Concentration-response evaluation of ToxCast compounds for multivariate activity patterns of neural network function.

The US Environmental Protection Agency's ToxCast program has generated toxicity data for thousands of chemicals but does not adequately assess potential neurotoxicity. Networks of neurons grown on microelectrode arrays (MEAs) offer an efficient approach to screen compounds for neuroactivity and distinguish between compound effects on firing, bursting, and connectivity patterns. Previously, single concentrations of the ToxCast Phase II library were screened for effects on mean firing rate (MFR) in rat primary cortical networks. Here, we expand this approach by retesting 384 of those compounds (including 222 active in the previous screen) in concentration-response across 43 network activity parameters to evaluate neural network function. Using hierarchical clustering and machine learning methods on the full suite of chemical-parameter response data, we identified 15 network activity parameters crucial in characterizing activity of 237 compounds that were response actives ("hits"). Recognized neurotoxic compounds in this network function assay were often more potent compared to other ToxCast assays. Of these chemical-parameter responses, we identified three k-means clusters of chemical-parameter activity (i.e., multivariate MEA response patterns). Next, we evaluated the MEA clusters for enrichment of chemical features using a subset of ToxPrint chemotypes, revealing chemical structural features that distinguished the MEA clusters. Finally, we assessed distribution of neurotoxicants with known pharmacology within the clusters and found that compounds segregated differentially. Collectively, these results demonstrate that multivariate MEA activity patterns can efficiently screen for diverse chemical activities relevant to neurotoxicity, and that response patterns may have predictive value related to chemical structural features.