Validation of Zebrafish Embryotoxicity Test (ZET) as a Qualified Alternative Assay For Its Regulatory Use.
The new ICH S5 (R3) guideline on reproductive toxicology proposes the use of alternative testing assays to minimize the use of animals. The guide does not recommend specific assays and provides a Reference Compound List that contains 29 compounds that have been shown to induce specific malformation or embryo-fetal lethality plus 3 negative compounds that can be used to support the qualification of an alternative assay.
Our research focused on the predictivity of the zebrafish developmental toxicity assay to further validate this model for its regulatory use by testing the 32 compounds indicated in the new ICH S5 guideline. To determine the teratogenic risk of these chemicals, the presence of morphological alterations was analyzed at two different stages and a teratogenic index (TI) was established as the ratio between LC50/EC50. Developmental toxicity was well predicted for Carbamazepine, 5-Fluorouracil, Hydroxyurea, Isotretinoin, Methotrexate, Tretinoin and Valproic acid. However, morphology was not or barely affected after exposure to Phenytoin, Pomalidomide and Thalidomide, although the limited solubility of these compounds in water restricted the concentrations that could be assayed. In the case of Aspirin, mortality was the main toxicity manifestation detected but this compound was not classified as teratogenic based on the threshold established to differentiate teratogenic of non-specific toxic compounds. At last, no toxicity was caused by Cetirizine, Saxagliptin and Vildagliptin, the 3 negative compounds selected in the guide. These are promising results that need to be further confirmed testing all proposed compounds.
Finally, our objective is also to evaluate the internal concentration of these chemicals in zebrafish embryos and compare results with the mammalian exposures provided in the guide to determine the ability of this assay to predict mammalian embryotoxic exposure.