The COST Manual of Laboratory Animal Care and Use Refinement, Reduction, and Research The COST Manual of Laboratory Animal Care and Use Refinement, Reduction, and Research Edited by Bryan Howard Timo Nevalainen Gemma Perretta COST is supported by the EU RTD Framework programme. ESF provides the COST Office through an EC contract. CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York The COST Manual of Laboratory Animal Care and Use Refinement, Reduction, and Research Edited by Bryan Howard Timo Nevalainen Gemma Perretta COST is supported by the EU RTD Framework programme. ESF provides the COST Office through an EC contract. CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York The COST Manual of Laboratory Animal Care and Use Refinement, Reduction, and Research Edited by Bryan Howard Timo Nevalainen Gemma Perretta COST is supported by the EU RTD Framework programme. ESF provides the COST Office through an EC contract. CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York The COST Manual of Laboratory Animal Care and Use Refinement, Reduction, and Research Edited by Bryan Howard Timo Nevalainen Gemma Perretta COST is supported by the EU RTD Framework programme. ESF provides the COST Office through an EC contract. CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York The Open Access version of this book, available at www.taylorfrancis.com, has been made available under a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 license. Neither the COST Office nor any person acting on its behalf is responsible for the use which might be made of the infor- mation contained in this publication. The COST Office is not responsible for the external websites referred to in this publication. 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Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com v Contents COST and COST Action B-24 .........................................................................................................vii Preface...............................................................................................................................................ix Editors ...............................................................................................................................................xi Contributors ................................................................................................................................... xiii Editorial Board of the Manual .......................................................................................................xvii 1 Chapter Introduction ..................................................................................................................1 Bryan Howard, United Kingdom 2 Chapter Design and Oversight of Laboratory Animal Facilities ...............................................7 Dag Sørensen, Norway; Heinz Brandstetter, Germany; Nikolaos Kostomitsopoulos, Greece; and Richard Fosse, France 3 Chapter Housing and Care of Laboratory Animals ................................................................. 29 Hanna-Marja Voipio, Finland; Ping-Ping Tsai, Germany; Heinz Brandstetter, Germany; Marcel Gyger, Switzerland; Hansjoachim Hackbarth, Germany; Axel Kornerup Hansen, Denmark; and Thomas Krohn, Denmark 4 Chapter Animal Needs and Environmental Refinement ......................................................... 75 Vera Baumans, The Netherlands; Hanna Augustsson, Sweden; and Gemma Perretta, Italy 5 Chapter Ethical Evaluation of Scientific Procedures: Recommendations for Ethics Committees .............................................................................................................. 101 Rony Kalman, Israel; I. Anna S. Olsson, Portugal; Claudio Bernardi, Italy; Frank van den Broek, The Netherlands; Aurora Brønstad, Norway; Istvan Gyertyan, Hungary; Aavo Lang, Estonia; Katerina Marinou, Greece; and Walter Zeller, Switzerland 6 Chapter Reduction by Careful Design and Statistical Analysis ............................................ 131 Michael Festing, United Kingdom 7 Chapter Animal Models: Selecting and Preparing Animals for a Study............................... 151 Patrick Hardy, France and Sarah Wolfensohn, United Kingdom 8 Chapter Creation of Genetically Modified Animals .............................................................. 179 Belen Pintado, Spain and Marian van Roon, The Netherlands vi Contents 9 Chapter Management of Genetically Modified Rodents .......................................................205 Jan-Bas Prins, The Netherlands 1 Chapter 0 Impact of Handling, Radiotelemetry, and Food Restriction .................................... 227 Timo Nevalainen, Finland: Marlies Leenaars, The Netherlands; Vladiana Crljen, Croatia; Lars Friis Mikkelsen, Denmark; Ismene Dontas, Greece; Bart Savenije, The Netherlands; Carlijn Hooijmans, The Netherlands; and Merel Ritskes-Hoitinga, The Netherlands 1 Chapter 1 Basic Procedures: Dosing, Sampling and Immunisation ......................................... 257 Ismene Dontas, Greece; Jann Hau, Denmark; Katerina Marinou, Greece; and Timo Nevalainen, Finland 1 Chapter 2 Imaging Techniques ................................................................................................. 287 Aurora Brønstad, Norway and Ismene Dontas, Greece 1 Chapter 3 Anaesthesia and Analgesia ....................................................................................... 313 Patricia Hedenqvist, Sweden and Paul Flecknell, United Kingdom 1 Chapter 4 Use of Humane Endpoints to Minimise Suffering................................................... 333 Coenraad Hendriksen, The Netherlands; David Morton, United Kingdom; and Klaus Cussler, Germany 1 Chapter 5 Euthanasia ................................................................................................................ 355 Luis Antunes, Portugal 1 Chapter 6 Education, Training, and Competence ..................................................................... 369 Bryan Howard, United Kingdom; Katey Howard, Spain; and Peter Sandøe, Denmark 1 Chapter 7 Animal Experimentation and Open Communication .............................................. 391 Ann-Christine Eklöf, Sweden; Anne-Grethe Berg, Norway; and Jon Richmond, United Kingdom Index ..............................................................................................................................................405 vii COST and COST Action B-24 COST is an intergovernmental framework for European Cooperation in Science and Technology, which promotes and coordinates nationally funded research in Europe. COST provides funds for research networks (Actions) and in this way helps consolidate European research investment and opens the European research area to cooperation worldwide. COST is solely funded from a specific part of the EU RTD Framework Programmes. The finan- cial support that COST provides to Actions is used for coordination and networking activities, while the research itself is funded at the national/EU level. In this way, COST reaches out to over 30,000 researchers across Europe and levers approximately EUR 2 billion of research funding, although its direct support is less than 1% of this sum. One of the key scientific domains that COST covers (out of a total of nine) is the Domain of Biomedicine and Molecular Biosciences (BMBS), which at the time of writing (June 2010) is funding a total of 29 Actions. This manual summarises the output, over a period of four years, of BMBS COST Action B-24 on “Laboratory Animal Science and Welfare”. The remit of Action B-24 was to increase knowledge and awareness of the scientific uses of laboratory animals within the context of the Three Rs, and to promote the conduct of high-quality research. The Action began its activi- ties in March 2004 and came to an end in April 2009. However, many participants have links with national associations and with the Federation of European Laboratory Animal Science Associations (FELASA) with which the Action has collaborated closely throughout. It is antici- pated that FELASA will continue to serve as a coordinating body for the progress of labora- tory animal science and the Three Rs within Europe. Twenty-four countries participated in the Action, as shown in Table 0.1. Participants of Action B-24 have published many papers in specialist journals and offer this man- ual, as a collated source of up-to-date information about ethically sound approaches to the care and scientific use of animals. It draws heavily from the widely accepted principles of Russell and Burch, usually known as the Three Rs. We hope that this volume will be of value to those who wish to benchmark or improve practices relating to the scientific use of animals, and will serve as a conve- nient source of evidence-based information about good practices and laboratory animal science and welfare to all those engaged in this field, including educators and regulatory/oversight authorities. For more information: http://www.cost.eu Dr. Kalliopi Kostelidou Cluster Leader, Cluster of Life Sciences Science Officer, BMBS Domain TABLE 0.1 Nations Participating in Cost Action B-24 Austria Germany The Netherlands Belgium Greece Norway Croatia Hungary Portugal Czech Republic Ireland Spain Denmark Israel Sweden Estonia Italy Switzerland Finland Lithuania Turkey France Malta United Kingdom ix Preface A revised Appendix A to the European Convention ETS 123 came into force in 2007. This represented a significant change to baseline standards for the care of laboratory animals within Europe. Subsequent adoption of these guidelines by the European Commission and development of a new Directive to replace 86/609, marks greater emphasis on the Three Rs in laboratory animal science. In 2004, COST (European Cooperation in Science and Technology) established Action B-24 (Laboratory Animal Science and Welfare) as an initiative to increase knowledge necessary for the ethically and scientifically sustainable use of laboratory animals in research. The Action attracted 24 Member States, each represented by one or two members, who contributed to on-going dialogue and research about the most humane and effective way of using laboratory animals for scientific purposes. One result of this initiative has been the preparation of this text— The COST Manual of Laboratory Animal Care and Use: Refinement, Reduction, and Research . The manual is a joint effort involving many authors and it represents a truly international perspective on best practice, as currently perceived. Although 15 different COST countries have each contributed one or more authors in preparation of the text, the information contained includes a great deal of less formal input by other members of the COST Action. Although it is not possible to recognise everybody who has participated in preparation of the manual, the editors are extremely grateful for the support received from them and also from a number of colleagues who, though not directly related to the Action, nonetheless contributed material where specific expertise was not immediately available. The manual can be viewed as an international venture, made possible by the funding and support of COST. The reason for not addressing “Replacement” in this manual is that the underlying technologies involve different scientific disciplines and often require specialisms that are beyond the reach of laboratory animal scientists. Omission in no way reflects a view of the editors that Replacement is in any way less of a priority in laboratory animal science than Reduction and Refinement. Indeed, we firmly believe that Replacement is the ultimate objective for laboratory animal science, although we are of the opinion that science should not be sidestepped simply because appropriate replace- ment techniques are not yet currently available. In circumstances where no replacement strategy is capable of generating the scientific information necessary to test the hypothesis being addressed and where the work proposed can be shown to have important applications, then every measure needs to be taken to minimise the number of animals used and the impact of procedures on each of those animals. Only the more commonly used laboratory animal species are considered: rodents and rabbits. Our intention is to offer a one-stop source of best practice, which will be of value to personnel responsible for the care and welfare of animals and scientists conducting activities related to the use of animals for scientific purposes. It is also addressed to those with management responsibili- ties including facility engineers, architects and lay persons involved with ethical review, and the interested general reader. The manual presents perceived best practices and, as far as possible, the contents are evidence- based. In the many cases where the evidence is poor, the authors have endeavoured to demonstrate the rationale underlying their contentions by including relevant references. The emphasis is not on describing current practices, but rather what is seen as good practice; moreover it is not our intention to tell the reader what to do, but rather to offer options and provide advice on what not to do. Each chapter follows a similar structure, comprising six sections, the first of which, “Objectives”, outlines the breadth of what the chapter is addressing. This is followed by “Key Factors”, which x Preface summarises the central issues that underpin good practice. Each chapter ends with a short section, “Questions Unresolved”, which identifies areas of uncertainty and may serve as a prompt to those wishing to undertake investigations directed at progressing knowledge and application within par- ticular fields. COST offers the manual as a benchmark against which practices can be judged, and it is hoped in this way to provide a mechanism for the advancement of laboratory animal science at a time when the regulatory paradigm is changing in the same direction. xi Editors Bryan Howard retired from the post of Director of Animal Welfare at the University of Sheffield in 2005 but retains an active interest in promoting the Three Rs in relation to the scientific use of ani- mals. He qualified as a veterinary surgeon from the University of Glasgow in 1962 and completed a Doctorate in neurophysiology at the University of Edinburgh in 1966. After a period working on the welfare of poultry at slaughter, he took up posts as visiting professor of Veterinary Physiology at several universities in the Middle East. Since returning to the UK in 1986, he completed the Royal College of Veterinary Surgeons Certificate in Laboratory Animal Science followed by a Master of Science (with distinction) in Laboratory Animal Science at the University of London, and M Ed in Teaching and Learning for University Lecturers; he remains a Fellow of the Higher Education Academy of UK. Apart from pursuing research into best practice for the care of laboratory species, he has been President of the Laboratory Animal Science Association, a Board member of the UK National Centre for the Three Rs (NC3Rs) and Chair of the Trustees and the Council of the Universities Federation for Animal Welfare (UFAW). He was also a member of the Governing Board of COST Action B-24. He is cur- rently a member of the European Board of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International), the Board for Accreditation of Training estab- lished by the Federation of European Laboratory Animal Science Associations (FELASA) and of the Reduction Committee of the Fund for the Replacement of Animals in Medical Experiments (FRAME). In addition Howard has contributed internationally to a large number of training courses designed to advance laboratory animal science and welfare and has participated in the development of printed and web-based guidance for best practice in these areas. Timo Nevalainen earned his Doctor of Veterinary Medicine in 1972 in Helsinki, graduated from the MS program in Laboratory Animal Medicine at the Pennsylvania State University in 1976, and defended his PhD thesis in physiology (1984) in Kuopio. His professional career started as a physiology teacher, from where he progressed to being named director of the National Laboratory Animal Center, both at the University of Kuopio, Finland. Since 1990 he has served as the Professor in Laboratory Animal Science and Welfare both at the University of Kuopio (now University of Eastern Finland) and at the University of Helsinki. While on sabbatical leaves, he has worked as a visiting professor at the University of Cincinnati and at the Pennsylvania State University. He has had an active role in scientific and professional associations; he is a past president of both Scand-LAS and FELASA. He has chaired an EU COST Action B-24 and is involved with various other EU activities, such as ethics review of the Seventh Framework Program. In 2009 he joined the ILAR Council. He has a keen interest in training the future generation of scientists; in addi- tion to being instrumental in establishing competence education of all categories in his country of origin, he has organised six competence courses for scientists in the Baltic countries and Russia starting immediately after the collapse of the Soviet Union. Since then, he widened this activity to teaching such courses in Thailand. He has had a key role in establishing FELASA education guide- lines of all four categories, and FELASA Accreditation of training; most recently he was invited to join the FELASA Accreditation Board. The common theme of his research emphasises the Refinement and Reduction alternatives, and their interplay in the use of laboratory animals. More recently, he has become interested in a third dimension; that is, to examine how those two Rs alternatives can also achieve better science. Currently his group is actively pursuing various aspects of bedding, evaluation of variants of the most common procedures (handling, IG-gavage, blood sampling, IP-injection, identification), comparing xii Editors efficacy of cage furniture, impact of group size and a novel solution to a “poorly controlled variable” called ad libitum feeding suitable for use in group-housed rats. Gemma Perretta is the head of the Division of Special Zoology and Animal Models of the Italian National Research Council in Rome. She graduated in veterinary medicine and specialised in labo- ratory animal science and medicine at the University of Milan for which she also served as professor from 1993 through 2006. Over the past 25 years, Perretta has developed relevant competence in the field of laboratory animal welfare and science and gained wide knowledge on international guidelines and regula- tions on the protection of animals. She has been an invited expert in several commissions and working groups and has provided technical and scientific support to public regulatory authorities at national and European level. As a participant in national and international commissions, she has been involved in the promotion and implementation of high-quality standards in laboratory animal care with particular focus on education and training requirements and recommendations for staff working with animals. She is a former president of the Federation of European Laboratory Animal Science Associations (FELASA) and the Italian Association for Laboratory Animal Science (AISAL) and serves as spe- cial consultant for the International Council for Laboratory Animal Science (ICLAS). Perretta has been vice-chair of the Management Committee in the EU COST Action B-24 (Laboratory Animal Science and Welfare). xiii Contributors Luis Antunes* Veterinary Sciences Department Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal lantunes@utad.pt Hanna Augustsson Sweden Vera Baumans* Department of Animals in Science and Society Division of Animal Welfare and Laboratory Animal Science Utrecht University The Netherlands v.baumans@uu.nl Anne-Grethe Trønsdal Berg Nord-Trøndelag University College Norway Claudio Bernardi Italy Heinz Brandstetter Germany Aurora Brønstad* Vivarium, Haukeland sykehus Faculty of Medicine and Dentistry University of Bergen Bergen, Norway aurora.bronstad@ffhs.uib.no Vladiana Crljen Croatia Klaus Cussler Germany * Senior author. Ismene Dontas* School of Medicine University of Athens Athens, Greece idontas@med.uoa.gr Ann-Christine Eklöf* Researchlab Q2:09 Karolinska Institutet Stockholm, Sweden Ann-Christine.Eklof@ki.se Michael Festing* c/o Understanding Animal Research London United Kingdom michaelfesting@aol.com Paul Flecknell United Kingdom Richard Fosse France Istran Gyertyan Hungary Marcel Gyger Switzerland Hansjoachim Hackbarth Germany Axel Kornerup Hansen Denmark Patrick Hardy* AFSTAL (Association Française des Sciences et Techniques de l’Animal de Laboratoire) Paris, France afstal@free.fr Jann Hau Denmark xiv Contributors Patricia Hedenquist* Unit for Comparative Physiology and Medicine Department of Large Animal Clinical Sciences Swedish University of Agricultural Sciences (SLU) Uppsala, Sweden Patricia.Hedenqvist@kv.slu.se Coenraad Hendriksen* Netherlands Vaccine Institute (NVI) Bilthoven The Netherlands Coenraad.Hendriksen@nvi-vaccin.nl and Department of Animals in Science and Society Utrecht University Utrecht, The Netherlands Carlijn Hooijmans The Netherlands Bryan Howard* Formerly of University of Sheffield United Kingdom B.R.Howard@sheffield.ac.uk Katey Howard Spain Rony Kalman* Authority for Animal Facilities Hebrew University Israel ronyk@huji.ac.il Nikolaos Kostomitsopoulos Greece Thomas Krohn Denmark Aavo Lang Estonia Marlies Leenaars The Netherlands Katerina Marinou Greece Lars Friis Mikkelsen Denmark David Morton* Formerly University of Birmingham United Kingdom d.b.morton@bham.ac.uk Timo Nevalainen* Laboratory Animal Center University of Eastern Finland Kuopio, Finland Timo.Nevalainen@uef.fi I. Anna S. Olsson* Institute for Molecular and Cell Biology—IBMC Porto, Portugal olsson@ibmc.up.pt Gemma Perretta Institute of Neurobiology and Molecular Medicine National Research Council Rome, Italy ims@casaccia.enea.it Belen Pintado* CSIC Centro Nacional de Biotecnología Madrid, Spain bpintado@cnb.csic.es Jan-Bas Prins* Leiden University Medical Centre Leiden, The Netherlands j.b.prins@lumc.nl Jon Richmond United Kingdom Merel Ritskes-Hoitinga The Netherlands Peter Sandøe Denmark Contributors xv Bart Savenije The Netherlands Dag R. Sørensen* Centre for Comparative Medicine Rikshospitalet Oslo University Hospital Oslo, Norway dagrs@rr-research.no Ping-Ping Tsai Germany Frank van den Brook The Netherlands Marian van Roon The Netherlands Hanna-Marja Voipio* Laboratory Animal Centre University of Oulu Oulu, Finland Hanna-Marja.Voipio@oulu.fi Sarah Wolfensohn United Kingdom Walter Zeller Switzerland xvii Editorial Board of the Manual Heinz Brandstetter Bryan Howard (Chair) Nikolaos Kostomitsopoulos Aavo Lang Timo Nevalainen Anna Olsson Gemma Perretta Jan-Bas Prins Annie Reber Merel Ritskes-Hoitinga Hanna-Marja Voipio Johannes Wilbertz 1 1 Introduction Bryan Howard, United Kingdom 1.1 PuBLiC CoNCErNs The use of animals in scientific experimental and testing laboratories within Europe is the subject of considerable public concern. Although a small proportion of the population believes that animal testing can never be morally justified, a similarly small proportion believes that animal testing presents no ethical difficulties. The great majority of people recognise that some animal experi- mentation may be necessary, but dislike it. The outcomes of surveys of public attitude are strongly influenced by the nature of the questions and the way they are asked. For example, a poll conducted in UK in 1999 (Ipsos Mori 1999) found that of 1014 people questioned, just over a quarter stated that animal experimentation was acceptable, providing there was no alternative; the number rose to two-thirds if the research was for medical purposes. In the same survey, 39% of respondents to another question said they did not support the use of animals for any experiments. These findings were broadly confirmed by a similar survey of 1,125 people in 2002. Both of these surveys were commissioned by bodies that supported biomedical research. In contrast a poll conducted in 2003 by the British Union for the Abolition of Vivisection found that three-quarters of respondents believed that the British Government should, as a matter of principle, prohibit experi- ments on live animals (via The Nuffield Council on Bioethics 2005). In 2005 a poll carried out on behalf of a major UK newspaper found that only about 10% of the UK population would support painful experiments on mice for testing cosmetics, but that over 60% would support such experiments to develop a treatment for leukaemia (Daily Telegraph 2006). Providing experiments did not cause pain, acceptance for using mice rose to over 80%. Many of the differences between the findings of these polls in UK are influenced by the precise way in which the question is asked and the caveats that respondents place on their answers (Ipsos Mori 2006). For the public to become convinced that animal experimentation is ethically justified, not only must there be a clear beneficial outcome, but there should be no means of acquiring the information without using live animals, experiments should involve the smallest possible number of animals and these must be cared for and used in the most humane way possible. These issues translate to the three Rs of Russell and Burch (1959). Similar findings have arisen from polls carried out in several other European countries, but perhaps the most rigorous examination of public opinion has taken place in Switzerland. Under the Swiss constitution, the Government is obliged to call a national referendum if it receives a petition containing at least 100,000 citizens’ signatures. The collection of signatures and subsequent refer- endum encourage considerable debate about the key issues. Referenda relating to the use of labora- tory animals in Switzerland have been conducted in 1985 and 1992; the first, seeking an outright CoNTENTs 1.1 Public Concerns ........................................................................................................................1 1.2 Replacement..............................................................................................................................2 1.3 New Technical Advances..........................................................................................................3 1.4 The Culture of Care ..................................................................................................................4 References ..........................................................................................................................................5