The data presented here support the application of the Stemina devTOXqP platform for predictive toxicology and further demonstrate its value in ToxCast as a novel resource that can generate testable hypotheses aimed at characterizing potential pathways for teratogenicity and HTS prioritization of environmental chemicals for an exposure-based assessment of developmental hazard. The dataset from the Stemina (STM) assay is annotated in the ToxCast portfolio as STM. Major findings from the analysis of ToxCast_STM dataset include (1) 19% of 1065 chemicals yielded a prediction of developmental toxicity, (2) assay performance reached 79%-82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with in vivo animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain. The results of this study will be useful to improving our ability to predict in vivo developmental toxicants based on in vitro data and in silico models. This dataset is associated with the following publication: Zurlinden, T., K. Saili, N. Rush, P. Kothiya, R. Judson, K. Houck, E. Hunter, N. Baker, J. Palmer, R. Thomas, and T. Knudsen. Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 174(2): 189-209, (2020).

Background: High-content imaging (HCI) allows simultaneous measurement of multiple cellular phenotypic changes and is an important tool for evaluating the biological activity of chemicals. Objectives: Our goal was to analyze dynamic cellular changes using HCI to identify the “tipping point” at which the cells did not show recovery towards a normal phenotypic state. Methods: HCI was used to evaluate the effects of 967 chemicals (in concentrations ranging from 0.4 to 200 μM) on HepG2 cells over a 72-hr exposure period. The HCI end points included p53, c-Jun, histone H2A.x, α-tubulin, histone H3, alpha tubulin, mitochondrial membrane potential, mitochondrial mass, cell cycle arrest, nuclear size, and cell number. A computational model was developed to interpret HCI responses as cell-state trajectories. Results: Analysis of cell-state trajectories showed that 336 chemicals produced tipping points and that HepG2 cells were resilient to the effects of 334 chemicals up to the highest concentration (200 μM) and duration (72 hr) tested. Tipping points were identified as concentration-dependent transitions in system recovery, and the corresponding critical concentrations were generally between 5 and 15 times (25th and 75th percentiles, respectively) lower than the concentration that produced any significant effect on HepG2 cells. The remaining 297 chemicals require more data before they can be placed in either of these categories. Conclusions: These findings show the utility of HCI data for reconstructing cell state trajectories and provide insight into the adaptation and resilience of in vitro cellular systems based on tipping points. Cellular tipping points could be used to define a point of departure for risk-based prioritization of environmental chemicals. This dataset is associated with the following publication: Shah , I., W. Setzer , J. Jack, K. Houck , R. Judson , T. Knudsen , J. Liu, M. Martin , D. Reif, A.M. Richard , R.S. Thomas , K. Crofton , D.J. Dix , and R.J. Kavlock. (Envir. Health Perspect.) Using ToxCast data to reconstruct dynamic cell state trajectories and estimate toxicological points of departure. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA, 1-33, (2015).

Background: High-content imaging (HCI) allows simultaneous measurement of multiple cellular phenotypic changes and is an important tool for evaluating the biological activity of chemicals. Objectives: Our goal was to analyze dynamic cellular changes using HCI to identify the “tipping point” at which the cells did not show recovery towards a normal phenotypic state. Methods: HCI was used to evaluate the effects of 967 chemicals (in concentrations ranging from 0.4 to 200 μM) on HepG2 cells over a 72-hr exposure period. The HCI end points included p53, c-Jun, histone H2A.x, α-tubulin, histone H3, alpha tubulin, mitochondrial membrane potential, mitochondrial mass, cell cycle arrest, nuclear size, and cell number. A computational model was developed to interpret HCI responses as cell-state trajectories. Results: Analysis of cell-state trajectories showed that 336 chemicals produced tipping points and that HepG2 cells were resilient to the effects of 334 chemicals up to the highest concentration (200 μM) and duration (72 hr) tested. Tipping points were identified as concentration-dependent transitions in system recovery, and the corresponding critical concentrations were generally between 5 and 15 times (25th and 75th percentiles, respectively) lower than the concentration that produced any significant effect on HepG2 cells. The remaining 297 chemicals require more data before they can be placed in either of these categories. Conclusions: These findings show the utility of HCI data for reconstructing cell state trajectories and provide insight into the adaptation and resilience of in vitro cellular systems based on tipping points. Cellular tipping points could be used to define a point of departure for risk-based prioritization of environmental chemicals. This dataset is associated with the following publication: Shah , I., W. Setzer , J. Jack, K. Houck , R. Judson , T. Knudsen , J. Liu, M. Martin , D. Reif, A.M. Richard , R.S. Thomas , K. Crofton , D.J. Dix , and R.J. Kavlock. (Envir. Health Perspect.) Using ToxCast data to reconstruct dynamic cell state trajectories and estimate toxicological points of departure. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA, 1-33, (2015).

We differentiated human induced pluripotent stem cells (hiPSCs) to embryonic endoderm and sought to identify a tipping point at which the developing system did not recover from perturbations caused by exposure to a known teratogen, all-trans retinoic acid (ATRA). Differentiating iPSC-derived endoderm was exposed to five concentrations of ATRA between 0.001 and 10 µM at 6h, 96h, or 192h and assessed for forkhead box A2 (FOXA2) protein expression and global gene transcript expression measured by RNA-sequencing. A tipping point of 17±11 nM was identified where patterns of differentially expressed genes supported a shift in the developmental trajectory away from embryonic endoderm in favor of mesoderm and extraembryonic endoderm. Five concentrations of all-trans retinoic acid (ATRA) between 0.001 and 10 µM were compared to time-matched 0.1% DMSO controls at three timepoints (6h, 96h, and 192h) in differentiating endoderm. Two biological replicates were used. Undifferentiated controls (not in DMSO) were also included in duplicate as internal controls for 6h, 96h, and 144h. This dataset is associated with the following publication: Saili, K., T. Antonijevic, T. Zurlinden, I. Shah, C. Deisenroth, and T. Knudsen. Molecular characterization of a toxicological tipping point during human stem cell differentiation. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 91(January 2020): 1-13, (2020).

httk: High-Throughput Toxicokinetics Functions and data tables for simulation and statistical analysis of chemical toxicokinetics ("TK") using data obtained from relatively high throughput, in vitro studies. Both physiologically-based ("PBTK") and empirical (e.g., one compartment) "TK" models can be parameterized for several hundred chemicals and multiple species. These models are solved efficiently, often using compiled (C-based) code. A Monte Carlo sampler is included for simulating biological variability and measurement limitations. Functions are also provided for exporting "PBTK" models to "SBML" and "JARNAC" for use with other simulation software. These functions and data provide a set of tools for in vitro-in vivo extrapolation ("IVIVE") of high throughput screening data (e.g., ToxCast) to real-world exposures via reverse dosimetry (also known as "RTK"). This dataset is associated with the following publication: Pearce, R., W. Setzer, J. Davis, and J. Wambaugh. Evaluation and Calibration of High-Throughput Predictions of Chemical Distribution to Tissues. JOURNAL OF PHARMACOKINETICS AND PHARMACODYNAMICS. Springer, New York, NY, USA, 44(6): 549-565, (2017).

httk: High-Throughput Toxicokinetics Functions and data tables for simulation and statistical analysis of chemical toxicokinetics ("TK") using data obtained from relatively high throughput, in vitro studies. Both physiologically-based ("PBTK") and empirical (e.g., one compartment) "TK" models can be parameterized for several hundred chemicals and multiple species. These models are solved efficiently, often using compiled (C-based) code. A Monte Carlo sampler is included for simulating biological variability and measurement limitations. Functions are also provided for exporting "PBTK" models to "SBML" and "JARNAC" for use with other simulation software. These functions and data provide a set of tools for in vitro-in vivo extrapolation ("IVIVE") of high throughput screening data (e.g., ToxCast) to real-world exposures via reverse dosimetry (also known as "RTK"). This dataset is associated with the following publication: Pearce, R., W. Setzer, J. Davis, and J. Wambaugh. Evaluation and Calibration of High-Throughput Predictions of Chemical Distribution to Tissues. JOURNAL OF PHARMACOKINETICS AND PHARMACODYNAMICS. Springer, New York, NY, USA, 44(6): 549-565, (2017).

HT-H295R data was downloaded using the ToxCast pipeline (tcpl) R package and is publicly available. Multi-concentration level 0 data from invitrodb (version 3.1) were downloaded and converted from g/ml into micromolar concentrations prior to calculation of mMds and data simulation (Supplemental Data 1). This dataset is associated with the following publication: Haggard, D., W. Setzer, R. Judson, and K. Friedman. Development of a prioritization method for chemical-mediated effects on steroidogenesis using an integrated statistical analysis of high-throughput H295R data. REGULATORY TOXICOLOGY AND PHARMACOLOGY. Elsevier Science Ltd, New York, NY, USA, 109: 104510, (2019).

HT-H295R data was downloaded using the ToxCast pipeline (tcpl) R package and is publicly available. Multi-concentration level 0 data from invitrodb (version 3.1) were downloaded and converted from g/ml into micromolar concentrations prior to calculation of mMds and data simulation (Supplemental Data 1). This dataset is associated with the following publication: Haggard, D., W. Setzer, R. Judson, and K. Friedman. Development of a prioritization method for chemical-mediated effects on steroidogenesis using an integrated statistical analysis of high-throughput H295R data. REGULATORY TOXICOLOGY AND PHARMACOLOGY. Elsevier Science Ltd, New York, NY, USA, 109: 104510, (2019).

This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. This dataset is not publicly accessible because: no EPA data; all the data generated by external organizations; EPA coauthors. It can be accessed through the following means: Data generated by external organizations. Format: N/A. This dataset is associated with the following publication: Ellis-Hutchings, R., R. Settivari, A. McCoy, N. Kleinstreuer, J. Franzosa, T. Knudsen, and E. Carney. (REPRODUCTIVE TOXICOLOGY) EMBRYONIC VASCULAR DISRUPTION ADVERSE OUTCOMES: LINKING HIGH THROUGHPUT SIGNALING SIGNATURES WITH FUNCTIONAL CONSEQUENCES. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 70: 82-96, (2017).

This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. This dataset is not publicly accessible because: no EPA data; all the data generated by external organizations; EPA coauthors. It can be accessed through the following means: Data generated by external organizations. Format: N/A. This dataset is associated with the following publication: Ellis-Hutchings, R., R. Settivari, A. McCoy, N. Kleinstreuer, J. Franzosa, T. Knudsen, and E. Carney. (REPRODUCTIVE TOXICOLOGY) EMBRYONIC VASCULAR DISRUPTION ADVERSE OUTCOMES: LINKING HIGH THROUGHPUT SIGNALING SIGNATURES WITH FUNCTIONAL CONSEQUENCES. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 70: 82-96, (2017).