Citation:

Carstens, K., A. Carpenter, M. Martin, J. Harrill, T. Shafer, AND K. Friedman. Integrating Data From In Vitro New Approach Methodologies for Developmental Neurotoxicity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 187(1):62-79, (2022). https://doi.org/10.1093/toxsci/kfac018

Impact/Purpose:

The primary goal of this work is to assess the performance of the developmental neurotoxicity new approach methodologies (DNT NAMs) to extend the biological coverage of targeted high-throughput screening assays for putative hazard of interest for chemical safety assessment. This work importantly extends analysis of the DNT NAMs beyond the Agency Issue Paper presented to a scientific advisory panel in 2020, which was limited in scope to DNT NAMs and organophosphate insecticides. The analysis herein reveals patterns of responses for the 92 substances screened to date in both the microelectrode array neural network formation assay (MEA NFA) and the high content imaging (HCI) assay suite in neural cell lines. These patterns of response, or in vitro phenotypes, may help elucidate the in vitro biological effects of substances specific to DNT-relevant processes and function and further inform the hypothesis that these DNT NAMs form a sensitive and specific screen for putative DNT. This work applies computational approaches to further address three related hypotheses: (1) the DNT NAMs provide a sensitive indicator of  disruptions in key neurodevelopmental processes; (2) evaluating bioactivity that may be selective, i.e. occurring at concentrations less than observed cytotoxicity, may provide a more specific indicator of the functional processes involved in putative DNT bioactivity; and, (3) the available DNT NAM screening battery may optimally classify putative positive and negative reference chemicals identified previously from in vivo studies. The result of this work provides an analysis method for interpretation of these DNT NAMs for in vitro hazard identification and prioritization of chemicals for DNT bioactivity.   Evaluation of a battery of in vitro assays for DNT screening. (Journal article of 1.2.2.5)

Description:

In vivo developmental neurotoxicity (DNT) testing is resource intensive and lacks information on cellular processes affected by chemicals. To address this, DNT new approach methodologies (NAMs) are being evaluated, including: the microelectrode array neuronal network formation assay (NFA); and high-content imaging (HCI) to evaluate proliferation, apoptosis, neurite outgrowth, and synaptogenesis. This work addresses three hypotheses: (1) a broad screening battery provides a sensitive marker of DNT bioactivity; (2) selective bioactivity (occurring at non-cytotoxic concentrations) may indicate functional processes disrupted; and, (3) a subset of endpoints may optimally classify chemicals with in vivo evidence for DNT. The dataset was comprised of 92 chemicals screened in all 57 assay endpoints sourced from publicly available data, including a set of DNT NAM evaluation chemicals with putative positives (53) and negatives (13). The DNT NAM battery provides a sensitive marker of DNT bioactivity, particularly in cytotoxicity and network connectivity parameters. Hierarchical clustering suggested potency (including cytotoxicity) was important for classifying positive chemicals with high sensitivity (93%) but failed to distinguish patterns of disrupted functional processes. By contrast, clustering of selective values revealed informative patterns of differential activity but demonstrated lower sensitivity (74%). The false negatives were associated with several limitations, such as the maximal concentration tested or gaps in the biology captured by the current battery. This work demonstrates that this multi-dimensional assay suite provides a sensitive biomarker for DNT bioactivity, with selective activity providing possible insight into specific functional processes affected by chemical exposure and a basis for further research.  

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