Citation:

Knudsen, T. B., A. M. Richard, P. G. Kothiya, R. S. Judson, K. A. Houck, K. C. Crofton, R. S. Thomas, S. Little, E. S. Hunter, N. C. Baker, C. K. M. Leung, J. Palmer, A. Smith, M. Colwell, P. West, R. Burrier, AND L. Egnash. Evaluation of 1066 ToxCast Chemicals in a human stem cell assay for developmental toxicity (SOT). Presented at Society of Toxicology, San Diego, CA, March 21 - 26, 2015. https://doi.org/10.23645/epacomptox.5178703

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poster presented at SOT annual meeting in San Diego, CA on March 24, 2015

Description:

To increase the diversity of assays used to assess potential developmental toxicity, the ToxCast chemical library was screened in the Stemina devTOX quickPREDICT assay using human embryonic stem (hES) cells. A model for predicting teratogenicity was based on a training set of 23 pharmaceuticals (96% accurate, 92% sensitivity, 100% specificity) and an independent 13 pharma test set (77% accurate, 57% sensitivity, 100% specificity). In this 96-well plate platform, hES cells (undifferentiated H9 line) were exposed to 1,066 ToxCast compounds for 72h. We tested a subset of 127 chemicals and 13 sample repeats in 8-point concentration series, and the remaining 939 chemicals in a single concentration screen. Each compound was tested in triplicate with a positive-control plate reference (1 uM methotrexate) and a neutral 0.1% DMSO vehicle, to which final values were normalized. The maximum test concentration was set at 1, 10 or 100 uM based on ToxCast cytotoxicity determinations. Conditioned media from the final 24h treatment period was analyzed using LC-MS based metabolomic analysis to determine the ornithine/cystine (o/c) ratio. Cell viability was assessed using the CellTitre-FluorTM. The chemical library included internal (blinded) methotrexate, which gave o/c ratio and cell number measures similar to the positive-control reference. Preliminary analysis defined actives as an o/c ratio falling at or below 0.88 and viable cell titers at ¬>0.65 control values to indicate developmental toxicity potential. These cutoffs flagged 165 of 1066 compounds (15.5%) showing potential for developmental toxicity in a human system. Results in the concentration series indicate that strong teratogens are easily identified (LECs in parentheses) including trans-retinoic acid (3 nM), warfarin (3 nM), 5-fluorouracil (100 nM), methotrexate (100 nM), thalidomide (300 nM), and carbamazepine (3 uM). Many chemicals not yet classified were predicted positive, including TNP-470 (10 nM), pyridaben (30 nM), cladribine (300 nM), mirex (300 nM), maneb (3 uM), and 5HPP-33 (10 uM) among others. Cross-referencing these results with additional assays in ToxCast and the in vivo studies in ToxRefDB databases provides additional opportunity to profile the performance of this assay in predictive computational models for malformations, developmental delays, fetal weight reduction, resorptions and maternal developmental toxicity. [This abstract does not necessarily reflect US EPA policy]

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