3R methods

One of the most important spin-offs of our current research is the development of methods potentially suitable to replace or reduce the use of animals in Life Sciences. Animal experiments in science are necessary to gain mechanistic insight into biological processes, to assess (risk to) hazard of potentially harmful effects of chemicals and pharmaceuticals to humans and animals, and to ascertain the potency and safety of biologicals and vaccines. Every animal experiment performed in the Netherlands is done under rules and regulations that are described in the Dutch Act on the Welfare and Use of Animals for Sciences (Wet op de Dierproeven en Het Dierproevenbesluit). Currently, national laws of all the member states of the European Union are being revised and brought in line with the new EU Directive on the protection of animals used for scientific purposes.

Since 1986, the EU has had in place specific legislation covering the use of animals for scientific purposes. On 22 September 2010 the EU adopted Directive 2010/63/EU which updated and replaced the 1986 Directive 86/609/EEC on the protection of animals used for scientific purposes. The aim of the new Directive is to strengthen legislation, and improve the welfare of those animals still needed to be used, as well as to firmly anchor the principle of the Three Rs, to Replace, Reduce and Refine the use of animals, in EU legislation. Directive 2010/63/EU has taken full effect on 1 January 2013.

This 3R principle was first introduced by two scientists: Russel and Burch, who published their ideas in 1958 [1]. The principle of the 3Rs obligates researchers to use alternatives for the animal experiment, if available (Replacement). If an alternative is unavailable, one must consider refining the experiment, using for example anti-pain medication prior and after the operation; housing  enrichment and/or group housing; or handling the animals before the experiments to reduce in-experiment stress levels (Refinement). Meanwhile, before or while performing an experiment, one has to consider the possibility to reduce the number of animals as much as possible (Reduction). Power analysis to ensure valid group-size and the right experimental design to get the most relevant data out the experiment are examples of reducing strategies, as are computer simulations, which could also be a true replacement.

The INT group works on several 3R methods in a number of scientific areas. We have been working on the development and validation of methods suitable to assess the capacity and potency of chemicals to cause skin-allergy [2]. An important project with regard to the new EU Cosmetics Regulation, that states that it is not allowed to perform animal experiments for the testing of cosmetics and that it is not allowed to import cosmetics into the European Union, that have been tested on animals elsewhere.

Our research on 3R methods also includes the development of a non-animal method for the assessment of safety and potency of pertussis (whooping cough) vaccine [3] (PERTUSSAY project). The development and validation of a test-battery for the safety and efficacy testing of natural compounds (RAAK PRO, NAT-TEST project) and the development of a testing strategy to address the issue of developmental effects of certain chemicals, using non-mammalian species and cell culture.

Links to additional information:

Nederlands Kenniscentrum voor Alternatieven (in Dutch): www.nkca.nl

National Centre for the Replacement, Refinement & Reduction of animals in research: www.nc3rs.org.uk


1) Russell, W.M.S. and Burch, R.L., The Principles of Humane Experimental Technique. Methuen, London, 1959.
Reprinted by UFAW, 1992: 8 Hamilton Close, South Mimms, Potters Bar, Herts EN6 3QD England. ISBN 0 900767 78 2

2) Teunis et al. Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency. Toxicol In Vitro. 2013 Apr;27(3):1135-50. doi: 10.1016/j.tiv.2012.06.004. Epub 2012 Jun 19. PubMed, PMID: 22728231.

3) Vaessen et al. Identification of biomarkers to detect residual pertussis toxin using microarray analysis of human dendritic cells. In Press, Vaccine, 2013.

Author article: Marc Teunis, September 2013