Abstract
Being based within a Contract Research Organization (CRO), this thesis was focused on the field work; aiming at practical implementations with the goal to instruct and motivate peers to reach a feasible 3R (reduce, refine and replace) standard within the currently accepted and performed study models for regulatory developmental and
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reproductive toxicity (DART) testing. The objective was set to assess whether and how improvements on animal welfare in DART testing can be made by refinement, reduction and/or replacement of regulatory requested studies, without loss of integrity and validity of the study designs. The work was divided into three parts: The first part describes the implementation of refinement and reduction in current DART studies. This concerns refined blood sampling techniques which lead to less discomfort and reduced animal use. We evaluated the use of capillary microsampling (CMS) in juvenile rats in support of reduced animal usage. The goal was to study the best location for CMS in the juvenile rat with the least discomfort to the animal and least impact on the study outcome. Utilizing the optimal method could decrease the number of satellite juvenile animals by 75%. CMS can also be used in embryo-fetal development (EFD) studies in the rat, and would eliminate the need of satellite animals. However, in case CMS cannot be used, another method to omit satellite animals in EFD studies is the use of jugular vein blood sampling, which was successfully demonstrated in this thesis. The second part depicts the impact on refinement and reduction in recently updated DART guidelines. The efficacy of reproduction/developmental screening studies was assessed in a retrospective evaluation of 134 studies. It was concluded that reproduction/developmental screening studies are effective in providing unique data, especially considering the limited number of animals used. However, some simple additions would enrich their value in risk assessment even further. In addition, the thesis describes details on our experiences with implementation of OECD 443, the extended one-generation reproductive toxicity study (EOGRTS). The hallmark of the EOGRTS is that, based on certain criteria or triggers, selected offspring are assigned at weaning to different cohorts for further investigation of sexual maturation, reproductive organ integrity and function, neuropathological and behavioral endpoints, and/or immune function. Two chapters discuss the important aspects of this challenging study design. One chapter focuses on practical implementation, while another chapter offers guidance on science-based triggers for the extended evaluations. The third part provides a first step towards replacement in future DART testing through optimization of the zebrafish embryotoxicity test (ZET). Currently many different methodologies exist for the performance of the ZET. To assist in harmonization, this thesis proposes the most optimal study design based on literature and empirical data. Furthermore, our morphology scoring system was reported in detail to further enhance study design harmonization. In addition, one chapter describes the important role of internal zebrafish exposure on the predictability of the ZET. The impact of lipophilicity, solvent use and chorion presence on compound distribution within the zebrafish embryo were investigated.
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