design in laboratory animal research (Russell and Burch 1959). Over the years, the Three Rs have become an internationally accepted approach for researchers to apply when deciding to use animals in research and in designing humane animal research studies.

Replacement refers to methods that avoid using animals. The term includes absolute replacements (i.e., replacing animals with inanimate systems such as computer programs) as well as relative replacements (i.e., replacing animals such as vertebrates with animals that are lower on the phylogenetic scale).

Refinement refers to modifications of husbandry or experimental procedures to enhance animal well-being and minimize or eliminate pain and distress. While institutions and investigators should take all reasonable measures to eliminate pain and distress through refinement, IACUCs should understand that with some types of studies there may be either unforeseen or intended experimental outcomes that produce pain. These outcomes may or may not be eliminated based on the goals of the study.

Reduction involves strategies for obtaining comparable levels of information from the use of fewer animals or for maximizing the information obtained from a given number of animals (without increasing pain or distress) so that in the long run fewer animals are needed to acquire the same scientific information. This approach relies on an analysis of experimental design, applications of newer technologies, the use of appropriate statistical methods, and control of environmentally related variability in animal housing and study areas (see Appendix A).

Refinement and reduction goals should be balanced on a case-by-case basis. Principal investigators are strongly discouraged from advocating animal reuse as a reduction strategy, and reduction should not be a rationale for reusing an animal or animals that have already undergone experimental procedures especially if the well-being of the animals would be compromised. Studies that may result in severe or chronic pain or significant alterations in the animals’ ability to maintain normal physiology, or adequately respond to stressors, should include descriptions of appropriate humane endpoints or provide science-based justification for not using a particular, commonly accepted humane endpoint. Veterinary consultation must occur when pain or distress is beyond the level anticipated in the protocol description or when interventional control is not possible.


The Committee for the Update of the Guide believes that the terms set out below are important for a full understanding of the Guide. Accordingly, we have defined these terms and concepts to provide users of the Guide with additional assistance in implementing their responsibilities.

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