al.

test animal. The use of more animals on the reduction in the use of animals, provided grounds that they are smaller and less that there is a reasonable compromise on expensive is not scientifically justifiable. If a acceptable sample sizes. Such harmonisation test has to be conducted in a rodent and in a should provide an opportunity to review the non-rodent species and the test guideline design and sample sizes required in regula­ specifies the use of four dogs, then the use of tory experiments, since, in some cases, sam­ more than four rats cannot be scientifically ple sizes appear to have been decided in an justifiable. It is recognised that problems arbitrary manner, without taking statistical with a protocol may be encountered once the considerations into account. Where possible, experiment is under way. To prevent the the requirements should be formulated in continuation of unsuccessful animal experi­ terms of acceptable confidence intervals, ments without review, acceptable limits for rather than by specifying the numbers of failures in protocols, and the actions to be animals needed, so that where greater con­ taken if these occur. should be specified. trol of phenotypic variation is possible, the Careful attention should also be given to number of animals can be reduced. the type of endpoint to be used. Qualitative Efforts at international harmonisation endpoints (for example, dead/alive) often should target not only the protocols for par­ involve severe animal pain and distress, and tinliar tests, but also the specific require­ generally provide less information than do ments for those tests. This would reduce the quantitative measurements. numbers of animals used by minimising the array of tests required. Research strategy Non-regulatory experiments Relatively little consideration has been given There is evidence that poor experimental to research strategy f';ince Russell and Burch design and inappropriate statistical analysis (2) discussed the random screening ofpoten~ of experimental results is leading to ineffi­ tial new pharmaceutical agents under this cient use of animals and of scientific heading. Such screening is now largely done resources in toxicological research (36-38). by using in vitro systems, which is one rea­ This is in agreement with previous studies of son for the decrease in the numbers of ani­ statistical methods used in other areas of mals used in the last decade. However, biomedical research (37-41). However, more research strategy is also important in other investigation is needed to determine whether contexts. In particular, it may be necessary appropriate experimental design and statisti­ to carry out small pilot studies which can be cal analysis are employed in other areas of reviewed before committing animals and research, such as experimental surgery, resources to major experiments. The statisti­ pharmacology, biochemistry, experimental cal guidelines developed by Muller et ai. (35), immunology, and microbiology. which include a detailed discussion of possi­ In some cases, the level of statistical ble research strategies, should be brought to expertise appears to be so low that investiga­ the attention of biomedical investigators. tors are either unaware of the potential value of obtaining statistical advice, or they Experimental design and statistics are unable to obtain appropriate statistical Optimum experimental design and statisti­ advice, because there are so few biometri­ caL considerations may suggest that a partic­ cians with experience in their field of inter­ ular protocol employs an insufficient number est. of animals, and that more are needed to pro­ The "named statisticia.n" vide a satisfactory answer to the question In some countries, a "named veterinarian" being posed. However, this should still lead must be appointed to supervise some aspects to an overall reduction in animal use, since of laboratory animal welfare. In view of the experiments which use too few animals will importance of good experimental design and generally not achieve their desired objec­ appropriate statistical analysis in underpin­ tives, and will frequently need to be repeated ning high quality research, and the potential with a larger number of animals. savings in terms of the numbers of animals Regulatory tests which are used, consideration should be International harmonisation of protocols in given to the need for a full-time or part-time regulatory testing should lead to an overall "named statistician" to be associated with ECV AM: Workshop 11: Three Rs 848 research facilities. This statistician would mental procedures, post-operative pain, and undertake to be available to all investigators improper euthanasia techniques. Non-expE>r­ who needed advice on experimental design imental sources include: naturally occurring and statistical analysis of experimental data, infectious and non-infectious diseases. suh­ and possibly would have some statutory optimal environmental conditions, improper obligation to' meet regularly with investiga­ handling, stressful housing situations (for tors to discuss current research projects. example, social isolation, barren cages or pens), injuries sustained during fighting. and Education in statistics injuries associated with the housing or A basic understanding of experimental design and statistics is necessary for all sci­ caging. entists. For investigators with no previous Much potential pain and distress can be training in statistics, this level of expertise avoided or at least alleviated with the proper can probably be obtained from a course use of anaesthetics, analgesics and tran­ involving the equivalent of about 30 hours of quilisers, which is a critical component of lectures and associated practical work. any comprehensive programme of adequate However, an equivalent level of expertise veterinary care. Such a programme provides could also be obtained by reading and by for frequent observation of the animals by using computer-assisted learning tech­ trained veterinary staff, to detect and appro­ niques. Books by Cox (42) and Cochran & priately relieve pain and distress. However, a Cox (43) provide a good introduction to substantial number of animals used in experimental design; Cohen (44) deals specif­ research and testing experience unrelieved ically and in detail with the problem of deter­ pain or distress. All experimental protocols mining the appropriate size for an should be sufficiently detailed with regard to experiment. There are many texts on statis­ the type and severity of likely adverse tical methods, which can be used both for effects, the times of peak occurrence, learning purposes and as reference books humane endpoints, and the remedial actions (45-48). There is also a need for some bio­ to be taken. medical research workers to have a more In The Netherlands, a serious attempt has detailed training in biometrics/statistics, so been made to categorise animal experiments that they can act as consultants to other on the basis of the severity of pain and dis­ investigators in their own institutes. tress experienced. In 1993, 51.47< of the ani­ mals were reported to have experienced either no or minor discomfort, 26. Fk experi­ Refinement Alternatives enced moderate discomfort, and 22.51}(· expe­ rienced severe discomfort (with a fifth of Refinement alternatives encompass those these receiving drugs to prevent or relieve methods which alleviate or minimise poten: the pain or distress; 24), tial pain and distress, and which enhance The percentage of animals experiencing animal well-being. "Distress" is an aversive unrelieved pain and distress from non-exper­ state in which an animal is unable to adapt imental causes is not known. Recent completely to stressors and the resulting advances in science and technology, and in stress, and therefore shows maladaptive laboratory animal medicine, offer significant behaviour (49), The stressors may induce opportunities to develop alternative methods physiological, psychological or environmen­ which may further reduce or eliminate unre­ tal stress. "Pain" results from potential or lieved pain and distress. It is proposed that actual tissue damage, such as that caused by research, testing and education facilities are injury, surgery or disease, and can lead to encouraged to improve and optimise the distress. These terms and concepts have well-being of laboratory animals, for exam­ been defined and discussed previously ple, by: (49-51). Pain and distress can result from both a) The procurement and maintenance of experimental and non-experimental causes. animals free of pathogenic organisms; Potential sources of experimental pain and this requires effective vendor surveil­ distress include: improper or prolonged lance, quarantine, health monitoring, restraint, experimental infections, chemical­ disease investigation and preventive induced toxic effects, surgical and experi- medicine programmes. M. Balls et ai. 849 b) The provision of optimal caging and hus­ and aggressively disseminated (52). bandry procedures (appropriate to the physiological and behavioural needs of International harmonisation the species), such as avoiding caging ani­ Attitudes differ as to which scientific proce­ mals singly whenever feasible and practi­ dures lead to animal pain and distress, and cal. the severity of the pain and distress they c) The provision of optimal environmental cause. Efforts to identify these differences, to conditions, with minimal variations in determine the underlying reasons for them, temperature, humidity, etc. and to harmonise international standards as d) The provision of enrichment/exercise much as possible should be undertaken. programmes where they are appropriate and will be beneficial. Research support for refinement alternatives Need for a.ssessment measures of animal pain Very little research funding is available to and distress support efforts to investigate and refine experimental techniques and scientific pro­ At present, we do not have a convenient and cedures. Some ad hoc funding has supported standardised way of objectively assessing workshops on antibody production, adjuvant animal pain and distress. Rather, the assess­ use and infectious disease models. The BVA ment is generally based on subjective clinical Animal Welfare Foundation, FRAME, the signs of abnormal behaviour and appearM RSPCA and UFAW formed a Joint Working ance. The approach to animal pain and dis­ Group on Refinement in 1989, with the tress is to assume that a procedure which intention of setting up a series of workshops inflicts pain and distress in human beings to discuss ways in which common laboratory will inflict at least as much pain and distress procedures could be refined. The first and in animals, unless there is evidence to the second reports were on the removal of blood contrary (52, 53). from laboratory mammals and birds (61) and There are wide variations among different on refinements in rabbit husbandry (62), countries with respect to assessments of the M respectively. Nevertheless, no sustained extent of pain and distress caused by partic source of funding is available to support the ular husbandry and experimental relatively modest research projects which approaches. There are no internationally could provide th~ impetus to develop an defined standards on animal pain and disM appropriate measure 9f adverse effects on tress or agreed cut-off points, although some animals, and which could provide essential working guidelines have been produced and data on the actual impact of particular scien­ disseminated widely (for example, 54). There tific procedures and experimental tech­ are a number of specific national guidelines niques. Some research funding is available to on procedures which cause particular con­ support animal husbandry modifications and cern, such as those on cancer research proM environmental enrichment (especially for duced by the UK Coordinating Committee on primates, dogs and cats), but there is little Cancer Research (55), on antibody produc­ funding available to explore environmental tion (56), and on lethal endpoint screening enrichment in rodent housing, despite the tests for antimicrobial agents (57). The num­ fact that rodents constitute 85% of all labo­ ber of techniques or scientific procedures ratory animals used in experiments. that are commonly used and are of signifiM cance probably amount to no more than one Dissemination of information on refinement or two hundred. alternatives Before adverse effects on animals can be assessed, one must be able to recognise such. Scientists are not sufficiently aware of the effects via either behavioural or physiological concept of refinement alternatives and, in measures. Several such measures have been general, they do not recognise the impor­ suggested (for example, 58-60), but have not tance of refinement in their research. The been widely adopted. In order to prioritise concept of recognising, minimising and elim­ which husbandry and experimental proce­ inating pain and distress in laboratory ani­ dures need to be refined and modified, appro­ mals should be included in training priate objective measures of adverse effects programmes for all persons involved in the on animals need to be developed, validated care and use oflaboratory animals. Details of ECV AM Workshop 11: Three Rs 848 research facilities. This statistician would mental procedures, post-operative pain. and undertake to be available to all investigators improper euthanasia techniques. Non-exper­ who needed advice on experimental design imental sources include: naturally occurring and statistical analysis of experimental data, infectious and non-infectious diseases. suh­ and possibly would have some statutory optimal environmental conditions, improper obligation to· meet regularly with investiga­ handling, stressful housing situations (for tors to discuss current research projects. example, social isolation. barren cages or pens), injuries sustained during fighting. and Education in statistics injuries. associated with the housing or A basic understanding of experimental caging. design and statistics is necessary for all sci­ Much potential pain and distress can be entists. For investigators with no previous training in statistics, this level of expertise avoided or at least alleviated with the proper use of anaesthetics, analgesics and tran­ can probably be obtained from a course involving the equivalent of about 30 hours of quilisers, which is a critical component of lectures and associated practical work. any comprehensive programme of adequate However, an equivalent level of expertise veterinary care. Such a programme provides could also be obtained by reading and by for frequent observation of the animals by using computer-assisted learning tech­ trained veterinary staff, to detect and appro­ niques. Books by Cox (42) and Cochran & priately relieve pain and distress. However, a Cox (43) provide a good introduction to substantial number of animals used in experimental design; Cohen (44) deals specif­ research and testing experience unrelieved ically and in detail with the problem of deter­ pain or distress. All experimental protocols mining the appropriate size for an should be sufficiently detailed with regard to experiment. There are many texts on statis­ the type and severity of likely adverse tical methods, which can be used both for effects, the times of peak occurrence, learning purposes and as reference books humane endpoints, and the remedial actions (45-48). There is also a need for some bio­ to be taken. medical research workers to have a more In The Netherlands, a serious attempt has detailed training in biometrics/statistics, so been made to categorise animal experiments that they can act as consultants to other on the basis of the severity of pain and dis­ investigators in their own institutes. tress experienced. In 1993, 51.4'7r of the ani­ mals were reported to have experienced either no or minor discomfort. 26.1(Yr- experi­ Refinement Alternatives enced moderate discomfort. and 22.50'r· expe­ rienced severe discomfort (with a fifth of Refinement alternatives encompass those these receiving drugs to prevent or relieve methods which alleviate or minimise poten: the pain or distress; 24), tial pain and distress, and which enhance The percentage of animals experiencing animal well-being. "Distress" is an aversive unrelieved pain and distress from non-exper­ state in which an animal is unable to adapt imental causes is not known. Recent completely to stressors and the resulting advances in science and technology, and in stress, and therefore shows maladaptive laboratory animal medicine, offer significant behaviour (49). The stressors may induce opportunities to develop alternative methods physiological, psychological or environmen­ which may further reduce or eliminate unre­ tal stress. "Pain" results from potential or lieved pain and distress. It is proposed that actual tissue damage, such as that caused by research, testing and education facilities are injury, surgery or disease, and can lead to encouraged to improve and optimise the distress. These terms and concepts have well-being of laboratory animals, for exam­ been defined and discussed previously ple, by: (49-51). Pain and distress can result from both a) The procurement and maintenance of experimental and non-experimental causes. animals free of pathogenic organisms; Potential sources of experimental pain and this requires effective vendor surveil­ distress include: improper or prolonged lance, quarantine, health monitoring, restraint, experimental infections, chemical­ disease investigation and preventive induced toxic effects, surgical and experi- medicine programmes. ECVAM Workshop 11: Three Rs 850 refinement and animal welfare considera­ Replacement Alternatives tions should routinely be included in scien­ tific papers and publications (63). Replacement alternatives encompass those methods which permit a given purpose to be Refinement alternatives have multiple bene­ achieved without conducting experiments or fits, and the promotion of their implementa­ other scientific procedures on animals. tion should include the publication of Russell & Burch (2) distinguished between appropriate review articles which document relative replacement, for example, the the scientific, economic and humane bene­ humane killing of a vertebrate animal to pro­ fits. vide cells, tissues and/or organs for in vitro There is no readily available up-to-date studies, and absolute replacement, in which knowledge base on refinement. Techniques animals would not need to be used at all, for that are developed to refine a procedure are example, the culture of human and inverte­ frequently not reported in the scientific liter­ brate cells and tissues. ature, or are established simply as Standard It was discussed whether animal organ­ Operating Procedures (SOPs) within an atypic and primary cell culture should be institution. To establish "best practice"and classed as reduction, since an animal would to advance the implementation of refinement still have to be used, whereas serial cell cul­ alternatives, it is important to share such ture, for example, the use of permanent cell experience, data and SOPs. Sharing of data lines, would be classed as replacement. It and theories is normally accomplished via was thought to be preferable to continue to the scientific literature, but there has been a use Russell and Burch's approach, partly marked lack of opportunity to discuss and because the use of different terms for non­ provide information on refinement alterna­ human vertebrate primary cultures on the tives in the main biological journals. one hand, and human and invertebrate pri­ Since the pioneering work of Chance (64), mary cultures and non-human vertebrate there have been several publications on the serial cell cultures on the other, would lead assessment of distress and refinement proce­ to confusion, and partly because the humane dures (2, 33), which should be made as killing of an animal does not represent an widely known as possible (65-67). In particu­ experiment or a regulated scientific proce­ lar, experimenters should be familiar with dure in most countries. the important general survey by Claassen Nevertheless, statistics on ~nimal use (68). As pointed out by Gardner & Gardner should include the numbers of animals killed (69), acceptance of the very real need for specifically for the purpose of providing cells more research in distress assessment and and tissues for in vitro studies, and for the refinement should not discourage us from production of subcellular fractions, such as ,making urgently needed immediate improve­ liver S9 preparations for use in metabolism ments in cases where there are obvious phys­ and mutagenicity studies. Account should iological or behavioural indications of stress, also be taken of the culling of animals sur­ and therefore of distress, due to factors such plus to requirements, which can be very high as overcrowding, social isolation or restraint. in the breeding and supply of rodents. Replacement alternative methods and Auditing system approaches Auditing of the implementation of refine­ The range of replacement alternative meth­ ment alternatives at the institutional level is ods and approaches includes the following rarely carried out, although it can easily be (27, 70, 71): done by requiring investigators to provide a) The improved storage, exchange and use basic information for review by ethics com­ of information about animal experiments mittees/IACUCs, or by independent review already carried out, so that unnecessary by animal welfare officers, laboratory animal repetition of animal procedures can be veterinarians, or government inspectors. avoided. Such data can significant.ly enhance the bl The use of physical and chemical tech­ development of further refinements, and niques, and of predictions based on the raise awareness of the concept of refinement physical and chemical properties of mole­ and its importance. cules. 851 M. Balls et al. c) The use of mathematical and computer tal biomedical research and in regulatory models, including: i) modelling of quantita­ testing (72), In the former case, scientific tive structure-activity relationships, i.e. methodology evolves mainly through an taking advantage of correlations between informal publication and peer-review accep­ molecular structure and biological activity tance and/or improvement process. In the in the prediction of the potential desired latter case, however, formal validation of the and undesired effects of series of related replacement alternative method in terms of chemicals; ii) molecular modelling and the its relevance and reliability for its stated pur­ use of computer graphics, for example in pose is likely to be necessary (18, 73), since actively designing drugs and other chemi­ national or international laws, guidelines or cals for specific purposes; and iii) modelling regulations will need to be modified, if the of biochemical, physiological, pharmaco­ non-animal test is to gain wide acceptance as logical, toxicological and behavioural sys­ a replacement for the animal test. In addi­ tems and processes. tion, as practised at present, regulatory test­ d) The use of "lower" organisms with limited ing often requires the induction of adverse sentience and/or not protected by legisla­ effects, and even of considerable animal suf­ tion controlling animal experiments, fering, which are integral to the test proce­ including invertebrates, . plants and dure and are therefore unavoidable. microorganisms; for example, the use of It follows from this that those of us who bacteria in genotoxicity testing. are concerned that the Three Rs concept e) The use of the early developmental stages should be implemented as fully as possible, of vertebrates before they reach the point should welcome the trend toward the use of at which their use in experiments and non-animal methods in fundamental other scientific procedures is regulated. research. However, in the case of regulatory D The use of in vitro methods, including sub­ efficacy and toxicity testing, research specifi­ cellular fractions, short-term maintenance cally aimed at providing validated replace­ of tissue slices, cell suspensions and per­ fused organs, and tissue culture proper ments for the currently accepted animal test (cell and organotypic culture), including procedures and testing strategies should be human tissue culture. conducted, and the value of such research g) Human studies, including the use of should be recognised by the scientific com­ human volunteers, post-marketing surveil­ munity at large. This would be fully consis­ lance and epidemiology; for example, sldn tent with one of the hopes expressed by patch testing in humans before marketing, Russell and Burch in the conclusion to their and monitoring consumer response after book, i.e. that their efforts "would stimulate marketing, as alternatives to the animal some experimentalists to devote special testing of cosmetic products. attention to the subject"(2). A great deal of effort is being put into the In many areas of the biomedical sciences, in development and evaluation of replacement vitro methods are increasingly used as the alternative methods for use in testing, by methods of choice in place of animal studies, industry and academia, often with the finan­ not because they provide precisely the same cial support of animal welfare organisations information, but because they offer the best and/or government funds specifically ear­ scientific approach to tackling the questions marked for this purpose. Until now, the rate being asked. An example of this would be the of progress has been slow, partly because a use of tissue, cell and subcellular preparations series of barriers must be overcome, in addi­ in vitro for screening candidate compounds for tion to the legislative/regulatory barrier pharmacological activity. The information pro­ referred to earlier (74). The most important vided by the in vitro methods may ultimately of these are the validation barrier and the have an outcome similar to that provided by scientific barrier. the animal studies used in the past (for exam­ The validation barrier ple, the identification oflead compounds). The validation of new tests and testing strategies in terms of assessing their rele­ Replacement alternatives in research and vance and reliability is difficult, and the hur­ testing dles placed in the path of replacement A distinction also needs to be made between alternatives must necessarily be high, if mis­ the replacement of animal use in fundamen- takes are to be avoided which could have dis~ ~E~C~V~AM~~W~o~rk~s~h~OEP~l~l~,T~h~r~e~e~Rs~ ______~8.52

astrous human health and environmental use of rodents and other laboratory mam­ consequences and thus delay the achieve· mals in toxicity testing, are used because ment of our objectives (75). However, these "in their general physiological and pharma­ hurdles must be fair, especially as the ani­ cological properties" they are "more consis­ mal tests we are seeking to replace have not tently like us than are other organisms". themselves been subjected to formal, inde­ High discrimination models, on the other pendent and objective evaluation in terms hand, "reproduce one particular property of of their relevance, reliability and applicabil­ the original, in which we happen to be inter­ ity. Validation should be seen as a continu­ ested". The Limulus amoebocyte lysate OllS process, and the principles and criteria (LAL) test is one such model. The use of dis­ involved and the correct practices to be fol­ crimination models in toxicity testing, for lowed are still being debated (18, 73, 76), example, is represented by the currently One of the greatest problems in planning available in vitro systems and other replace­ validation studies in the area of toxicity ment alternatives, which are more suitable testing is finding in vivo data of sufficiently for answering a specific question about the high quality for use in evaluating the pre­ mechanism of a toxic effect or toxic dictive value of the results obtained in in response in a particular cell type than for vitro tests. This has led to recommendations answering the more general question: «Is that an International Reference Chemical this chemical likely to be toxic in ways Data Bank be established 06, 77), to pro­ which we cannot envisage?" vide open-access listings of chemicals, Russell and Burch warned of the high backed by first-class toxicological data fidelity fallacy and of the danger of expect­ reviews, safety advice and a source of chem­ ing discrimination in particular circum­ icals of known purity. The European Centre stances from models which show high for the Ecotoxicology and Toxicology of fidelity in other, more general, terms (2), Chemicals (ECETOC) has established task They pointed out that the fidelity of mam­ forces for providing chemicals for use in val­ mals as models for man is greatly overesti­ idation studies on alternatives to the Draize mated, and concluded that the assumption eye and skin irritation/corrosivity tests (for that "mammals are always the best models" example, 78). The US National Toxicology for man "is maintained with special stub­ Program (NTP) publishes the results of all borness in some special fields (such as that of its studies, both electronically on the of toxicity testing)". They went on to say Internet and in book form. that the most important consequence of the Of course, the most appropriate way to high fidelity myth is that it "ignores all the assess a new in vitro method is not neces­ advantages of correlation ", whereby "the sarily to use in vivo data from laboratory responses of two utterly different systems animals as the reference standard, but to may be correlated with perfect regularity", compare results from both methods to those despite other differences between them. obtained in human (clinical) studies. The argument about fidelity, discrimination Unfortunately, finding human data of suffi­ and correlation test systems is still going on cient quality is a major problem, and those today. data which do exist are often proprietary. Ultimately, there is only one way forward Moreover, generating new human data is - the development and acceptance of fraught with ethical and logistical consider­ replacement alternatives for both research ations. and testing must be based on a sufficient understanding of the molecular and cellular mechanistic basis of what is being studied The scientific barrier or measured, i.e. on sound science. Hence, Replacement alternative methods must be the current trend toward a more mechanis­ based on good science, and extravagant tic approach should be welcomed, ·encour~ claims which cannot be substantiated must aged and financially supported. not be made about them. One of the most Several specific recommendations relat­ fascinating sections of Russell and Burch's ing to the development and use of replace­ book is their discussion on the relative mer­ ment alternatives in production and testing its of fidelity and discrimination models (2). are given at the end of this report (see High fidelity models, as exemplified by the Conclusions and Recommendations). 853 M. Balls et al.

Education and Training c) directors of animal facilities and animal welfare officers; The successful implementation of the Three d) national and regional inspectors; and Rs heavily depends upon the e~ucation at;d e) members of ethics committees/IACUCs training of those persons Involved. l~ (scientists and lay representatives). research and testing. A distinction must InI­ tially be made between "education" and Course content a.nd format "training". Education is defined here as the didactic presentation of the information and The type of education and training referred theories of animal use that will contribute to to below is intended primarily for students the development of proper attitudes toward preparing for an advanced degree in biom~d­ the use of animals in scientific procedures. ical research. Such courses should emphaSise Training is defined as the acquisition ofprac­ that the aim of the training is to improve sci­ tical knowledge and skills directly associated entific quality as well as to educa~e scientists with animal handling and procedures. Both with respect to the legal reqUlrem~nt for education and training are necessary for the using alternatives, whenever posslble. ~ implementation of all of the Three Rs. . description of the course on ammal experI­ The objective of the educatIOn and tram­ mentation and alternatives currently offered ing is to provide sufficient information to at Utrecht University in The Netherlands allow scientists to conduct animal proce­ has been published recently (79). In addition, the Federation of European Laboratory dures to high standards of both science ~nd animal welfare, following proper evaluatIOn Animal Science Associations (FELASA) of the scientific and ethical considerations Working Group on Education in Europe, and which should govern the use of alliaborat?ry the National Research Council in the USA, animals. All people involved in performmg have published guidelines for the education animal experiments should have appropriate and training of persons working with lab~ra­ practical training, to help ensure that they tory animals (80, 811. All of these publica­ are technically competent to carry out the tions serve as excellent prototypes for the procedures. Most countries require that development of courses in other countries. those conducting animal research be compe­ There are several ways in which a course tent and trained in the techniques that they on animal experimentation and alternatives are using or plan to use. Training for those in can be packaged. The first is the standard written syllabus with accompanying refer­ professions that require expe~tise in animal handling should he accomplished through ence materials (for example, 82). apprenticeships (that is, "on-the-job" train­ Demonstration videos can also be used. Other possible formats include interactive ing) and, in such cases, animals sh?uld o~ly he used in order to perfect speCIfic skIlls CD-ROM and the Internet, the use of both of which cannot be achieved in any other way. which is becoming more widespread. These The education and training should con­ latter two formats have the potential to tribute to a scientist's ability to design exper­ reach a greater number of scientists. iments properly and to plan research strategies, to become competent in ~ni~al Education in ethology handling and the performance of sCIentIfic It is clear that the progress of implementing procedures, to make decisions with re.gard to reduction and refinement alternatives the ethics of using animals in experIments, depends largely on the ability of scientists to and to determine whether alternatives are observe and understand the behaviour and available. The concept of refinement alterna­ needs of their laboratory animals. At pre­ tives and the obligation to make them pub­ sent it would seem that many experimenters licly ~vailable, should also be emphasised in are lacking in ethological equipment as education and training programmes. ~s they are in statistical knowledge ..Fo: ex~n:­ The workshop participants identified five pie, many experimenters have an mdlscnml­ separate groups for which education and training are necessary: nate reaction to work with mazes, some regarding it as harmless and some as stress­ a) animal technicians; ful. In fact, if a rat is put in a maze when not b) scientists, including laboratory animal particularly hungry, he is given an .interest­ veterinarians; ing opportunity to exercise the speCIal talent ECVAM Workshop 11: Three Rs 854 of his species, that of making a cerebral map driven by political and social forces exclu· of his spatial surroundings. Such a rat, sively, rather than being relevant to scien· exploring the maze thoroughly, will learn it, tilic issues (71). This is partly due to a lack of so that later, if the maze is altered, he can appreciation of the basis of the Three Rs con· soon spot new blind alleys or short cuts. If a cept as proposed by Russell and Burch, i.e. rat is put in a maze when severely hungry, that scientific excellence and the greatest he will run it with anxiety and distress, humanity in the use of laboratory animals becoming conditioned to one route and stick· are inextricably linked, and of the great ing to it thereafter, even when conditions are potential value of alternative methods (87), changed and it no longer leads to reward. It also stems from a defensive attitude These two cases have been used as classical among some scientists, perhaps resulting examples of relaxed and stressful behaviour, from the campaigns of some antivivisection respectively, by Russell (83). organisations and from insufficient dialogue One very useful guideline for mammalian among the scientific and animal protection behaviour supplied by ethology is this: any­ communities (88). thing that leads to learning is harmless or Scientists should be better informed about positively enjoyable; anything that leads to the Three Rs concept, and should be encour· conditioning involves some stress and, there· aged to see it as an opportunity, rather than fore, distress. A very effective short course in as a threat. At the conclusion of their book, ethological observation was designed some Russell and Burch expressed the hope that years ago for medical students by Chance & experimenters would be stimulated "to Mackintosh (84). It originally required the devote special attention to the subject ... to use of two rats, but could easily be adapted to work in full awareness of its existence and a video. possibilities"(2L For some, including the par· ticipants in this workshop, the Three Rs has Replacement alternatives in education become a subdiscipline within the biological Students should not be forced to use animals sciences, but this is not generally recognised. for any purpose. Replacement alternatives Such recognition should be encouraged and for use in education have been described by promoted through scientific societies, acade· Fosse (85). These include models, films and mic journals and funding agencies. Various videotapes of procedures, interactive com· governments have already given a lead, by puter programs, software simulations, creating such bodies and centres as ECV AM, courseware on compact discs and interactive ICCV AM and ZEBET, the German Federal laser discs, and virtual reality programs. The Government's Centre for Documentation NORINA database contains information on and Evaluation of Alternative Methods to those alternatives which are available (86). Animal .Experiments. The journals specifi· Resources are required to produce these cally devoted to the Three Rs, such as materials and to make them more widely AATEX, ALTEX, TAR and ATLA, should be available. made more readily available to scientists by their institutional libraries.

Informing Scientists about the Three Rs Concept Informing the General Public

The use of the term alternatives to encom· Although many members of the general pub­ pass all of the Three Rs is now widely lic are concerned about the use of animals in accepted in many countries, and is incorpo· research, testing and education, it is clear rated into the names of various centres, for that they are not typically well-informed example, CAAT, ECVAM and the NCA, and about the Three Rs approach to th~ ethical journals, such as AATEX (Alternatives to issues raised by animal experimentation. Animal Testing and Experimentation - There are several reasons for this: the Three Japan), ALTEX (Alternativel! zu Rs approach has not been embraced by many Tierexperimenten - Switzerland), TAR (The of the protagonists in the vivisection contro· Alternatives Report - USA), and ATLA versy (neither scientists nor animal protec· (Alternatives To Laboratory Animals - UK). tionists); some individuals and organisations However, some scientists see its use as being who have embraced the Three Rs have not 855 M. Balls et al.

made a point of informing the public of this social studies and biology syllabuses. This approach; and those who have made the has led many organisations to produce spe­ effort have often found the media unrecep­ cial publications, and FRAME was invited by tive, preferring instead to portray confronta­ an educational publisher to produce two tions between those holding diametrically booklets to convey an objective, middle­ opposed views. Indeed, it is easier for all con­ ground position on animal experimentation cerned to convey a message in the form of and on alternatives (93, 94). With the sup­ simple slogans, such as Stop Animal port of industrial companies, a copy of each Experiments (89) or Most People See a Rat­ booklet was given free of charge to every sec­ We See a Cure {or Cancer (90), than it is to ondary school in Britain. It is particularly explain the principles of reduction, refine­ important that material for schools and col­ ment and replacement as a blueprint for ree­ leges should be designed to help young peo­ oncili ng the interests of science and animal ple make up their own minds on this issue welfare. (the approach adopted, for example, in the Why should supporters of the Three Rs newsletter GAATALYST, which is produced consider informing the public about this by GAAT for 11-14 year olds), rather than to approach? Their efforts can reach members seek to persuade them. In addition, while of the public who are sympathetic, and who more-detailed material might usefully be can then offer moral, financial and political provided for teachers, care should be taken support to the cause. We know that members not to be seen to want to instruct them in of the general public are hearing about more how to teach their subjects. confrontational approaches to the animal One very successful way for organisations research controversy; unless they also hear to encourage individual students to become about the Three Rs, our approach will be per­ actively involved in the Three Rs is the pro­ ceived as being irrelevant to this issue. vision of support for temporary employment Many organisations concerned with pro­ in laboratories or in the offices of the organ­ moting the Three Rs have sought to inform isations themselves. Replacement alterna­ the public about their activities, and about tive methods can be very suitable for the alternatives approach in general (for undergraduate, as well as graduate, research example, 91 J. These efforts have entailed projects. producing leaOets, brochures and other writ­ There are occasions when concerted action ten material (92); taking advantage of media by groups of organisations with similar opportunities to appear on radio or television philosophies and policies could be the best programmes, or in print; speaking to stu­ way of getting a message across to a large dents and community groups; and placing audience. For example, advertisements could advertisements. The Internet is providing be placed in newspapers or magazines in the organisations with a new opportunity to light of specific events. Organisations which make their message widely and freely avail­ were willing to join in such concerted actions able. An unexplored possibility is for one or might also agree on a common logo, which more organisations to independently pro­ would become more widely recognised the duce a high quality video programme, which more it was used. can be shown on educational television sta­ tions and be distributed to interested indi­ viduals and organisations. Past efforts· to inform the general public Special Considerations about the issues relating to alternatives have had mixed outcomes. Accordingly, pro-alter­ Vaccines and other immunobiologicals natives organisations should devise and The development, production and quality implement such efforts carefully, with due control of vaccines and other immunobiolog­ consideration to past successes and failures. icals was recognised as being an important Perhaps the most important specific tar­ area for the implementation of the Three Rs, get audiences within the general public are since large numbers of animals are used teachers and students in schools and col­ which usually suffer significant pain and dis· leges. In the UK, there has been a surge of tress as a result of the experimental proce· interest since animal experimentation dures employed. Most of the animal tests became a subject in secondary (high) school undertaken are documented in either a com- ECVAM Workshop 11: Three Rs 856 pendium, such as the European be made to minimise the numbers of mice Pharmacopoeia, or in guidelines produced by used for pertussis vaccine testing by standar­ national control authorities and interna­ dising the procedures employed. tional regulatory bodies (for example, by the It was emphasised that the development of World Health Organisation), as well as in reduction, refinement and replacement national product licences. alternative methods is the responsibility of For a variety of reasons, interest in both all those involved in the production and qual­ the Three Rs and in vaccine quality control ity control of vaccines and other immunobio­ strategies has grown in the past decade. This logicals (that is, both industry and has led to a reduction in the use of animals governm€mt). for vaccine quality control purposes. Reviews of recent developments, and recommenda­ Transgenic animals tions for further initiatives, have been pub­ The production, breeding and lise of animals lished (95, 96). genetically modified via the application of Steps should be taken to facilitate further transgenic techniques will undoubtedly implementation of the Three Rs concept increase significantly in the future. This new with respect to the development, production technology offers great potential scientific and quality control of immunobiologicals, benefit, in terms of the understanding of especially in relation to testing require­ fundamental biological processes, the ments. Potency tests based on a challenge nature, diagnosis and treatment of various procedure are of particular concern. Lethal diseases, the production of useful biological endpoint potency tests for tetanus and diph­ products, and the husbandry of disease-resis­ theria vaccines are still documented in some tant animals. Animal transgenesis may also pharmacopoeias, and are therefore still per­ contribute to the implementation of the formed in some countries, although alterna­ Three Rs, by reducing animal numbers, by tive non-lethal endpoint tests are also permitting the replacement of more-sentient permitted and are widely used by more species with less·sentient species, by reduc­ advanced countries. Lethality endpoints ing animal suffering in other ways, and by should be replaced with other assessment increasing the relevance of laboratory ani­ measures. Validated in vitro test systems, mal use. For example, in the USA, several such as enzyme-linked immunosorbent transgenic mouse models are currently being assays (ELISAs) or serological systems based investigated by the NTP with respect to on in vitro models, should be employed, to their usefulness in evaluating the potential reduce and refine the use of animals. When carcinogenicity and mutagenicity of chemi­ challenge of experimental anim.als is cals (98). Carcinogenicity studies employing unavoidable, clear clinical symptoms should these models are completed in six months or be taken as being equivalent to the lethality less, compared with the standard two-year endpoint, and animals showing such symp­ bioassay, and involve fewer animals per dose toms should then be killed humanely (951. group. In addition, the animals are not sub· A proposed alternative approach for esti­ ject to age-related morbidity and mortality. mating potency (97) should be evaluated. In Despite these kinds of developments, some this approach, a distinction is made between: observers are of the opinion that the use of a) estimation of the immunogenic potency of transgenic animals will lead to an overall the first few batches obtained from a seed increase in animal use, rat-her than to a lot; and b) monitoring the consistency of sub­ decrease. There is also legitimate concern sequent batches. The use of animals is lim­ that the control of the production, breeding ited to the first few batches, while and use of transgenic animals is not ade­ monitoring the consistency of the quality of quately provided for in current legislation for subsequent batches is undertaken by using the protection of laboratory animals. For in vitro methods (971. example, there is as yet no broad agreement Although there is no in vitro model avail­ on whether only the creation of transgenic able for replacing the Abnormal Toxicity animals, andlor their breeding, and/or their Test in vaccine safety testing, the value of use in specific experiments should be treated this test is questionable, since its lack of as regulated procedures, or whether trans­ specificity hampers reliable interpretation of genic animals should be protected through­ the results obtained (95). Efforts should also out their lives, whether or not they are ever 857 M. Balls et al. used in specific experiments. The system Secondly, cats, dogs and horses have been adopted in the UK, whereby strict controls bred over many centuries to be companion are applied to the breeding of transgenics animals for human beings, and it can be until it can be shown that no special health argued that, since they have been bred to be and husbandry problems are involved, is to dependent on us, we have a specific moral be recommended. obligation to them. That is certainly how the Molecular geneticists involved in produc~ vast majority of members of the general pub­ ing transgenic animals must always be aware lic would be likely to view the situation, and of the animal welfare implications of their both animal experimentation and the Three experiments, and of their legal and moral Rs must be viewed in a social, as well as in a responsibilities (99, IDOl. Concern that this scientific, context. Nevertheless, there is a is not always the case has been highlighted strong counter-argument that all laboratory by the occurrence of unexpected, sometimes animals should be given equal protection, very severe, adverse effects in transgenic ani M and that all forms of speciesism should be mals. There are also ethical concerns about avoided. transferring genes between species, particu­ larly when the genes involved are human Benefit and suffering! genes (101). Moore & Mepham (102) have suggested recently that the actual and poten­ It is implicit, if not explicit, in all the regula­ tial implications of the increased use of tions governing laboratory animal use that transgenic animals may result in the need the purpose of the work must be worthwhile, for a fundamental reappraisal of the mecha· and that it must be viewed in relation to the nisms whereby permission to perform scien· suffering caused to the animals used. tific procedures on animals is considered and The British 1986 Act requires that the granted. likelihood of a beneficial outcome of a pro­ posed programme of work is assessed in Special protection for selected animals advance, and that, where it would involve A fundamental tenet of the refinement prin· vertebrate animals which might be caused to ciple is that the least sentient species suit· suffer pain, distress and/or lasting harm, a able for the proposed experimental work weighing of likely benefit versus likely suf­ should always be selected. However, there is fering must be made, before permission is no general agreement on how sentience can given for the experimental work to proceed be measured or how degrees of sentience can (16,27, 103). be compared. For example, is a marmoset Cost-benefit apaIysis is part of our every­ more sentient than a rat? In some legisla· day lives, and it involves judgement on the tion, 'Special consideration is given to non· basis of evidence, rather than automatic human primates, cats and dogs. In Britain, decisions based on precise measurements. this special consideration is also extended to Applying this kind of approach should not be Equidae. It can be argued that there are two new to scientists, since it is an essential part different justifications for this approach. of the peer· review system for granting Firstly, in the case of the higher primates research funding. (Old World monkeys and Hominids), what­ Who should be involved in assessing and ever is meant by "sentience" or, in the words weighing benefit and suffering will depend of Directive 86(609(EEC, "degree of neuro­ on the particUlar system for regulating ani­ physiological sensitivity"(12), it is commonly mal experimentation. The ultimate moral accepted that these animals are placed at the responsibility should lie with the scientists top end of any spectrum or scale. They there­ who propose to perform the studies. fore deserve special consideration, both in However, institutions, funding agencies, gov­ their own right, and also because, if we can· ernments and the community in general not ensure the proper application of labora­ should all have some input (27). In the UK, tory animal protection laws and regulations the Home Office Inspector would discuss this to them, we are unlikely to be successful in question with a project licence appJicant, and providing adequate care for rodents, birds would advise the Home Secretary, with and fish. whom the ultimate legal responsibility lies

I The word "suffering" has been used as a generic term for "undergoing, experiencing or being subjected to pain, distress and lasting harm n. ECV AM Workshop 11: Three Rs 858

(104). In other countries, IACUCs or ani­ irritancy test, and to all procedures which mal ethics committees contribute to the result in substantially severe effects. discussion or even make decisions. This is a topic which deserves further consideration, especially with respect to Concluding Remarks the ways in 'which benefit and suffering should be assessed and weighed. Four The workshop participants unanimously main schemes have been proposed or are reaffirmed the principles put forward by in use: that suggested in Lives in the Russell & Burch (2), that humane science is Balance, the report of the Institute of good science and that this is best achieved Medical Ethics (UK) Working Party (27); by vigorous application of the Three Rs: the Dutch model developed by the reduction alternatives, refinement alterna· Department of Animal Problems, Leiden tives and replacement alternatives. Thus, University (105); a model proposed by the only acceptable animal experiment is Porter (106); and the system already used one which uses the smallest possible number by the British Home Office Inspectorate of animals and causes the least possible pain for the assessment and weighing of bene· or distress which is consistent with the fit and suffering, which was described in achievement of a justifiable scientific pur· the 1993 report of the APC (107). pose, and which is necessary because there It is widely agreed that there are levels is no other way of achieving that purpose. of animal suffering which could never be Any proposed experiments on animals justified on scientific grounds, but there should be subjected to prior and effective is no agreement as to what those levels expert review by an ethics committee or an are. It is certainly possible to perform equivalent body. The Three Rs should be procedures in one country which would seen as a challenge and as an opportunity not be permitted in another. This is a for reaping benefits of every kind - scien· tific, economic and humanitarian - not as a matter which deserves urgent interna· threat. tional discussion and agreement. It was proposed that, if funds could be obtained, an animal welfare information The setting of targets unit for Europe should be established at Sheringham, under the direction of Rex One way of making progress is to agree Burch; this would liaise with the corre· that specific targets should be met, espe­ sponding centre in the USA. Such an initia­ cially if a date for meeting a target is also tive could help meet some of the specific agreed. Whether or not a target is met, recommendations made in this report. as the fact that it exists can influence policy well as playing an important role in the edu­ and stimulate action, as has been seen in cation of the general public. response to the Sixth Amendment It was agreed that contact would be main­ (Directive 931351EEC [108)) to the tained by the workshop participants, and European Union (EU) Cosmetics that a further meeting of the Sheringham Directive, Directive 7617681EEC (109, Group would take place on the occasion of 110). the Second World Congress on Alternatives In 1993, the Member States of the EU and Animal Use in the Life Sciences, to be agreed that everything possible should be held in Utrecht in October 1996. One of the done to achieve a reduction of 50% in the principal aims of this meeting will be to use of vertebrate animals for experimental review the steps taken by the participants in and other scientific purposes by the year the workshop, individually and collectively, 2000 (l1ll. It has also recently been sug­ to see that its report and recommendations gested that agreement should be sought in have been publicised and implemented. In the EU on phasing out the use of non­ addition, it was agreed that an informal human primates as laboratory animals by meeting of societies and organisations com­ the year 2005 (112). Further targets which mitted to the implementation of the Three could be suggested would be an end to Rs should be arranged at the Second World potency and toxicity tests involving lethal Congress, with a view to the possible forma­ endpoints, to the use of the Draize eye tion of an international federation. 859 M. Balls et at.

Conclusions and Recommendations 8. There should also be international dis­ cussion and agreement on what levels of General animal suffering should not be permit­ ted in any circumstances, regardless of 1. Existing laboratory animal protection any likely or potential benefits. laws should be fully implemented. 9. It is the responsibility of the investigator 2. All countries should have a legal frame­ to choose and justify, on scientific and work which actively incorporates the animal welfare grounds, the animal Three Rs into all animal-based research, species and strain which is most suitable testing and education. for the proposed investigation. 3. There should be formal and informal 10. It is unacceptable to export scientific mechanisms for the education and train­ work involving laboratory animals to ing of academic, industrial and govern­ avoid scientifically realistic, but more ment scientists and officials in the Three stringent, animal welfare codes. Rs, to ensure compliance with the spirit and letter of laboratory animal protec­ 11. Discussions should be encouraged at the tion legislation and regulations. national and international level with a view to setting targets and time limits 4. Before proposing any programme of for the achievement of specific goals in work involving laboratory animals, sci­ the reduction, refinement and replace­ entists should ask themselves whether ment of the use of vertebrate animals in the project is worth doing in the first experiments and other scientific proce­ place, and, if so, whether the problem dures. could be approached in a different way, for example by using in vitro methods or 12. There is a need for the involvement in animals of lower sentience. animal welfare issues of more people with initial training in the life sciences 5. Any proposed experiments on animals and postgraduate training in should be subjected to prior and effective biometry/statistics. expert review, both for scientific merit and animal welfare considerations. All scientists and institutions concerned Reduction altenwtives should take steps to ensure that proposed 13. In cases where a choice between species programmes of work involving animals is possible, tHere is generally no scien­ which do not have to pass through an tific justification for using more of the external peer-review process are never­ smaller species than of the larger one. theless subjected to effective evaluation for scientific merit and necessity. 14. Research strategy should be considered carefully, with a view to reducing the 6. Institutions should be required to numbers of animals used. The example appoint one or more persons responsible of Hendriksen ef al. (32), in choosing for ensuring that the Three Rs are fully strains of laboratory mice in order to taken into consideration when pro­ minimise the numbers needed in specific grammes of work on animals are pro­ biological assays, should be followed for posed and during the experimental work those assays which use large numbers of itself. animals and which are unlikely to be 7. It should be recognised that the assess­ replaced with in vitro alternatives in the ment and weighing of the likely benefit near future. and likely animal suffering involved in a 15. The design of regulatory testing proce­ proposed programme of work is an dures, including the sample sizes essential part of the process whereby required, should be reviewed regularly, permission for the work to proceed is/is possibly as part of international harmon­ not granted. Further studies should be isation. undertaken on how this assessment and weighing could be conducted, as a basis 16. Substantial reduction in animal use for international agreement and har­ could be achieved by further harmonis­ monisation. ing toxicity testing regulations, for ECVAM: Workshop 11: Three Rs 860

example, with respect to group sizes, quality of research data. dose levels and the length of studies. 26. Journal editors should be encouraged to 17. In view of the uncertainties inherent in include a separate consideration of "extrapolating" to humans, the need for Animals and Procedures within the very high precision in data provided by Materials and Methods section of the animal experiments should be reconsid· articles they publish. ered. Replacement alternatives 18. There is evidence that some non· regula­ tory animal experiments are poorly 27. Statistics on animal use should include designed an~ incorrectly analysed. As a the numbers of animals killed specifi­ minimum, all research workers should cally for the purpose of providing cells have adequate training in experimental and tissues for in vitro studies. design and the proper use of statistical methods. 28. In the case of regulatory efficacy and toxicity testing, research specifically 19. The concept of the "named statistician" aimed at providing validated replace­ as an essential part of the regulatory ment alternatives for the currently framework of animal experimentation accepted animal procedures should be should be explored. conducted.

Refinement alternatives 29. The development and acceptance of replacement alternatives for both 20. An international data bank on refine­ research and testing must be based on ment alternatives should be developed. an understanding of the molecular and 21. The validation process should include cellular mechanistic basis of the phe. the evaluation of refinement alternative nomenon being studied or measured. procedures, particularly in relation to The current trend toward a more mech­ regulatory testing. anistic approach should be welcomed, encouraged and financially supported. 22. There should be internationally agreed guidelines for the categorisation of ani­ 30. Monoclonal antibodies should only be mal pain, distress and other adverse produced by using in vitro methods, effects, including agreement on physio­ unless a convincing scientific case can be logical and behavioural signs for the made for using the mouse ascites tech­ recognition of adverse effects and for nique. their measurement. 31. New in vitro methods for the production 23. Working parties should be set up, on an of hormones and other biological prod~ international, collaborative basis, to ucts, which would result in purer prep­ develop codes of practice and guidelines arations, should be sought. Efforts of best practice for specific animal hus­ should be made to determine whether in bandry (welfare) and research proce· vivo bioassays for the safety and efficacy dures. When such codes and guidelines testing of hormones and related prod­ have been developed and agreed, adher­ ucts can be replaced by using a combina~ ence to them should be mandatory. tion of physicochemical and in vitro tests. 24. Individuals and institutions should be responsible to their national authorities 32. The animal welfare and ethical issues for prospective and retrospective assess­ pertaining to the procurement of fetal ments of the nature and levels of adverse and neonatal calf sera should be investi­ effects likely to be experienced or actu· gated. The development of fullJ defined ally experienced by animals in each pro­ substitutes which could replace the use gramme of work. of serum when culturing cells should be encouraged. 25. Research on refinement and welfare aspects should be encouraged and 33. Since it is unlikely that an animal test funded, including studies on the effects could be replaced with a single in vitro of minimising pain and distress on the test, the development, evaluation and 86[ M. Balls et al.

optimisation of testing strategies and and promote research and the imple­ integrated testing schemes should be a mentation of the Three Rs through fund­ high priority. ing and education. These centres should be networked, to facilitate coordination 34. Human cells and tissues should be used in preference to those isolated from lab­ and information exchange. oratory animals whenever possible. 41. Government, industrial and academic However, it is recognised that there are scientists should be encouraged to ethical, safety, legal and logistical prob­ become involved to a greater extent in lems which may prevent the widespread the development, validation and imple­ use of human tissues. mentation of alternative methods.

Educahon and training 42. Funding agencies should allocate funds specifically for research on all of the 35. A clear distinction needs to be made Three Rs. between education which aims to con­ 43. The concept and availability of the Three tribute to the development of proper Rs, in the context of excellence and attitudes toward the use of animals, and the training of individuals who will be humanity in scientific research, should practicalIy involved in animal experi­ be incorporated into graduate education and into training programmes which mentation itselC be it by conducting also cover experimental design, animal experiments, by contracting or permit­ welfare issues and statistics. ting experiments, or by caring for the animals used. 44. The editors of appropriate academic 36. The responsible authorities should journals should be encouraged to intro­ require that all those with any practical duce regular and specific consideration involvement should take accredited of progress in the Three Rs in relation to courses which em phasise the importance the subject areas covered by their jour­ of all of the Three Rs, and the legal nals. requirement to use alternatives when­ 45. The officers of learned societies should ever possible. Emphasis should be placed be encouraged to establish subdisci­ on "best practice", for the sake of both plines within their organisations to con­ 8cientific quality and the welfare of the sider the Three Rs aspects of their animals used. disciplines, thereby encouraging recogni­ tion of the scientific and humanitarian 37. Permission to conduct experimental pro­ importance of the Three Rs concept. cedures on animals should be based not only on general training, but also on spe­ Informing the general public about the cific training and evidence of compe­ Three Rs tence, which should be reassessed regularly. 46. Organisations concerned in promoting all types of alternative methods should 38. A Three Rs education and training data­ devote some of their energies to inform­ base should be established, so that all ing the general public of the nature and concerned can have easy access to infor­ importance of the Three Rs approach. mation and advice on the availability of relevant literature. 47. Such organisations should consider joint efforts, as a means of improving quality, 39. School students and undergraduates avoiding pitfalls, and sharing costs. should not be forced to conduct proce­ When appropriate, concerted advertis­ dures on animals as part of their ing, mailings and other actions should be courses, but, should, where necessary, be devised and implemented, to take advan­ provided with alternative options. tage of timely, high profile issues, whether to encourage positive develop­ Informing scientists about the Three Rs ments or to forestall negative develop· concept ments. 40. National, regional and international cen­ 48. Priority should be given to providing tres should be established to facilitate materials for teachers. ECVAM Workshop 11: Three Rs 862

49. Organisers of workshops, conferences or caused by mutagens or transgenic and other meetings on the Three Rs, techniques, are afforded the same level should, when appropriate, consider pub­ of protection throughout their lives as licising the outcome of such meetings. that which would be appropriate had Lay summaries of the proceedings of that level of suffering been induced by meetings on alternatives should, when any other method or technique. appropriate and feasible, be published. 57. There should be a review of the systems currently used for evaluating the ethical Special considerations acceptability of gene transfer into ani­ 50. Animal welfare considerations should be mals on a case-by-case hasis, with partic­ an essential part of the evaluation of the ular emphasis on the transfer of human acceptability of proposed new regulatory genes into animals. test guidelines, or modifications of exist­ 58. The higher primates, cats, dogs and ing guidelines. horses should continue to be regarded as 51. When a new regulatory test guideline animals deserving special consideration. has been accepted, which involves fewer animals or less animal suffering, it should not be optional, but should be Acknowledgement required in preference to any other method. The workshop participants would like to thank Mr & Mrs B.H. Annett, of AMA 52. Wherever possible, quantitative end­ Services, London, UK, for undertaking all points should be used. Death and other the organisational aspects of the workshop. qualitative endpoints are often inhu­ mane, and provide less information than quantitative measurements. It is inex­ cusable to use lethal endpoint acute tox­ References icity tests (for example, LD50 tests), 1. Anon. (1994). ECVAM News & Views.ATIA 22. when a non-lethal endpoint test (for 7-11. example, the Fixed Dose Procedure) has 2. Russell, W.M.S & Burch, R.L. (19591. The been formally accepted as an alternative Principles of HI/mane Experimental Techniqlle. OEeD test guideline method. 238pp. London: Methuen. 3. Russell, W.M.s & Burch, R.L. (19921. The 53. When challenge of experimental animals Principles of Humane Experimental Technique. is unavoidable as, for example, in vaccine 238pp. Potters Bar, Herts .. UK: UFAW. 4. Russell, W.M.S. (1995). The development of the potency testing, clear clinical symptoms Three Rs concept. AT1A 23. 298-304. should be taken as being equivalent to 5. Anon. (1957). UFAW Symposium on Humane the lethality endpoint, and animals Technique in the Laboratory. Collected Papers showing such symptoms should then be of the taboratory Animals Bureau 6, 5-81. 6. Hume, C.W. {l9621. The vivisection controversy killed humanely. in Britain. In Man anel Beast (ed. C.W. Hume), pp. 57-70. Potters Bar, Herts, UK: UFAW. 54. A proposed alternative approach for esti­ 7. Annett, B.H. (1995), The Fund for the mating vaccine potency should be evalu­ Replacement of Animals in Medical ated, which involves limiting the use of Experiments (FRAME): the first 25 yeal's. animals to the first few batches, while AnA 23, 19-32. 8. Anon. (1876). An Al't fo Amend the Law monitoring the consistency of the qual­ Relating to Cruelty to Animals, 8pp. London: ity 'of subsequent batches by using in HMSO. vitro methods. 9. Hollands, C. (1995). Achieving the unachiev­ able: a review of animals in politics. ATIA 23, 55. Since it is of questionable value, the use 33-38. of the Abnormal Toxicity Test in vaccine 10. Smyth, D. (1978). Alternatives to. Animal safety testing should be discontinued. Experiments. 218pp. London: Scalar Press. 11. Anon. (1986). European Convention for the 56. Urgent consideration should be given to Protection of Vertebrate Animals Used for the revision of laws and regulations con­ Experimental and Olher Scientifil' Pllrposes, 51pp. Strasbourg: Council of Europe. cerning the use of animals, so that, 12. Anon. (1986), Council Directive of24 November where necessary, animals with a delete­ 1986 on the approximation of laws, regulations rious phenotype, whether spontaneous and administrative provisions of the Member 863 M. Balls et at.

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