Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) for Regulatory Testing—Protocol Optimization and Historical Control Data

The Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) is a promising and innovative method with potential to replace the screening of teratogenic substances in mammals during preclinical development. However, a harmonized and validated protocol does not exist for the ZEDTA, and data on the background incidence of spontaneous malformations are not readily accessible. Therefore, the aim of this research was twofold: (1) to optimize the ZEDTA protocol and (2) to generate historical control data. The most optimal results were achieved by exposing zebrafish larvae in 24-well plates at a temperature of 26 °C in combination with the renewal of test solutions after 48 h of exposure. Furthermore, the use of 0.5% <i>v</i>/<i>v</i> DMSO did not induce more malformations or mortality than exposure to standard ISO medium. In total, 26 valid experiments were conducted using the optimized ZEDTA protocol. An overall mortality of 3.5% was recorded after 96 h of exposure. Malformations were observed in 7.6% of all surviving larvae. The most frequently observed abnormalities included yolk sac deformation (4.0%), followed by tail (2.8%), heart (2.6%), and head malformations (1.6%). The optimized protocol was considered effective in supporting an optimal development rate of exposed zebrafish larvae, with low mortality and minimal background malformations. These findings indicate a low level of confounding factors and high reliability of results, making an essential step in the refinement of ZEDTA toward global harmonization and regulatory acceptance.