Using Caenorhabditis elegans and zebrafish embryos (Danio rerio) in a combined approach to screen chemicals for developmental (neuro)toxicity and reproductive toxicity

• zebrafish embryos and C. elegans allow for fast DART and DNT screening • Of 36 chemicals tested, triazoles and PFOS caused DART in these models • Compared to CLP the combined models predict DART with higher sensitivity than alone Testing of chemicals for (neuro)developmental and reproductive toxicity traditionally relies on animal models, but ethical concerns and high costs have driven interest in exploring new approach methodologies (NAMs). In this study, two whole organism models, zebrafish ( Danio rerio ) embryos and nematodes ( Caenorhabditis elegans ), were used in combination to predict the (neuro)developmental and/or reproductive toxicity of 16 triazoles, 9 triazines and 11 per- and polyfluoroalkyl substances (PFAS), some of which are known reproductive toxicants. Zebrafish embryos were additionally used to screen for thyroid hormone system disruption. To evaluate regulatory relevance, the results were compared with the harmonized classification under the European Classification, Labelling and Packaging (CLP) regulation for reproductive toxicity. All tested endpoints were affected by at least one test compound, and each chemical class showed distinct hazard profiles in C. elegans and/or zebrafish. The combination of affected endpoints determined in the different model species demonstrated 75% sensitivity compared to CLP harmonized classification, while using zebrafish embryos or C. elegans alone resulted in 63% and 50% sensitivity, respectively. The increased sensitivity supports the complementary use of zebrafish embryos and C. elegans as screening models to prioritize compounds for further testing.