Refinement

Russell and Burch’s principle of refinement has endured from its inception in the 1950s, to appear in today’s EU Directive

Bryan Howard

In the first issue of PiLAS, the subject of inhumanity was reviewed, as propounded by Russell and Burch in their book, The Principles of Humane Experimental Technique.1 Although it is often considered that strict adherence to the Three Rs will satisfy concerns about inhumanity arising from the use of animals in scientific investigations, and although Russell and Burch considered the Three Rs as a means of “the removal of inhumanity”, they emphasised the pre-eminent need for replacement, and, where that is not possible, the application of the principles of reduction and refinement. Indeed, Part II of that excellent text places these three topics in just that sequence. This is currently codified in Article 4 of the new European directive (Directive 2010/63/EU),2 which requires Member States to “ensure refinement of breeding, accommodation and care, and of methods used in procedures, eliminating or reducing to the minimum any possible pain, suffering, distress or lasting harm to the animals”.

Russell and Burch’s definition of refinement
Specifically, Russell and Burch defined refinement as “any decrease in the incidence or severity of inhumane procedures applied to those animals which… have to be used”. They acknowledged it, not only as an ethical imperative, but also as a prerequisite for successful animal experimentation. They also emphasised the critical role of those persons in charge of experimental animals — this places obligations not only on those responsible for their day-to-day care, but also on those designing and those conducting experiments which involve living animals. In order to meet obligations such as these, the competence of the staff involved with all aspects of the care and use of laboratory animals is paramount — a point emphasised by Russell in his review of progress in 2005.3 To some extent, the welfare of animals is inevitably compromised by the need to care for them under laboratory conditions; consequently, the time for which animals are maintained within research facilities should be as short as possible, so that any such negative effects are of the shortest possible duration. In addition to identifying a need to provide the highest standards of care, it is also important to minimise the impact of procedures carried out on animals. Taken together, this can be seen as arguing for the use of younger animals, for the designing of investigations to be as short as practically possible, and for limits to be placed on unavoidable suffering by the application of humane endpoints.4 This is also in accord with Russell and Burch’s assertion that “Refinement will inevitably increase efficacy, and may incidentally entail reduction as well”, which, as a consequence, leads to “more efficient” science. In relation to refinement, Russell and Burch identified two broad categories of scientific investigation: studies directed at acquiring knowledge about the mechanisms and consequences of pain and distress (stressful investigations) and all other studies (neutral investigations). In this latter category, animal stress and distress is inevitably an uncontrolled variable, which will ultimately introduce error and impair the efficiency of the investigation. They saw this as an area in which there was considerable scope for refinement, and strongly urged the elimination of “contingent inhumanity”, by not only ensuring the very best standards of husbandry, but also by carefully considering experimental design and the conduct of investigations themselves. In the case of stressful investigations, Russell and Burch acknowledged that there is an apparent conflict between the needs of humanity and efficiency. However, they demonstrated that, by the careful selection of an appropriate model, experiments can be designed so as to minimise the impact on individual animals, whilst still revealing the underlying physiological mechanisms and facilitating the development of treatment strategies. Although at the time when they were writing, knowledge of general anaesthetics was relatively limited, they clearly appreciated the importance of focusing on specific measures required to provide the scientific answers.

A broader context
Speaking personally, I see refinement in a rather broader context, that applies not only to the experience of animals subjected to experimentation, but also to the scientific methodology used in the laboratory. Lack of precision due to faulty technique, or use of inappropriate equipment, may result in false scientific findings, setting back the field of investigation and sometimes requiring studies to be repeated. For example, the use of poorly validated or out-of-date materials or chemical agents (anaesthetics, pharmaceutical agents, test substances, etc.), or uncalibrated and badly maintained measuring equipment, constitute unquantifiable variables that introduce unrecoverable errors. The impact of experimental error on the reliability of scientific results is probably greater in most cases than the variability among the animals themselves. Similarly, it is essential that the scientist, in publishing experimental findings, should provide details of any refinement techniques developed and implemented during the course of the study, so that others working in similar fields can adopt or modify them as appropriate.
Many journals now encourage the inclusion of such material, which may be incorporated in printed publications or on the publisher’s website.

The ongoing search for refinement opportunities
In their book, Russell and Burch pointed out that, to a large extent, refinement “can be reduced in principle to the matter of choice between procedures for a given objective”. Article 13 of EU Directive 2010/63/EU2 requires that Member States shall ensure that in choosing between procedures, those which cause the least pain, suffering, distress or lasting harm, and are most likely to provide satisfactory results, shall be selected. It should be noted here that opportunities to introduce refinements may often be found in techniques adopted in the pursuit of scientific procedures, rather than in the entire procedure itself — for example, the offering of a small ‘treat’, such as a morsel of dried fruit to a mouse or rat, immediately after the conduct of a moderately stressful procedure.
Opportunities for offering rewards of this nature should always be sought, but never introduced if it can be shown that they are likely to interfere with interpretation of the scientific findings. Another strategy illustrating this approach, is the prior training of animals to undergo or perform procedures with which they might otherwise be reluctant to comply. Often, the use of rewards in association with training may substantially reduce the stress that an animal demonstrates when the procedure is next carried out.

Developments since the 1950s
In the opinion of Russell and Burch, areas in which refinement was desperately required included the induction and maintenance of anaesthesia and the conduct of euthanasia. In the case of the former, very considerable advances have taken place since 1959,5 and there can be very little justification for researchers not to employ anaesthetic regimes that are every bit as safe and effective as those used in human medical practice. Unfortunately, there is still debate about the most humane methods of killing animals,6 and it remains incumbent upon those required to kill experimental animals, to ensure that this is done expeditiously and humanely.

Also, since the publication of their book, sophisticated equipment has become available, which permits the refinement of many chronic investigations that require the monitoring of physiological and behavioural parameters or the parenteral administration of medicines or test substances over prolonged periods. Non-invasive monitoring techniques include magnetic resonance imaging (MRI), positron emission tomography (PET), X-ray and ultrasonic imaging.7
Each of these methods requires an animal to be immobile whilst imaging is carried out, so brief periods of anaesthesia may be necessary. The impact of this requirement on the overall well-being of the animal needs to be carefully considered, before such approaches are adopted. Techniques such as microdialysis and subcutaneous or intraperitoneal implantation of permeable micro-cages facilitate, respectively, the collection of tissue fluid samples and the examination of therapeutic strategies, with minimal impact on the host animal. There is also a range of implantable micropumps, varying from osmotic capsules to refillable programmable micropumps, that facilitate the keeping of animals in social groups within their home cages during prolonged periods of investigation. Such devices may facilitate reduction, as well as refinement, by permitting animals to act as their own experimental controls, so that there is no need for a separate control group.However, such ‘longitudinal studies’ extend the lifetime experience of each experimental animal, so,before adopting these studies, it is important that a clear evaluation is made of the impact of such approaches on the animals’ welfare. It is unacceptable to subject fewer animals to greater suffering as a means of achieving reduction. However, provided that measures are taken to maximise each animal’s habituation to its care and scientific routines, and to minimise the adverse impacts arising from them, refinement can also arise as a consequence of minimising the number of animals that need to be so habituated.

References
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, xiv + 238pp. London, UK: Methuen.
2 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
3 Russell, W.M.S. (2005). The Three Rs: Past, present and future. Animal Welfare 14, 279–286.
4 NC3Rs (undated). Humane endpoints. London, UK: National Centre for the Replacement, Refinement and Reduction of Animals in Research. Available at: http://www.nc3rs.org.uk/category.asp?catID=21 (Accessed 10.07.13).
5 Flecknell, P. (2009). Laboratory Animal Anaesthesia, 3rd Edition, xxii + 304pp. London, UK: Academic Press.
6 AVMA (2013). Guidelines for the Euthanasia of Animals, 102pp. Schaumburg, IL, USA: American Veterinary Medical Association.
7 Brønstad, A. & Dontas, I. (2011). Imaging techniques. In The COST Manual of Laboratory Animal Care and Use: Refinement, Reduction, and Research (ed. B. Howard, T. Nevalainen & G. Perretta), pp. 287–311 Boca Raton, FL, USA: CRC Press, Taylor & Francis Group.

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