Leveraging the developmental neurotoxicity in vitro battery: lessons learned interpreting assay outputs and...

The developing brain is highly sensitive to chemical insults due to the complexity and precise timing of neurodevelopmental processes. In vitro assays targeting key neurodevelopmental events offer insights into developmental neurotoxicity (DNT) pathways and represent an important advance in alternative testing strategies. The Organisation for Economic Co-operation and Development (OECD) published initial recommendations on the design and evaluation of a DNT in vitro battery (DNT IVB) for screening chemical effects on neurodevelopment. Thus far, there has been no characterization of the regulatory application of this new testing battery due to uncertainties in assay performance, interpretation, and contextualization of bioactivity. To gain experience with evaluating assay performance and interpretation of the DNT IVB, a subset of representative, commercially available assays were used to evaluate eight natural compounds, each with established dietary background exposure and no assumed DNT risk under "normal" dietary intake: EGCG, folic acid, vanillin, limonene, vitamin E, curcumin, genistein, and caffeine. 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.