Effects of bisphenol E on thyroid hormone system and developmental neurotoxicity-sensitive endpoints in zebrafish embryos - a new approach methodologies-based evaluation.

Endocrine-disrupting chemicals (EDCs), including some bisphenols, are of increasing concern. Aligned with the 3Rs (Reduction, Replacement, Refinement of animal experiments) and the EU's roadmap to phase out animal testing, zebrafish eleutheroembryos are key in developing New Approach Methodologies (NAMs) as they are non-protected until five days post-fertilization. While bisphenol A is restricted in the EU, the effects of bisphenol E (BPE) on thyroid hormone system (THS) sensitive endpoints and developmental neurotoxicity (DNT) remain unclear. We investigated whether BPE disrupts THS-sensitive endpoints and induces DNT in zebrafish eleutheroembryos. Based on adverse outcome pathways (AOPs): AOP 364 and AOP 157, we assessed eye morphology, inner plexiform layer (IPL), retinal pigmentary layer (RPE), posterior swim bladder inflation and swimming performance, and transcriptional analysis of THS- and DNT-related genes. We hypothesized that BPE would affect THS-sensitive endpoints, such as RPE and posterior swim bladder inflation, impairing swimming performance, and exert DNT effects through THS-mediated mechanisms. BPE impairs RPE without affecting overall eye development. However, transcriptional analysis of THS-related genes did not support a THS-mediated mechanism. BPE exposure also impaired posterior swim bladder inflation, which was identified as the primary contributor to impaired swimming performance. No effects were seen on quantitative brain measurements, leaving the sensitivity of zebrafish eleutheroembryos for brain morphological assessment unclear. Nevertheless, molecular DNT markers were detected, which could indirectly contribute to impaired swimming performance. Overall, our results demonstrate that the zebrafish eleutheroembryo is a valuable NAM model for assessing THS effects and DNT, and that BPE induces DNT and THS effects.