1. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 1 Introduction to the Three Rs Concept Marlies Halder European Commission Joint Research Centre Institute for Health & Consumer Protection ECVAM 21020 Ispra, Italy e-mail: marlies.halder@jrc.it

2. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 2 The Start 1954:Scientific study of humane technique in laboratory animal experiments launched by Charles Hume (Universities Federation for Animal Welfare, UFAW) 1955-work carried out by W.M.S. Russell & R.L. Burch 1959 first public discussion of the Three Rs concept “This deserves to become a classic for all time, and we have great hopes that it will inaugurate a new field of systematic study. We hope that others will follow up the lead it has given, and that a generalised study of humane technique, as a systematic component of the methodology of research, will come to be considered essential to the training of a biologist” Charles Hume, 1959

3. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 3 The Three Rs of Russell & Burch The Three Rs concept embraces reduction as a means of lowering “the number of animals used to obtain information of a given amount and precision”, refinement as any development leading to a “decrease in the incidence or severity of procedures applied to those animals which have to be used”, and replacement as “any scientific method employing non- sentient material which may replace methods which use conscious living vertebrates”. W M S Russell & R L Burch The Principles of Humane Experimental Technique Methuen, London (1959)

4. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 4 The Evolution of the Three Rs Concept > 1970 various privately funded organsiation started to foster the Three Rs concept, e.g. FRAME, AWI, HSUS 1978 – Three Rs definition of alternatives Alternatives to animal experimentation include: “all procedures which can completely replace the need for animal experiments, reduce the number of animals required, or diminish the amount of pain or distress suffered by animals in meeting the essential needs of man and other animals”. David Smyth Alternatives to Animal Experiments Scolar Press, London (1978)

5. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 5 The Adoption of the Three Rs Concept into Legislation Examples for EU Member States 1977The Netherlands Act on Animal Experimentation 1986UK Animals Scientific Procedures Act 1987Germany Tierschutzgesetz International 1986Council of Europe Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes European Union 1986Directive 86/609/EEC, on the Approximation of Laws, Regulations and Administrative Provisions of the Member States Regarding the Protection of Animals Used for Experimental and Other Scientific Purposes

6. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 6 Directive 86/609/EEC on the Use of Laboratory Animals Article 7 2. An experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available. 3. When an experiment has to be performed, the choice of species shall be carefully considered and, where necessary, explained to the authority. In a choice between experiments, those which use the minimum number of animals, involve animals with the lowest degree of neurophysiological sensitivity, cause the least pain, suffering, distress or lasting harm and which are most likely to provide satisfactory results shall be selected. Experiments on animals taken from the wild may not be carried out unless experiments on other animals would not suffice for the aims of the experiment. 4. All experiments shall be designed to avoid distress and unnecessary pain and suffering to the experimental animals. They shall be subject to the provisions laid down in Article 8. The measures set out in Article 9 shall be taken in all cases. Replace Reduce Refine

7. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 7 Directive 86/609/EEC on the Use of Laboratory Animals Article 23: The Commission and Member States should encourage research into the development and validation of alternative techniques which could provide the same level of information as that obtained in experiments using animals, but which involve fewer animals or which entail less painful procedures, and shall take such other steps as they consider appropriate to encourage research in this field. Establishment of ECVAM in 1991

8. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 8 7 th Amendment to the Cosmetics Directive –ban to use animals for finished product since 2003 –ban to use animals for safety testing of ingredients/formulations from 2009 for certain endpoints & 2013 complete ban –only replacement methods Registration Evaluation Authorisation of Chemicals (REACH) –Several articles refer to the use of laboratory animals, alternative methods, reduction, refinement and replacement - namely articles 1, 13, 37, 40 OECD guidance document on the use of humane endpoints European Pharmacopoeia..... and many more Directives/Guidance Documents Referring to Three Rs Methods

9. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 9 Examples of Implementation of the Three Rs Quality control of vaccines each lot/batch is checked for safety and potency before release, often in comparison to a reference vaccine might involve animal tests, e.g. inactivated vaccines some of the test inflict high distress & pain, e.g. immunisation-challenge and endpoint “death” safety tests ensure that a product is not contaminated with toxin, virus, pyrogen etc and adequately attenuated/inactivated potency tests ensure that the product induces the same biological activity as a reference preparation

10. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 10 Quality control of vaccines - Reduction Single-dilution tests instead of multi-dilution tests introduced for tetanus, erysipelas, rabies vaccines Upstream testing introduced for various safety tests Reduction of group size/groups optimise statistical methods & requirements for a valid assay Frequency of testing production of larger batches

11. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 11 Quality control of vaccines - Replacement Deletion of no longer relevant tests abnormal toxicity test for human vaccines target animal safety test for veterinary vaccines only on first 10 batches Specific toxicity tests use of cell culture methods, e.g. diphtheria, clostridial vaccines Extraneous agents testing of live vaccines use of cell cultures, hatched eggs instead of e.g. chicks or mice Potency testing antigen quantification for e.g. hepatitis, Newcastle disease vaccine

12. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 12 Potency tests –Immunisation-challenge Death Survival Vaccine ImmunisationChallenge Used for a number of inactivated vaccines e.g. rabies, pertussis, leptospiral

13. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 13 Potency tests –Immunisation + antibody quantification Serological methods, e.g. ELISA, ToBI, Vero cell assay, MAT for tetanus, diphtheria and leptospiral vaccines Vaccine ImmunisationSerum Immunochemical method

14. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 14 Potency tests –Antigen quantification Used for vaccines with well-defined antigens such as hepatitis, polysaccharide vaccines, leptospiral dog vaccine, Newcastle disease Vaccine Immunochemical method

15. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 15 Use of humane (non-lethal) endpoints requirement of European Pharmacopoeia as soon as significant clinical signs develop, animals are killed Tetanus: mouse showing paralysis Photo by C. Hendriksen Quality control of vaccines - Refinement

16. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 16 HELP - Group developed humane endpoints for potency testing of rabies endpoints are incorporated in the Ph.Eur. monographs for veterinary and human rabies vaccines potency testing of rabies vaccines: –groups of mice are immunised with a series of vaccine dilutions –after immunisation period, animals are challenged with rabies virus –non-protected animals develop rabies –endpoint is death humane endpoints: body weight & clinical signs ECVAM workshop 48 – Three Rs approaches in the QC of rabies vaccines Quality control of vaccines - Refinement

17. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 17 Clinical signs of rabies (14 days observation time) Non-lethal endpoints: - onset of neurological signs (Stage 2) & 15% of body weight - Stage 3 reduces suffering by 2-3 days Photos by HELP group

18. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 18 Summary Reduce Refine Replace Less painful procedures; e.g. best practice Humane endpoints Less animals/group Better experimental design Less tests Use of existing information In silico methods (e.g. (Q)SAR) Less sentient organisms (e.g. some invertebrates, bacteria, plants) Embryos & foetal stages before sentience develops Cell cultures, tissues, isolated organs Physicochemical methods

19. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 19 ReduceRefineReplace Before you think about: Ask yourself: Should the work be done at all? Excessive animal suffering? Are there broader ethical implications? Is the work worth doing it? Would the successful outcome outweigh the animal suffering? Harm/benefit analysis

20. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 20 Rex Burch & William Russell Sheringham, June 1995 Thank you!

21. Ankara, 12-13 November 2007 – JRC Enlargement & Integration Workshop - Alternatives 2007 21 Netherlands Association for Laboratory Animal Science (NVP)* Humane endpoints in Laboratory Animal Experimentation – An interactive CD ROM for education and training purposes Avaiable for free! e-mail to Iris Boumans i.boumans@uu.nl * in collaboration with Laboratory Animals, NCA, ZonMw, SPI