Zebrafish embryotoxicity test (ZET) as an alternative method for screening the developmental toxicity of difficult-to-test substances

Studies are needed to explore the potential human toxicity of Substances of Unknown or Variable Composition, Complex Reaction Products, or Biological Materials (UVCBs), as well as difficult-to-test substances, including those with low water solubility. The zebrafish embryo has been proposed as a new approach methodology (NAM) that enables the study of the embryotoxic potential of chemicals. Eight difficult-to-test substances, including multiple UVCBs and substances with low water solubility, were tested in zebrafish embryos by chemical incubation and microinjection, initially to determine the conditions and concentrations at which malformations and lethality were induced. A definitive experiment was subsequently conducted, and teratogenic index (TI) values were calculated based on the computation of lethality and embryotoxicity parameters (LC50, EC50, and NOAEL). Each of these substances had previously been tested in validated mammalian assays to assess reproductive or embryotoxicity. The zebrafish assay correctly predicted all three test substances that were not embryotoxic in mammalian reproductive or developmental assays. The zebrafish assay correctly predicted three of the five remaining substances that were classified for developmental toxicity based on prior mammalian assays using the incubation method but not the microinjection method. The two substances that were not correctly predicted by the zebrafish embryo assay produced less severe effects in the mammalian developmental screening assays. The use of the zebrafish developmental toxicity assay for screening UVCBs and difficult-to-test substances allowed the successful determination of their embryotoxic potential. The assay can help prioritize the testing of substances or as part of a weight of evidence in regulatory submissions.