Zebrafish embryotoxicity test for developmental (neuro)toxicity: Demo case of an integrated screening approach system using anti-epileptic drugs

- The zebrafish embryotoxicity test (ZET) was applied using integrated multiple endpoints.
- Morphology, motor activity, histopathology, kinetics and gene expression were studied.
- A series of anti-epileptic drugs showed specific sensitivity for each endpoint.
- This test strategy may contribute to elucidate the mode of action for developmental toxicity in ZET.
- This test strategy may contribute to better define the applicability domain of the ZET.

To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro)toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60 μM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180 μM. For ETH, all endpoints showed similar sensitivity (6.6 mM), whereas MA was the most sensitive parameter for LEV (40 mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET.