U.S. Environmental Protection Agency Office of Research and Development EPA www.epa.gov EVALUATION OF 1066 TOXCAST CHEMICALS IN A HUMAN STEM CELL ASSAY FOR DEVELOPMENTAL TOXICITY TB Knudsen1, AM Richard1, PG Kothiya1, RS Judson1, KA Houck1, KC Crofton1, RS Thomas1, S Little1, ES Hunter2, NC Baker3, CKM Leung1,4, JA Palmer5, AM Smith5, MR Colwell5, PR West5, RE Burrier5, LA Egnash5 1. National Center Computational Toxicology, US EPA, Research Triangle Park, NC 27711 (RTP), 2. NHEERL, US EPA-RTP, 3. Lockheed Martin-RTP, 4. ORISE-RTP, 5. Stemina Biomarker Discovery, Inc., Madison, WI 53719. A. INTRODUCTION B. METHODS C. METRICS OF ASSAY QUALITY Thomas B. Knudsen l knudsen.thomas@epa.gov l 919-541-9776 EPA’s ToxCast program has generated data on a battery of 821 in vitro endpoints for 1066 compounds including pharmaceuticals, natural products, pesticidal active ingredients, consumer use chemicals and industrial ingredients [1]. To increase the diversity of in vitro assays used to assess developmental toxicity, the ToxCast library was evaluated in the Stemina ‘devTOX quickPREDICT’ (qP) platform [2]. This assay measures two small molecules (ornithine, cystine) in medium conditioned by human embryonic stem (hES) cells yielding an ornithine:cystine ratio (o/c ratio) indicative of an imbalance in metabolism predictive for teratogenicity in a human system. Here, we provide a preliminary evaluation of the results focusing on metrics of assay quality, performance, and predictivity. Platform: Metabolomic analysis of the hES cell secretome for predictive developmental toxicity (devTOX platform) was reported in 2010 [3]. A 2011 pilot study conducted with 11 ToxCast chemicals predicted developmental toxicity in concordance with animal data with 83% accuracy [4]. In 2013, the Stemina ‘devTOX-qP’ platform was developed as a high throughput screening (HTS) assay for developmental toxicity testing [2]. The model was trained with 23 pharmaceuticals (96% accurate). An independent 13 pharmaceutical test set with known (human) teratogenicity was 77% accurate. Dosing: H9 cells (WA09 line, WiCell Research Institute) were cultured in 96-well plates. Each experimental plate included methotrexate (MTX) reference controls as calibration standards for negative- (5 nM) and positive- (1 uM) response as well as media blanks and 0.1% DMSO vehicle. Undifferentiated cells were exposed for 72h to test compound (blinded and in triplicate) with media and test compound replacement every 24h; maximum test concentration (MTC) for single concentration screen and/or 8-point conc. series set at 1-, 10- or 100-uM based on ToxCast cytotoxicity burst (TC-Cyto-Burst) [1] or compound availability. Evaluation: Cell-conditioned media from the final 24h treatment period was analyzed by LC- MS to determine ornithine/cystine (o/c) ratio. Concurrent cell viability was assessed with the CellTiter-FluorTM assay (Promega). The cytotoxicity Relative Fluorescence Unit (RFU) was background corrected and normalized to mean RFU of the neutral control (0.1% DMSO). Teratogen Index (TI) was defined by the o/c ratio, using the default threshold value < 0.88 and concurrent cell viability (RFU values for test compound relative to DMSO control). D. METRICS OF ASSAY PERFORMANCE and PREDICTIVITY LEFT • DMSO control (n=415) • MTX-negative reference (5 nM, n=208) • MTX-positive reference (1 uM, n=207, 210) • MTX-sample in ToxCast (3 nM, 1 uM, n=1) RIGHT • MTX-sample crosses TI threshold at 0.38 uM • ToxCast cytotox burst for MTX = 0.69 uM Quality Standards. Methotrexate (MTX) in the ToxCast library (blinded) gave ornithine/cystine (o/c ratio) and cell viability (cv) measures identical to the calibration standards. DISCLAIMER: The views expressed in this article are those of the authors and do not necessarily reflect the views of policies of the USEPA. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Replicate Samples. Concentration (8-point) resp