The zebrafish embryo is already being used for teratogenicity testing, i.e. in the Zebrafish Developmental Toxicity Assay (ZEDTA). However, in vitro data of our research group and in vivo data from other labs indicate that the zebrafish embryo shows no or low bioactivity. As such proteratogens, i.e. compounds that require bioactivation to exert their teratogenic potential, may be missed in the ZEDTA and lead to false negative results. Currently, rat exogenous metabolic activation system (MAS) is used to optimize the ZEDTA but it is controversial as it causes embryotoxicity by itself and its metabolic profile may be different than in man. The temperature of the co-incubation system may contribute to the observed embryotoxicity. Zebrafish embryos develop optimally between 26,5-28,5°C and do not tolerate the physiological temperature of rat MAS (38-39°C). Therefore, 32°C is often used in the coincubation system as a compromise. However, very recently we assessed temperature effects on zebrafish embryonic development and observed that 32,5°C causes embryotoxicty. Therefore, the aim of this research project is to develop a novel coincubation system (MAS) that functions at a non-embryotoxic temperature. Instead of rat MAS, we will evaluate in a first phase the bioactivity and embryotoxicity of human MAS (to obtain a human relevant metabolic profile) at different temperatures and of zebrafish MAS at 28,5°C (physiological condition for embryonic development). Regarding bioactivity, we will focus on xenobiotic metabolizing CYP families (i.e. CYP 1-3) and limit ourselves to fluorogenic substrates that are relevant to man and covering approx. 95% of human xenobiotic metabolism. Metabolite concentrations will be determined in the supernatans over-time by detection of the fluorescent signal. In a second phase we will evaluate biotransformation in the ZEDTA in presence and absence (controls) of hMAS and zMAS. This will be achieved by exposing early zebrafish embryos to the same substrates of the first phase. For the co-incubation, we will select the optimal temperature from the first phase. Metabolite concentrations will be determined over-time in the embryo and in the supernatans by detection of the fluorescent signal.