The 3Rs - first described by W. Russell and R. Burch in the book the 'The Principles of Humane Experimental Techniques' (1959) – are guiding principles for a more ethical use of animals in laboratory testing preserving, and often improving, the quality and reliability of the experimental data. The 3Rs are: 1) Replacement (use of non-animal over animal methods, whenever possible, to achieve the same scientific aims); 2) Reduction (using the lowest number of animals necessary to achieve reliable scientific results); 3) Refinement (use of methods that alleviate or minimize potential pain, suffering or distress, and enhance the wellbeing of the animals used). Replacement can be intended then as the use of alternative and/or complementary methods that allows to replace and/or accompany some methods or techniques traditionally used. Reduction is achieved by means of appropriate experimental design and precise statistical assessment of the smallest sample size useful for a particular experimental protocol, Refinement must not only aim at minimizing – or preventing – pain and distress from experimental procedures, but also enhance the welfare of each animal throughout all of its life span, as much as possible and to as many animals as possible.
The 3Rs are the overarching principle of Directive 2010/63/EU on the protection of animals used for scientific (1), as patent in Article 4. Knowledge of the '3Rs' is required by the personnel taking part (at any level) in projects involving in vivo models (Annex 5), and any researcher presenting a project application to the national Competent Authority must explain in which way he/she will apply the 3Rs Principle through the different stages of his/her project (Annex 6).
The '3Rs' are not always independent one from another, and both negative and positive interactions can occur. (2, 3) For example, Refinement can minimize procedure-related stress – a confounding factor in animal experiments – and hence improve the signal/'noise' ratio, allowing a Reduction of the sample size necessary to identify meaningful differences between groups. A negative interaction can occur when animal numbers are increased to preserve or enhance animal welfare. This is the case of some experiments where a given substance must be administered in the water or food. This implies that the cage – rather than the animal – is the experimental unit, so choosing to house animals in pairs over single housing will increase animal numbers, but prevent much distress from social isolation.
Which 'R' to choose? There is no definitive rule to follow, and the decision has to be taken on a 'case-bycase' basis. However, it is our view that the degree of welfare of the animals involved should always be given particular consideration.