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Re-testing of false negative compounds in new approach methods (NAMs) for developmental neurotoxicity screening
Citation:
Shafer, T., T. Freudenrich, K. Wallace, M. Mayil Vahanan, AND K. Carstens. Re-testing of false negative compounds in new approach methods (NAMs) for developmental neurotoxicity screening. SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25544425
Impact/Purpose:
Presentation to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity
Description:
Background and Purpose: Recently, the Organization of Economic Cooperation and Development (OECD) published an “Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity In Vitro Testing Battery (DNT-IVB)” that describes a battery of 17 in vitro assays that can be used to evaluate potential DNT hazard of chemicals. The overall performance of the battery in terms of sensitivity (correct identification of compounds with evidence of DNT hazard) ranged from 61-83%, indicating that some compounds with evidence of DNT hazard in vivo were not bioactive in the DNT-IVB, suggesting that these might be “false negative” compounds. There are numerous reasons for false negative responses in an assay: compound age/instability, compound insolubility, testing concentration ranges that are too low, or inactive/less active enantiomers, among others. "The current study identified 19 putative "false negative" compounds from the results of the six EPA assays that are included in the DNT-IVB, and re-tested them in those same six assays (hNP1 proliferation/apoptosis; Icell gluta neurite outgrowth; neurite outgrowth, neurite maturation, synaptogenesis, and network formation in primary cortical neurons). Methods: Nineteen compounds were identified as putative “false negative” compounds in the EPA assays of the DNT-IVB (Carstens et al., Toxicol Sci. 2022;187:62-79). New chemicals were obtained from appropriate vendors and fresh stock solutions of each compound were prepared, aliquoted, and stored frozen. Separate aliquots were thawed prior to each experiment. The exception was Na valproate, which was prepared fresh in media prior to each experiment. Briefly, high-content imaging (HCI) approaches were used to evaluate the effects of compound exposure on hNP1 proliferation (BrdU incorporation) and apoptosis (caspase 3 activation), as well as neurite outgrowth (betaIII tubulin) in human neuroprogenitor (Icell gluta) and rat cortical neurons. Neurite maturation (MAP2) and synaptogenesis (synapsin) were evaluated between 5 and 12 days in vitro (DIV) with HCI approaches while network formation over DIV 0-12 was evaluated using microelectrode array (MEA) recordings. All compounds were tested in half-log increments, generally from 0.003 to 30 µM; higher concentrations were tested when pharmacokinetic information indicated in vivo effects at higher blood concentrations. Results: Of the 19 compounds identified as putative “false negatives”, 15 compounds had activity in one or more of the EPA assays when retested using fresh solutions, higher concentration ranges and/or more active enantiomers. The four compounds that were still without activity were thalidomide, carbamazepine, terbutaline hemisulfate and caffeine. Rat cortical neurite outgrowth and network formation assays detected the most compounds at 13 and 10 chemicals with positive responses, respectively. All 15 active compounds were detected by one or both of these assays. Only 1 compound (hydroxyurea) was active in proliferation and apoptosis assays with hNP1 cells. Conclusions: These data indicate that compounds that were initially identified as false negative compounds were active when rescreened using fresh solutions and considering appropriate concentration ranges, active enantiomers, and other factors. Pending confirmation of the activity following pipelining through the ToxCast pipeline, reconsideration of the bioactivity of these compounds will improve the sensitivity of both the individual assays, as well as the overall sensitivity of the entire battery. Further, this illustrates the necessity to consider compound solubility, stability, stereoactivity and appropriate concentration ranges whenever possible. (This abstract does not reflect EPA policy.)
URLs/Downloads:
DOI: Re-testing of false negative compounds in new approach methods (NAMs) for developmental neurotoxicity screening
SHAFER_SOT_POSTER_2024_V4.PDF (PDF, NA pp, 1090.39 KB, about PDF)