Standardization and the 3Rconcept in animal testing - effects and benefits of refinement in two test protocols

The focus of this thesis is the impact of housing- and husbandry conditions and experimental procedures on the outcome of animal studies. The aim was 1) to improve the quality of experimental data by limiting confounding factors, either in a qualitative or quantitative way, and 2) to reduce the variance of experimental results. The ultimate goal was to suggest ways by which these variables can be eliminated or minimized, to improve quality of research and to allow for a reduction in animal numbers and/or refinement of the experimental protocols. In Part I of this thesis we studied whether refined housing conditions (single- and social housing) of rats in a one-generation endocrine disrupter study (OECD Test Guideline 415) exposed to tetrabromobisphenol A (TBBPA; a brominated flame retardant) influence standardization thereby affecting the validity, reliability and reproducibility of test-results. To this end immune- and endocrine parameters were used as well as histopathology, body- and organ weights and neurobehavioral parameters. The results challenged the general idea that social housing of rats increases variance. Significant differences between housing conditions were rarely noted, while single housing was able to modify the effect of TBBPA, and TBBPA was found to mask or enhance housing effects. Overall, the data from part I indicate that no scientific reasons seem to exist to withhold rats from social housing in toxicological assays. In Part II of this thesis we studied the effects of modified living conditions of rabbits with the aim to reduce variation in experimental outcome. We did so by allowing animals to exhibit a more natural expression of species-specific behaviour in a model for monitoring whole cell pertussis (wP) vaccine-induced adverse effects. Several factors, which were considered to be potentially stressful, such as housing- and husbandry conditions, as well as the human-rabbit interaction were hypothesized to cause this large variation. From this study we concluded that modifications such as species-specific housing, nutrition, husbandry and adjustment of the environment to the behavioural needs of animals and handling resulted in i) a reset of normal circadian body temperature and diminished effects of experimental- and routine procedures on body temperature, and ii) decreased variation in experimental outcome. This allowed us to discriminate between wP vaccine induced adverse effects. Furthermore, handled animals were more tame, less emotional and more cooperative during experimental procedures and less aggressive towards group mates