U.S. Environmental Protection Agency Office of Research and Development EPA www.epa.gov  Calculation of Potency Ratio (PR): The lower 5th percentile of concentrations that caused a 50% of maximal response (AC50) in NSR assays was compared to the 5th percentile AC50 in non-NSR assays.  PR < 0 indicated greater potency in NSR assays for 103/238 = 43.3% of neuroactive substances with sufficient data. Summary 88% of neuroactives substances are active in >=1 ToxCast assay endpoint; 72% are active in >=1 NSR assay endpoint. Lack of detection in the remaining 12% of neuroactive substances may be due problematic physicochemical properties for some. The number and lack of testing of the remaining undetected neuroactive substances seems insufficient to reveal a gap in the biological space for the detection of neuroactivity. Most whole-cell neuronal assay endpoints detect a greater percentage of neuroactive than other substances tested. Trends for metals, pyrethroids, and organochlorines suggest that NSR assays may detect activity at lower concentrations than other assays. Conclusions Overall, the majority of neuroactive substances evaluated here were detected by ToxCast assays. NSR assays will likely play a role in detecting neuroactive substances at sufficiently sensitive concentrations. Future directions Use in vivo to in vitro extrapolation to compare concentrations at which neurological effects are seen in vivo to the concentrations at which activity is observed in vitro. Assess the activity of neuroactive substances with a known mechanism of action in appropriate target-based assays. Evaluation of the ToxCast Assay Suite for the Detection of Neuroactivity Carpenter, A. F.1,2*, Carstens, K. E.1,2, Martin, M. M.1, Shafer, T. J.1, Paul Friedman, K.1 1 Center for Computational Toxicology and Exposure, ORD, US EPA, RTP, NC 27711 2 Oak Ridge Institute for Science and Education, Oak Ridge, TN, 37830 Amy F. Carpenter l carpenter.amy@epa.gov This poster does not reflect US EPA policy. A.F.C. was supported by appointment to the Research Participation Program of the U.S. Environmental Protection Agency, Office of Research and Development, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. EPA. Neuroactive substances not detected in ToxCast are largely volatile or have not been extensively tested in NSR assays Potency in NSR versus other assays varies by substance 4023/P721 Computational Toxicology I March 29, 2022 10:45am - 12:30pm Society of Toxicology Annual Meeting & ToxExpo 88% of neuroactive substances are detected in >=1 ToxCast endpoint. 80% of other tested substances are also detected in >= 1 ToxCast endpoint. NSR assay endpoints provide comparable sensitivity with higher specificity for neuroactive substances. Difference in % neuroactive vs. other substances detected increases with higher hit count threshold. 1. Russell S Thomas et al., “The Next Generation Blueprint of Computational Toxicology at the U.S. Environmental Protection Agency,” Toxicological Sciences 169, no. 2 (June 2019): 317–332. https://doi.org/10.1093/toxsci/kfz058. 2. Human Protein Atlas proteinatlas.org 3. Mathias Uhlén et al., “Tissue-based map of the human proteome,” Science 347, no. 6220 (January 2015). https://doi.org/10.1126/science.1260419.  Wilcoxon signed rank test: Tested for shift in distribution of PRs towards greater potency in NSR or non-NSR assays by chemical class.  PR’s for organophosphates shifted ~0.5 log10 µM towards greater potency in non-NSR assays.  Several organochlorines, pyrethroids, and metals have PR < 0, though trend is not statistically significant after Bonferroni adjustment by chemical class. 𝑃𝑃𝑃𝑃= log10 5%𝑖𝑖𝑖𝑖𝑖𝑖𝐴𝐴𝐴𝐴50 𝑁𝑁𝑁𝑁𝑁𝑁𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 5%𝑖𝑖𝑖𝑖𝑖𝑖𝐴𝐴𝐴𝐴50 𝑛𝑛𝑛𝑛𝑛𝑛𝑁𝑁𝑁𝑁𝑁𝑁𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 Concluding remarks ToxCast detects activity from 88% of neuroactive substances Most whole-cell NSR endpoints appear qualitatively but not quantita