The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e., abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although CYP1—except CYP1A—and SULT1ST1 were shown to be already mature in early embryonic development.

With the US Environment Protection Agency reducing requests for (and funding of) mammalian studies alongside the proposed elimination of requests by 2035, there is an urgent need for fully validated New Approach Methods (NAMs) to fill the resultant gap for safety assessment of agrochemicals. One promising NAM for assessing the potential for human prenatal developmental toxicity potential is the Zebrafish Embryo Developmental Toxicity Assessment, a bioassay that has been used by the pharmaceutical industry for more than a decade in early-stage drug safety assessment. Despite its promise, little data has been generated to assess the validity of ZEDTA for assessing Developmental and Reproductive Toxicity of new agrochemical products. Addressing this knowledge gap, we tested 67 compounds (insecticides, herbicides and fungicides) spanning multiple different chemical groupings and mechanisms of action. ZEDTA assay results were compared with the European Chemicals Agency (ECHA) Classification and Labelling (C&L) for mammalian hazard classification and with publicly available data to determine the ZEDTA's translation power. Overall, the ZEDTA assay had an effective detection capability of 65 % for sensitivity and 64 % for specificity as compared with the ECHA-C&L classification and publicly available data. Comparing the ZEDTA data there were both strengths and weaknesses in alignments for across the different chemical classes and chemical mechanisms of action. Overall, the data generated, show the performance of the ZEDTA assay was comparable with other bioassays highlighted as alternatives for mammalian assessment and holds good promise as a NAM for screening agrochemical prenatal developmental toxicity during new product human safety assessment.

Establishing anaesthesia for ensuring both animal welfare and compatibility with protocols required for different areas of scientific research is vital. Zebrafish ( Danio rerio ) are one of the most used animal models in research; however, little is known about the appropriateness of anaesthetic used for this species, especially for embryo-larval life stages. Using a combination of whole-brain functional imaging, quantification of cardiovascular performance, and behaviour, we explore the efficacy and tolerability of six widely used fish anaesthetics (2-phenoxyethanol, benzocaine, etomidate, MS222, isoeugenol and quinaldine sulfate) in larval zebrafish. We show that MS222 and quinaldine sulfate are the most suitable for achieving deep anaesthesia, while etomidate is better suited for studies focused on the cardiovascular system. Only quinaldine sulfate was found to be aversive. Our findings aid researchers for selecting the most suitable anaesthetic compounds and concentrations for their specific research goals, and the refinement of studies using anaesthesia in larval zebrafish.