In 1959, Russell and Burch formulated three principles to reduce the numbers of animals used in experimentation. These are known as the Three Rs: refinement, reduction, and replacement (absolute or relative).1 The validation process of regulatory testing has over the years incorporated many methods that support one or more of these principles for two main reasons: (1) only regulated and standardized animal tests that are repeatedly carried out over a long period of time (typical timeframe 12 years) and tests that have enormous costs warrant a formal validation process to be accepted as such; and (2) in the area of safety assessments, it is especially difficult to abandon an established test because safety standards could be lowered. In other areas of research and development, especially in the agent discovery phase, replacement of older with more advanced methods is more common due to constant pressure for more predictive and less costly tests.

In the case of developing countermeasures for bioterrorism agents where absolute dependence on animal models for efficacy testing serves to replace human clinical trials, the Three Rs provide a good framework to reduce animal use and minimize the animals’pain and distress.2 Because adoption of alternatives was driven by both animal welfare considerations and scientific advances in our understanding of biological phenomena, their utilization is often in the best interest of the study. Although alternatives do not compensate for the lack of clinical efficacy trials with human participants, they can enable technologies from which more information is gained than that gained by animal tests alone. In addition, they reduce the use of precious and expensive resources and reduce animal pain and distress, often resulting in improved quality of research outcomes (NCR 2008, 2009, 2011; Wolfer et al. 2004).

Applying the Principle of Refinement

The best possible treatment of animals starts with attention to husbandry (AWA; 7 USC § 2131-2159). Social housing and enriched environment within the cages, especially for the highly social nonhuman primates often used in these studies, while taking into consideration the scientific needs of the study, represent key strategies to avoid or reduce distress3 (NRC 2008). The use of analgesics and anesthetics is mandatory, not only for the alleviation of pain and distress, but also because it represents a more realistic approximation to the treatment of human patients.4,5

The development and use of protocol-appropriate humane endpoints, especially the early termination of studies at time points that indicate that animals are unlikely to recover, minimizes pain


1 The utility and applicability of the Three Rs has been described in various documents. For additional information, see the Guide for the Care and Use of Laboratory Animals, Eighth Edition and references therein (NRC 2011), and the website for the National Centre for the Replacement, Refinement and Reduction of Animals in Research (

2 The usefulness of the Three Rs and the employment of alternatives in regulatory safety testing are not discussed here as safety testing does not fall under the auspices of the Animal Rule.

3 The ability to provide enriched environment or social housing in biocontainment facilities may be difficult, extremely limited, or impossible. Although it is a necessary husbandry and animal welfare provision, methods to enrich the environment or house social animals in a biocontainment facility have not been studied.

4 It should be noted that the quality of care for human patients is not universally identical and that analgesics and anesthetics may not be available in natural outbreak settings. Therefore, different animal research protocols may be needed for the development of treatments under these conditions.

5 Non-administration of analgesics should be scientifically justifiable and accepted by an Institutional Animal Care and Use Committee (IACUC; Animal Welfare Act; 7 USC § 2131-2159) and employed as rarely as possible.

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