1. ‘EUROPE GOES ALTERNATIVE’ Animal Testing and Alternative Methods -
a Scientific Perspective
Coenraad F.M.Hendriksen
Netherlands Centre Alternatives to Animal Use (NCA), Utrecht University
& Netherlands Vaccine Institute (NVI)

2. Animal Testing and Alternative Methods - a scientific perspective
Topics of presentation
science and alternatives -
the way to go!
Other reasons for going
alternative
3Rs recommendations
Conclusion
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

3. Science and Alternatives - the way to go!
Treatment
Incubation time
minutes  years
phenotype
NCA | The Netherlands Centre Alternatives to Animal Use
(Timo Breit)
Scientific and regulatory evolution: from ‘what’ to ‘why’ (1)
Until now questions are based on ‘what’ :
- what is the toxicity of a chemical?
- what is the potency of a vaccine?
- what is the safety of a pharmaceutical?
Future: ‘why’ questions will dominate:
- why is a chemical toxic?
- why is a batch of vaccine potent?
- why is a pharmaceutical not safe?
Conference ‘Europe Goes Alternative’, Brussels,

4. Science and Alternatives - the way to go!
Paradigm shift in Science and regulatory testing: from ‘what’ to ‘why’ (2)
‘What’ questions
Parameter Endpoint (animal model)
vaccine potency protection after challenge
chemical toxicity increase tumour frequency
drug safety clinical signs
‘Why’ questions
Endpoint Model (e.g.)
3-D configuration antigen physiochemical
DNA or DNA repair change
at molecular level tissue culture
interaction at receptor level tissue culture
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

5. Science and Alternatives - the way to go!
New developments
Micro-array analysis:
genes
NCA | The Netherlands Centre Alternatives to Animal Use
Examples
- omics technologies & bioinformatics
embryonic stem cells
in silico modelling tools
improvements in tissue culture techniques
systems biology
etc.
Conference ‘Europe Goes Alternative’, Brussels,

6. Science and Alternatives - the way to go!
Paradigm shift in science and regulatory testing: from ‘what’ to ‘why’ (3)
Past/’present’
‘Future’
Conclusion:
Future will give a shift in research and regulatory testing paradigms: non-animal models will increasingly being used to generate answers to the ‘why’ questions (mechanistic info) while the animal model will be used to verify and confirm data from non-animal studies
R & D start
NCA | The Netherlands Centre Alternatives to Animal Use
End of research objective
Conference ‘Europe Goes Alternative’, Brussels,

7. Other reasons for Europe to go alternative
Animal use & concerns
Moral concerns
Economic and pragmatic concerns
Scientific concerns
(In)validation concerns
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

8. Other reasons for Europe to go alternative
Societies acceptance of animal use for specific purposes
Animal use & concerns
Moral concerns
Economic and pragmatic concerns
Scientific concerns
(In)validation concerns %
NCA | The Netherlands Centre Alternatives to Animal Use
(Aldhous et al. 1999)
Conference ‘Europe Goes Alternative’, Brussels,

9. Other reasons for Europe to go alternative
Animal use & concerns
Moral concerns
Economic and pragmatic concerns
Scientific concerns
(In)validation concerns
Increased legislation and regulation concerning laboratory animal use
Revision of Council
Directive 86/609/EEC
Appendix A of the
European Convention
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

10. Other reasons for Europe to go alternative
Animal use & concerns
Moral concerns
Economic and pragmatic concerns
Scientific concerns
(In)validation concerns
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

11. Europe goes alternative - Future trend in Lab. Animal use?
NCA | The Netherlands Centre Alternatives to Animal Use
year
year
Conference ‘Europe Goes Alternative’, Brussels,

12. Europe goes Alternative: the way forward
Objective Conference
demonstrate that the European Union keeps animal welfare high on the political agenda and remains in the lead for animal protection,
show industry’s approach to actively promote research in alternative methods,
illustrate progress made in the area of alternative methods to animal tests at EU and intermational level,
identify further possibilities to improve development, validation and legal acceptance of alternative methods
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

13. Objective: identify further possibilities to improve development, validation and legal acceptance of alternative methods
Effective EU policy towards
‘Europe goes Alternative’
should include the following elements
Rex Burch
Bill Russell R
eality
NCA | The Netherlands Centre Alternatives to Animal Use R
ealism R
eaction
Conference ‘Europe Goes Alternative’, Brussels,

14. Reality: numbers and purposes of animal use
Focus of Commission concerning 3Rs research priorities
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

15. Reality: numbers and purposes of animal use
No. of animals used in EU-MS in 2002 (N total = ): purposes
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

16. Reality: alternatives and the 3Rs principle
Replacement:
‘Desirable as replacement is, it would be a mistake to put all our humanitarian eggs in this basket alone.’
(Russell & Burch, 1959)
Replacement of animal tests by methods not using animals
Reduction: reducing the numbers of animals needed to answer a scientific question
Reduction:
‘….of all modes of progress it is the one most obviously, immediately, and universally advantageous in terms of efficiency.’
(Russell & Burch, 1959)
NCA | The Netherlands Centre Alternatives to Animal Use
Refinement: reducing pain and distress in a particular animal experiment
Refinement:
‘Happy animals make good science.’
(Trevar Poole)
Conference ‘Europe Goes Alternative’, Brussels,

17. Objective: identify further possibilities to improve development, validation and legal acceptance of alternative methods
Reality
NCA | The Netherlands Centre Alternatives to Animal Use
Realism
Reaction
Conference ‘Europe Goes Alternative’, Brussels,

18. Realism: Proposed timeframe for full replacement of animal tests
Human Health effects Foreseeable time involved
for full replacement
_________________ _____________________
acute toxicity >
skin irritation >
skin corrosion <
eye irritation >
skin sensitisation >
skin absorption/penetration >
subacute/subchronic toxicity >> 2014 (not foreseeable)
genotoxicity/mutagenicity >
UV-induced effects >> 2019 (not foreseeable)
Toxicokinetics and biotransformation >> 2014 (not foreseeable)
Carcinogenicity >> 2014 (not foreseeable)
Reproductive toxicity >> 2014 (not foreseeable)
NCA | The Netherlands Centre Alternatives to Animal Use
(SCCNFP, 2004)
Conference ‘Europe Goes Alternative’, Brussels,

19. Realism: Legislative and Regulatory context
Sectorial regulations and decisions regulate
the activities of particular sector.
Horizontal legislation interacts with
aspects of sectorial regulations
Examples of conflict between sectorial European directives and EU legislation 86/609/EEC:
re-testing of biological products entering the European market
shellfish toxin testing policies
Other concerns
Slow progress in Harmonisation and/or mutual acceptance
Increased priority for risk minimisation
Regulations to animal testing and multilateral
agreements, e.g. Council Directive 86/609/EEC
NCA | The Netherlands Centre Alternatives to Animal Use
Horizontal legislation
Council Directive
pharmaceuticals
Council Directive
vaccines
Council Directive
pesticides
Council Directive
chemicals
Sectorial regulation
(According to Schiffelers et.al.2005)
Conference ‘Europe Goes Alternative’, Brussels,

20. Objective: identify further possibilities to improve development, validation and legal acceptance of alternative methods
Reality
NCA | The Netherlands Centre Alternatives to Animal Use
Realism
Reaction
Conference ‘Europe Goes Alternative’, Brussels,

21. (John Maynard Keynes, 1883-1946)
Reaction: obstacles concerning implementation of validated alternatives
Council Directive 86/609/EEC on the protection of laboratory animals
Art. 7 (2)
An experiment shall not be performed if another scientifically satisfactory method of obtaining the results sought, not entailing the use of an animal, is reasonably and practically available
Art. 7 (3)
In a choice between experiments, those which use the minimum number of animals, cause the least pain, suffering, distress or lasting harm and which are most likely to provide satisfactory results shall be selected.
The difficulty lies not in the new ideas, but in escaping from the old ones
(John Maynard Keynes, )
NCA | The Netherlands Centre Alternatives to Animal Use
Examples:
- European Pharmacopoeia monographs for Diphtheria and Tetanus vaccine specify two possibilities for potency testing, substantially differing in the level of suffering from each other
- Production of monoclonal antibodies
Conference ‘Europe Goes Alternative’, Brussels,

22. Europe goes Alternative: the way forward
Objective Conference
demonstrate that the European Union keeps animal welfare high on the political agenda and remains in the lead for animal protection,
show industry’s approach to actively promote research in alternative methods,
illustrate progress made in the area of alternative methods to animal tests at EU and international level,
identify further possibilities to improve development, validation and legal acceptance of alternative methods
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

23. Alternatives developed and validated for regulatory testing in the last decade
Chemicals/biologicals
Animal model Alternative model
skin corrosivity test tissue culture tests
phototoxicity tests cell culture test
embryotoxicity tests cell culture test
skin sensitization test LLNA test
potency testing of tetanus Elisa test and ToBI test
potency test erysipelas Elisa test
vaccine
target animal safety test deletion
monoclonal antibody in vitro culture
production
Chemicals/biologicals
Animal model Alternative model
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

24. Alternatives developed and ‘validated’ for regulatory purposes in the last decade
Chemicals/biologicals
Animal model Alternative model
skin corrosivity test tissue culture tests
phototoxicity tests cell culture test
embryotoxicity tests cell culture test
skin sensitization test LLNA test
potency testing of Elisa test, ToBI test
tetanus vaccine
monoclonal antibody in vitro production
production
classical LD50 tests alternative approaches
pyrogenicity testing LAL test and in vitro model
percutaneous adsorption cell culture test
production inactivated in vitro culture
polio vaccine (monkeys)
abnormal toxicity test test deleted
specific tox, diphtheria cell culture test
vaccine
potency test diphtheria cell culture test
potency testing of diph single dose testing
theria and tetanus vaccine
potency test polio vaccine antigen quantification
Chemicals/biologicals
Animal model Alternative model
potency test hepatitis antigen quantification
vaccine
neurovirulence test PCR & transgenic animals
polio vaccine
potency test erysipelas Elisa test
potency test various clos Elisa tests
tridial vaccines
safety test erysipelas test deleted
potency test leptospirosis antigen quantification
target animal safety test
safety testing poultry in vitro methods
abnormal tox.test hormones test deleted
safety testing hormones test delted
potency test rDNA physico-chemical tests
hormones
lethal endpoints use of humane endpoints
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

25. Alternatives developed and validated for regulatory purposes in the last decade (tests developed in Europe are given in blue)
Chemicals/biologicals
Animal model Alternative model
skin corrosivity test tissue culture tests
phototoxicity tests cell culture test
embryotoxicity tests cell culture test
skin sensitization test LLNA test
potency testing of Elisa test, ToBI test
tetanus vaccine
monoclonal antibody in vitro production
production
classical LD50 tests alternative approaches
pyrogenicity testing LAL test and in vitro model
percutaneous adsorption cell culture test
production inactivated in vitro culture
polio vaccine (monkeys)
abnormal toxicity test test deleted
specific tox, diphtheria cell culture test
vaccine
potency test diphtheria cell culture test
potency testing of diph single dose testing
theria and tetanus vaccine
potency test polio vaccine antigen quantification
Chemicals/biologicals
Animal model Alternative model
potency test hepatitis antigen quantification
vaccine
neurovirulence test PCR & transgenic animals
polio vaccine
potency test erysipelas Elisa test
potency test various clos Elisa tests
tridial vaccines
safety test erysipelas test deleted
potency test leptospirosis antigen quantification
target animal safety test
safety testing poultry in vitro methods
abnormal tox.test hormones test deleted
safety testing hormones test delted
potency test rDNA physico-chemical tests
hormones
lethal endpoints use of humane endpoints
NCA | The Netherlands Centre Alternatives to Animal Use
Conference ‘Europe Goes Alternative’, Brussels,

26. Europe goes alternative!
Vision without action is daydream ~ Action without vision is nightmare --Japanese proverb
Recommendation: an independent advisory board to the Commission to identify
critical aspects of animal use,
priorities in 3Rs research
potential obstacles concerning implementation, and
potential conflicts between 86/609/EEC and other Directives

27. Thanks for your attention!!

29. if so, why do we still use animals?
Validated non-animal tests
Only 9 non animal tests endorsed as scientifically valid by ECVAM:
skin corrosivity (Rat TER, CORROSITEX, Episkin, Epiderm),
phototoxicity (3T3NRU),
embryotoxicity (EST, MM, whole embryo assay),
in vitro percutaneous absorption.
NCA | The Netherlands Centre Alternatives to Animal Use
Annual European Meeting Toxicology Forum, Brussels,

30. Ontwikkelingen in het gebruik van proefdieren
(De Boo)
NCA jubileum symposium, 25 november 2004