Introduction to the Three Rs Concept

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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_at_jrc.it
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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
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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 Technique
• Methuen, London (1959)

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The Evolution of the Three Rs Concept  
gt 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)
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The Adoption of the Three Rs Concept into
Legislation  
Examples for EU Member States 1977 The
Netherlands Act on Animal Experimentation 1986 UK
Animals Scientific Procedures Act 1987 Germany
Tierschutzgesetz International 1986 Council of
Europe Convention for the Protection of
Vertebrate Animals Used for Experimental and
Other Scientific Purposes European
Union 1986 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.

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Directives/Guidance Documents Referring to Three
Rs Methods

• 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

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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

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
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• Potency tests
• Antigen quantification

Vaccine Immunochemical method
Used for vaccines with well-defined antigens such
as hepatitis, polysaccharide vaccines,
leptospiral dog vaccine, Newcastle disease
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Quality control of vaccines - Refinement

• Use of humane (non-lethal) endpoints
• requirement of European Pharmacopoeia
• as soon as significant clinical signs develop,
  animals are killed
• Tetanus mouse showing paralysis

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Quality control of vaccines - Refinement

• 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

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• Clinical signs of rabies (14 days observation
  time)

Non-lethal endpoints - onset of neurological
signs (Stage 2) 15 of body weight- Stage
3reduces suffering by 2-3 days
Photos by HELP group
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Summary

• Less animals/group
• Better experimental design
• Less tests

Reduce

• Less painful procedures e.g. best practice
• Humane endpoints

Refine

• 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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Before you think about
Reduce
Refine
Replace

• 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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Thank you!
Rex Burch William Russell Sheringham, June 1995
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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_at_uu.nl
• in collaboration with Laboratory Animals, NCA,
  ZonMw, SPI