‘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)

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, 071105

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, 071105

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, 071105

Science and Alternatives - the way to go! New developments Micro-array analysis: 1.000-40.000 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, 071105

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, 071105

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, 071105

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, 071105

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, 071105

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, 071105

Europe goes alternative - Future trend in Lab. Animal use? NCA | The Netherlands Centre Alternatives to Animal Use year year Conference ‘Europe Goes Alternative’, Brussels, 071105

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, 071105

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, 071105

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, 071105

Reality: numbers and purposes of animal use No. of animals used in EU-MS in 2002 (N total = 10.731.020): purposes NCA | The Netherlands Centre Alternatives to Animal Use Conference ‘Europe Goes Alternative’, Brussels, 071105

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, 071105

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, 071105

Realism: Proposed timeframe for full replacement of animal tests Human Health effects Foreseeable time involved for full replacement _________________ _____________________ acute toxicity > 2014 skin irritation > 2014 skin corrosion < 2004 eye irritation > 2010 skin sensitisation > 2019 skin absorption/penetration > 2006 subacute/subchronic toxicity >> 2014 (not foreseeable) genotoxicity/mutagenicity > 2016 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, 071105

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, 071105

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, 071105

(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, 1883-1946) 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, 071105

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, 071105

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, 071105

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 3 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, 071105

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 3 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, 071105

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

Thanks for your attention!!

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, 091105

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