Development of a Zebrafish Embryo Teratogenicity Assay and Quantitative Prediction Model

BACKGROUND: A zebrafish (Danio rerio) teratogenicity assay has been developed and evaluated for its ability to predict the teratogenic potential of chemicals. METHODS: Zebrafish embryos were dechorionated and then exposed to a test solution from 4-6 hours post-fertilization, and embryos or larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. In preliminary experiments, the potential time points for assessment of compound-induced dysmorphology and general toxicity parameters were evaluated, and 5dpf was found to be the optimum developmental stage for evaluation. Additionally, a morphological scoring system was devised to identify the developmental no-observed-adverse-effect level (NOAEL). For assay evaluation, 4 compounds with adequate in vivo developmental toxicity data were chosen. The compound set represented diversity in teratogenic potencies, structural classes, and pharmacologic targets. For 31 test compounds, each was evaluated over a concentration range, while 3 others were insufficiently aqueous-soluble to be fully tested. For each of the 31 tested compounds, the 5 dpf NOAEL was determined, and the concentration resulting in 25% lethality (LC25) was calculated by curve-fitting. Teratogenic potential of each compound was predicted based on the ratio of the LC25 to the NOAFL. LC25/NOAEL ratios of 10 or greater were considered predictive of teratogenicity. RESULTS: The model Successfully categorized 87% of the compounds as teratogens or non-teratogens, with only 2 false-positives (dimethyl phthalate and a Bristol-Myers Squibb (BMS) investigative compound) and 2 false-negatives (valproic acid and a BMS compound). CONCLUSIONS: The results indicate that this assay is promising for screening compounds for teratogenic potential. Birth Defects Res (Part B) 89:66-77, 2010. (C) 2010 Wiley-Liss, Inc.