Module 03 - The Three Rs of Humane Animal Experimentation

Satisfying the Refinement Principle

Refinement has been the least glamorous of the Three Rs because it produces the least obvious changes in animal use if numbers are the most important statistic. The refinement of techniques has a significant role to play in both the reduction and replacement of animals in research, teaching and testing. Refined techniques will result in less variability and improve the outcome in terms of results obtained. For example, the introduction of new and safer anesthetic agents together with better training of investigators in their use has reduced the number of anesthetic deaths. The refinement of statistical analytical techniques has allowed investigators to use fewer animals without losing significant information.

Refinement has its greatest impact in the reduction of pain and distress in animals. Appropriate use of anesthetics, analgesics and other therapeutic measures are very important refinement measures in invasive studies. The refinement of husbandry, particularly by increasing the complexity of social and physical environments, has improved the well-being of research animals. The establishment of scientific and appropriate endpoints for many studies (e.g., vaccine testing) has meant that animals have had to suffer less without affecting confidence in the results.

There are many examples of refinements that have made a difference both to the animals (in terms of minimizing pain and distress), and the results of scientific investigations.

Husbandry. In the past research animals were often singly housed in cages or pens that provided very little substrate or space for normal behavioral activities. Most research animals are social in behavior and isolation is stressful for them.

The alternatives. Most animals may be kept in social groups in complex environments that allow them to behave in a normal manner. There are many reports documenting the beneficial effects of this type of husbandry. For example, rats living in a socially and physically complex environment develop a thicker cerebral cortex, with more dendritic connections compared with those that are kept in isolation. Young rabbits that were kept in small cages developed skeletal abnormalities because they were unable to hop and run during the time when their muscles and bones were maturing.

Alternatives to previously used blood sampling techniques. The retro-orbital sinus of some small species (particularly rodents) was a convenient site from which to collect fairly large samples of blood. The procedure had risks (e.g., the eye could be damaged, especially if samples were taken repeatedly), and was painful. Several alternatives have been developed, including blood sampling from the tail vein, the saphenous vein and the jugular vein. Although some skill is required to perform these efficiently, the risk of causing severe damage to the animal is greatly reduced.

Experiments that cause severe suffering or death. For studies involving vaccine testing, infectious diseases, tumours, organ rejection, etc., the endpoint for the animal may in the past have been death from the disease. As an animal approaches death, it stops eating and drinking and rapidly becomes dehydrated, and except in a small number of instances, death can be predicted to occur within a short period of time from the point at which the animal stops eating and drinking.

The alternatives. When an experiment is expected to cause severe suffering or the death of an animal, endpoints should be established to limit the extent of the suffering and to anticipate death. If possible, pilot studies should be used to demonstrate the earliest point at which the scientific goals are reached so that the experiment can be terminated before the animals suffer. At a minimum, the pilot studies should be used to determine which clinical signs are most appropriate to indicate that the endpoint has been reached or when the death of the animal becomes inevitable.

Toxicity testing. The LD₅₀ test was required by regulatory agencies as an assessment of toxicity of new compounds. The LD₅₀ is the dose that will kill 50% of the animals. Many animals were used to accurately find this dose although its relevance to human toxicity has not been established.

The alternatives. A number of refinements to toxicity testing have been developed and have become accepted as OECD guidelines. For acute toxicity testing the fixed dose procedure (Tg420); the acute toxic class method (Tg423) and the up and down procedure (Tg425) have now been accepted by OECD member countries. Fewer animals and earlier endpoints are part of the refinements. The LD50 test (Tg401) has now been withdrawn and regulatory agencies from the OECD member countries are required to accept data generated using one of the three alternative guidelines. In addition, recommendations from ICCVAM have been published describing how in vitro data may be used to select the starting dose for the test, further limiting the numbers of animals needed and increasing the predictivity of the data..