Supplementary file 1_Leveraging the developmental neurotoxicity in vitro battery: lessons learned interpreting assay outputs and integrating human exposure.docx

Of the compounds tested, four were found to have “specific hits” including EGCG, genistein, caffeine, and curcumin. Concentrations at which in vitro specific hits occurred were compared with published human plasma levels, revealing that in vitro bioactive concentrations for EGCG were within relevant human exposure levels. Conversely, for curcumin, genistein, and caffeine, any identified specific hits in the evaluated DNT IVB assays occurred at concentrations higher than reported human plasma levels indicating limited relevance for human risk assessment.

Moreover, our study highlights the need for well-defined performance criteria, reproducible reference controls, a standardized approach for hit calling, benchmark response determination, and interpretation across the battery to ensure reliability and reproducibility for bolstering readiness for regulatory acceptance. Overall, while the DNT IVB provides valuable insights and opportunities for screening applications to refine chemical safety evaluation, its effective use requires strengthened guidance on assay performance, data interpretation, and integration with exposure assessment to support robust application in human health risk assessment.</p>