In vivo high-throughput toxicity screening of brominated flame retardants using a Caenorhabditis elegans transcription factor RNAi platform

Brominated flame retardants (BFRs) such as tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCDD), and decabromodiphenyl ethane (DBDPE) are extensively used in a variety of consumer products, including electronics, textiles, and furniture. However, their environmental persistence and potential for neurodevelopmental toxicity have raised increasing concern. Legacy compounds such as TBBPA and HBCDD are undergoing regulation or being phased out, while alternatives like DBDPE remain poorly characterized, leaving uncertainties regarding their suitability as replacements. In order to rapidly fill knowledge gaps on these data poor substances and avoid regrettable substitutions, we established a high-throughput, mechanism-based in vivo toxicity screening platform. A RNAi approach on Caenorhabditis elegans Transcription Factors (TF) was used to assess biological pathways that were triggered by BFR exposure, leading to neurotoxicity (assessed via locomotion behavior). Using a 384-TF RNAi library, we identified 44 TFs modulating TBBPA-induced neurotoxicity. Pathway analyses (Reactome and CTD) highlighted retinoic acid receptor signaling as a key event, which mapped to four neurodevelopmental AOPs (AOP 520, 523, 532, and 533). Gene expression analysis of sex-1 and unc-55 confirmed retinoic acid signaling pathway activation. Application of the same framework to HBCDD and DBDPE revealed partially conserved behavioral and molecular responses, supporting the cross-chemical applicability of the TBBPA-derived AOP network. These findings demonstrate the utility of C. elegans-based TF RNAi screening as a fit-for-purpose New Approach Methodology (NAM) for mechanistic toxicology. By linking molecular initiating events to adverse outcomes, this strategy enables early hazard identification and read across strategy via AOP-informed, animal-free chemical risk assessment within next-generation risk assessment (NGRA) frameworks.