Navigation path

High level navigation

Page navigation

Additional tools

  • Instagram
  • Facebook
  • Twitter
  • YouTube
  • Print version
  • Decrease text
  • Increase text

Animals used for scientific purposes

Replacement, Reduction and Refinement – the "Three Rs"

What are the “Three Rs”?

The publication of "The Principles of Humane Experimental Technique"  by W.M.S. Russell and R.L. Burch in 1959 marks the birth of the principle of the “Three Rs”.

The authors proposed the principles of Replacement, Reduction and Refinement (the “Three Rs”) as the key strategies of a systematic framework aimed at achieving the goal of humane experimental techniques. Russell and Burch saw replacement as the ultimate goal for laboratory animal based research, education and testing, with the other two, reduction and refinement, being more readily achievable in the short term.


Replacement can be defined as methods, strategies or approaches which do not involve the use of live animals.

Replacement may be achieved through a number of tools or their combinations including

  • in vitro systems using tissues, whole cells or parts of cells
  • systems based on biochemical approaches, i.e. using synthetic (macro)molecules as proxies of (reactive) toxicity targets. Such methods are referred to as "in chimico"
  • computer-based models and approaches – often termed in silico
  • use of 'omics' technologies (e.g. transcriptomics, proteomics and metabonomics)
  • non-testing approaches such as 'read-across' technique


The concept of reduction covers any approach that will result in fewer animals being used to achieve the same objective, including maximising the information obtained per animal, reducing the number of animals used in the original procedure and/or  limiting or avoiding the subsequent use of additional animals.

The number of animals can also be reduced by performing procedures on animals more than once, where this does not detract from the scientific objective or result in poor animal welfare. However, the benefit of reusing animals should always be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal. As a result of this potential conflict, the reuse of animals should be considered on a case-by-case basis.


Image published courtesy of Novo Nordisk
Photo credit: Novo Nordisk

Today, the term refinement signifies the modification of any procedures or husbandry and care practices from the time the experimental animal is born until its death, so as to minimise the pain, suffering and distress experienced by the animal and enhance its well-being.

When an animal experiences pain, suffering or distress, there are often accompanying physiological changes which may increase the variability of scientific results. Refinement therefore is also likely to improve data quality and contribute to Reduction.

Refinement can also be achieved by moving from species that are considered more sentient to those less sentient. Examples: substituting a non-human primate by the use of a fish or substituting the use of fish with daphnia. These are both considered methods of refinement as they are likely to reduce the pain, suffering and distress experienced by the animal, however, still requiring the use of live animals.

The “Three Rs” in EU legislative framework

The principle of the “Three Rs” has been present in the EU legislation in spirit, from as early as 1986, when the first EU legislation for the protection of animals used for experimental and other scientific purposes was adopted. 

However, Directive 2010/63/EU on the protection of animals used for scientific purposes, for the first time in EU legislation spells out the principle of the “Three Rs” and makes it a firm legal requirement. The principles of Replacement, Reduction and Refinement must be considered systematically at all times when animals are used for scientific purposes in the EU. Under the Directive, the term "scientific purposes" covers all uses of animals for the purposes of basic, translation and applied research, regulatory testing and production as well as for the purposes of education and training.

Furthermore, the Directive ensures that its application goes beyond that of the original, narrower interpretation of the “Three Rs” only in the context of choice of methods. The Directive enlarges the Refinement also to cover all animal breeding and care – that is, to ensure refinement during housing, breeding and care even if the animal is not undergoing a scientific procedure.

What are alternative approaches?

The term ‘alternatives’ in this context includes all assays, tests, methods, techniques, tools, strategies and approaches etc. that contribute to the practical implementation of the “Three Rs”. That is

  • to obtain the required information without the use of live animals;
  • to reduce the numbers of animals whilst obtaining the same level of information;
  • to refine the use of live animals so as to cause less pain, distress or suffering, or improve the welfare of the animals.

Alternative approaches provide opportunities to advance the "Three Rs", but equally aim at developing better and more predictive scientific tools to protect human and animal health and the environment.