Post on 21-Dec-2015
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 ProtectionECVAM
21020 Ispra, Italye-mail: marlies.halder@jrc.it
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. Burch1959 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
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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 TechniqueMethuen, London (1959)
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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 needsof man and other animals”.
David SmythAlternatives to Animal Experiments
Scolar Press, London (1978)
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The Adoption of the Three Rs Concept into Legislation
Examples for EU Member States1977 The Netherlands Act on Animal Experimentation
1986 UK Animals Scientific Procedures Act
1987 Germany Tierschutzgesetz
International1986 Council of Europe Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes
European Union1986 Directive 86/609/EEC, on the Approximation of Laws, Regulations
and Administrative Provisions of the Member States Regarding the Protection of Animals Used for Experimental and Other Scientific Purposes
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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
Refine
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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
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• 7th 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
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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
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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
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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
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Potency tests
– Immunisation-challenge
Death
Survival
Vaccine Immunisation Challenge
Used for a number of inactivated vaccines e.g. rabies, pertussis, leptospiral
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Potency tests
– Immunisation + antibody quantification
Serological methods, e.g. ELISA, ToBI, Vero cell assay, MAT for tetanus, diphtheria and leptospiral vaccines
Vaccine Immunisation Serum Immunochemical method
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Potency tests
– Antigen quantification
Used for vaccines with well-defined antigens such as hepatitis, polysaccharide vaccines, leptospiral dog vaccine, Newcastle disease
Vaccine Immunochemical method
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• 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
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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
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Clinical signs of rabies (14 days observation time)
• Ruffled fur
• Hunched back
Stage 1
• Signs of paralysis
• Paresis
Stage 4
• Convulsions
• Shaky movements
• Trembling
Stage 3
• Slow movements
• Circular movements
Stage 2
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
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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
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Reduce Refine Replace
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
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Rex Burch & William RussellSheringham, June 1995
Thank you!
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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