Verification of In Vivo Estrogenic Activity for Four Per- and Polyfluoroalkyl Substances (PFAS) Identified as Estrogen Receptor Agonists via New Approach Methodologies.

Given concerns about potential toxicological hazards of the thousands of data-poor per- and polyfluorinated alkyl substances (PFAS) currently in commerce and detected in the environment, tiered testing strategies that employ high-throughput in vitro screening as an initial testing tier have been implemented. The present study evaluated the effectiveness of previous in vitro screening for identifying PFAS capable, or incapable, of inducing estrogenic responses in fish exposed in vivo. Fathead minnows (Pimephales promelas) were exposed for 96 h to five PFAS (perfluorooctanoic acid [PFOA]; 1H,1H,8H,8H-perfluorooctane-1,8-diol [FC8-diol]; 1H,1H,10H,10H-perfluorodecane-1,10-diol [FC10-diol]; 1H,1H,8H,8H-perfluoro-3,6-dioxaoctane-1,8-diol [FC8-DOD]; and perfluoro-2-methyl-3-oxahexanoic acid [HFPO-DA]) that showed varying levels of in vitro estrogenic potency. In agreement with in vitro screening results, exposure to FC8-diol, FC10-diol, and FC8-DOD caused concentration-dependent increases in the expression of transcript coding for vitellogenin and estrogen receptor alpha and reduced expression of insulin-like growth factor and apolipoprotein eb. Once differences in bioconcentration were accounted for, the rank order of potency in vivo matched that determined in vitro. These results provide a screening level benchmark for worst-case estimates of potential estrogenic hazards of PFAS and a basis for identifying structurally similar PFAS to scrutinize for putative estrogenic activity.

Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application.

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

Tox21BodyMap: a webtool to map chemical effects on the human body.

To support rapid chemical toxicity assessment and mechanistic hypothesis generation, here we present an intuitive webtool allowing a user to identify target organs in the human body where a substance is estimated to be more likely to produce effects. This tool, called Tox21BodyMap, incorporates results of 9,270 chemicals tested in the United States federal Tox21 research consortium in 971 high-throughput screening (HTS) assays whose targets were mapped onto human organs using organ-specific gene expression data. Via Tox21BodyMap's interactive tools, users can visualize chemical target specificity by organ system, and implement different filtering criteria by changing gene expression thresholds and activity concentration parameters. Dynamic network representations, data tables, and plots with comprehensive activity summaries across all Tox21 HTS assay targets provide an overall picture of chemical bioactivity. Tox21BodyMap webserver is available at https://sandbox.ntp.niehs.nih.gov/bodymap/.

The Tox21 10K Compound Library: Collaborative Chemistry Advancing Toxicology.

Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 "10K" chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure-activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical-activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

Quantitative Chemical Proteomics Reveals Interspecies Variations on Binding Schemes of L-FABP with Perfluorooctanesulfonate.

Evaluating interspecies toxicity variation is a long-standing challenge for chemical hazard assessment. This study developed a quantitative interspecies thermal shift assay (QITSA) for in situ, quantitative, and modest-throughput investigation of chemical-protein interactions in cell and tissue samples across species. By using liver fatty acid binding protein (L-FABP) as a case study, the QITSA method was benchmarked with six per- and polyfluoroalkyl substances, and thermal shifts (ΔTm) were inversely related to their dissociation constants (R2 = 0.98). The QITSA can also distinguish binding modes of chemicals exemplified by palmitic acid. The QITSA was applied to determine the interactions between perfluorooctanesulfonate (PFOS) and L-FABP in liver cells or tissues from humans, mice, rats, and zebrafish. The largest thermal stability enhancement by PFOS was observed for human L-FABP followed by the mouse, rat, and zebrafish. While endogenous ligands were revealed to partially contribute to the large interspecies variation, recombinant proteins were employed to confirm the high binding affinity of PFOS to human L-FABP, compared to the rat and mouse. This study implemented an experimental strategy to characterize chemical-protein interactions across species, and future application of QITSA to other chemical contaminants is of great interest.

Harmonized Cross-Species Assessment of Endocrine and Metabolic Disruptors by Ecotox FACTORIAL Assay.

Environmental pollution is a threat to humans and wildlife species. Of particular concern are endocrine disrupting chemicals (EDCs). An important target of EDCs is nuclear receptors (NRs) that control endocrine and metabolic responses through transcriptional regulation. Owing in part to structural differences of NRs, adverse effects of EDCs vary significantly among species. Here, we describe a multiplexed reporter assay (the Ecotox FACTORIAL) enabling parallel assessment of compounds' effects on estrogen, androgen, thyroid, and PPARγ receptors of representative mammals, birds, reptiles, amphibians, and fish. The Ecotox FACTORIAL is a single-well assay comprising a set of species-specific, one-hybrid GAL4-NR reporter constructs transiently transfected into test cells. To harmonize cross-species assessments, we used a combination of two approaches. First, we used the same type of test cells for all reporters; second, we implemented a parallel detection of reporter RNAs. The assay demonstrated excellent quality, reproducibility, and insignificant intra-assay variability. Importantly, the EC50 values for NR ligands were consistent with those reported for conventional assays. Using the assay allowed ranking the hazard potential of environmental pollutants (e.g., bisphenols, polycyclic aromatic hydrocarbons, and synthetic progestins) across species. Furthermore, the assay permitted detecting taxa-specific effects of surface water samples. Therefore, the Ecotox FACTORIAL enables harmonized assessment of the endocrine and metabolic disrupting activity of chemicals and surface water in humans as well as in wildlife species.

Nontarget Screening of Per- and Polyfluoroalkyl Substances Binding to Human Liver Fatty Acid Binding Protein.

More than 1000 per- and polyfluoroalkyl substances (PFASs) have been discovered by nontarget analysis (NTA), but their prioritization for health concerns is challenging. We developed a method by incorporating size-exclusion column co-elution (SECC) and NTA, to screen PFASs binding to human liver fatty acid binding protein (hL-FABP). Of 74 PFASs assessed, 20 were identified as hL-FABP ligands in which eight of them have high binding affinities. Increased PFAS binding affinities correlate with stronger responses in electrospray ionization (ESI-) and longer retention times on a C18 column. This is well explained by a mechanistic model, which revealed that both polar and hydrophobic interactions are crucial for binding affinities. Encouraged by this, we then developed an SECC method to identify hL-FABP ligands, and all eight high-affinity ligands were selectively captured from 74 PFASs. The method was further applied to an aqueous film-forming foam (AFFF) product in which 31 new hL-FABP ligands were identified. Suspect and nontargeted screening revealed these ligands as analogues of perfluorosulfonic acids and homologues of alkyl ether sulfates (C8- and C10/EOn, C8H17(C2H4O)nSO4-, and C10H21(C2H4O)nSO4-). The SECC method was then applied to AFFF-contaminated surface waters. In addition to perfluorooctanesulfonic acid and perfluorohexanesulfonic acid, eight other AFFF chemicals were discovered as novel ligands, including four C14- and C15/EOn. This study implemented a high-throughput method to prioritize PFASs and revealed the existence of many previously unknown hL-FABP ligands.

Selecting a minimal set of androgen receptor assays for screening chemicals.

Screening certain environmental chemicals for their ability to interact with endocrine targets, including the androgen receptor (AR), is an important global concern. We previously developed a model using a battery of eleven in vitro AR assays to predict in vivo AR activity. Here we describe a revised mathematical modeling approach that also incorporates data from newly available assays and demonstrate that subsets of assays can provide close to the same level of predictivity. These subset models are evaluated against the full model using 1820 chemicals, as well as in vitro and in vivo reference chemicals from the literature. Agonist batteries of as few as six assays and antagonist batteries of as few as five assays can yield balanced accuracies of 95% or better relative to the full model. Balanced accuracy for predicting reference chemicals is 100%. An approach is outlined for researchers to develop their own subset batteries to accurately detect AR activity using assays that map to the pathway of key molecular and cellular events involved in chemical-mediated AR activation and transcriptional activity. This work indicates in vitro bioactivity and in silico predictions that map to the AR pathway could be used in an integrated approach to testing and assessment for identifying chemicals that interact directly with the mammalian AR.

Potential Toxicity of Complex Mixtures in Surface Waters from a Nationwide Survey of United States Streams: Identifying in Vitro Bioactivities and Causative Chemicals.

While chemical analysis of contaminant mixtures remains an essential component of environmental monitoring, bioactivity-based assessments using in vitro systems increasingly are used in the detection of biological effects. Historically, in vitro assessments focused on a few biological pathways, for example, aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) activities. High-throughput screening (HTS) technologies have greatly increased the number of biological targets and processes that can be rapidly assessed. Here we screened extracts of surface waters from a nationwide survey of United States streams for bioactivities associated with 69 different end points using two multiplexed HTS assays. Bioactivity of extracts from 38 streams was evaluated and compared with concentrations of over 700 analytes to identify chemicals contributing to observed effects. Eleven primary biological end points were detected. Pregnane X receptor (PXR) and AhR-mediated activities were the most commonly detected. Measured chemicals did not completely account for AhR and PXR responses. Surface waters with AhR and PXR effects were associated with low intensity, developed land cover. Likewise, elevated bioactivities frequently associated with wastewater discharges included endocrine-related end points ER and glucocorticoid receptor. These results underscore the value of bioassay-based monitoring of environmental mixtures for detecting biological effects that could not be ascertained solely through chemical analyses.

Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates.

Methods are needed for rapid screening of environmental compounds for neurotoxicity, particularly ones that assess function. To demonstrate the utility of microelectrode array (MEA)-based approaches as a rapid neurotoxicity screening tool, 1055 chemicals from EPA's phase II ToxCast library were evaluated for effects on neural function and cell health. Primary cortical networks were grown on multi-well microelectrode array (mwMEA) plates. On day in vitro 13, baseline activity (40 min) was recorded prior to exposure to each compound (40 µM). Changes in spontaneous network activity [mean firing rate (MFR)] and cell viability (lactate dehydrogenase and CellTiter Blue) were assessed within the same well following compound exposure. Following exposure, 326 compounds altered (increased or decreased) normalized MFR beyond hit thresholds based on 2× the median absolute deviation of DMSO-treated wells. Pharmaceuticals, pesticides, fungicides, chemical intermediates, and herbicides accounted for 86% of the hits. Further, changes in MFR occurred in the absence of cytotoxicity, as only eight compounds decreased cell viability. ToxPrint chemotype analysis identified several structural domains (e.g., biphenyls and alkyl phenols) significantly enriched with MEA actives relative to the total test set. The top 5 enriched ToxPrint chemotypes were represented in 26% of the MEA hits, whereas the top 11 ToxPrints were represented in 34% of MEA hits. These results demonstrate that large-scale functional screening using neural networks on MEAs can fill a critical gap in assessment of neurotoxicity potential in ToxCast assay results. Further, a data-mining approach identified ToxPrint chemotypes enriched in the MEA-hit subset, which define initial structure-activity relationship inferences, establish potential mechanistic associations to other ToxCast assay endpoints, and provide working hypotheses for future studies.

Development and Validation of a Computational Model for Androgen Receptor Activity.

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can more rapidly and inexpensively identify potential androgen-active chemicals. We integrated 11 HTS ToxCast/Tox21 in vitro assays into a computational network model to distinguish true AR pathway activity from technology-specific assay interference. The in vitro HTS assays probed perturbations of the AR pathway at multiple points (receptor binding, coregulator recruitment, gene transcription, and protein production) and multiple cell types. Confirmatory in vitro antagonist assay data and cytotoxicity information were used as additional flags for potential nonspecific activity. Validating such alternative testing strategies requires high-quality reference data. We compiled 158 putative androgen-active and -inactive chemicals from a combination of international test method validation efforts and semiautomated systematic literature reviews. Detailed in vitro assay information and results were compiled into a single database using a standardized ontology. Reference chemical concentrations that activated or inhibited AR pathway activity were identified to establish a range of potencies with reproducible reference chemical results. Comparison with existing Tier 1 AR binding data from the U.S. EPA Endocrine Disruptor Screening Program revealed that the model identified binders at relevant test concentrations (<100 µM) and was more sensitive to antagonist activity. The AR pathway model based on the ToxCast/Tox21 assays had balanced accuracies of 95.2% for agonist (n = 29) and 97.5% for antagonist (n = 28) reference chemicals. Out of 1855 chemicals screened in the AR pathway model, 220 chemicals demonstrated AR agonist or antagonist activity and an additional 174 chemicals were predicted to have potential weak AR pathway activity.

An "EAR" on Environmental Surveillance and Monitoring: A Case Study on the Use of Exposure-Activity Ratios (EARs) to Prioritize Sites, Chemicals, and Bioactivities of Concern in Great Lakes Waters.

Current environmental monitoring approaches focus primarily on chemical occurrence. However, based on concentration alone, it can be difficult to identify which compounds may be of toxicological concern and should be prioritized for further monitoring, in-depth testing, or management. This can be problematic because toxicological characterization is lacking for many emerging contaminants. New sources of high-throughput screening (HTS) data, such as the ToxCast database, which contains information for over 9000 compounds screened through up to 1100 bioassays, are now available. Integrated analysis of chemical occurrence data with HTS data offers new opportunities to prioritize chemicals, sites, or biological effects for further investigation based on concentrations detected in the environment linked to relative potencies in pathway-based bioassays. As a case study, chemical occurrence data from a 2012 study in the Great Lakes Basin along with the ToxCast effects database were used to calculate exposure-activity ratios (EARs) as a prioritization tool. Technical considerations of data processing and use of the ToxCast database are presented and discussed. EAR prioritization identified multiple sites, biological pathways, and chemicals that warrant further investigation. Prioritized bioactivities from the EAR analysis were linked to discrete adverse outcome pathways to identify potential adverse outcomes and biomarkers for use in subsequent monitoring efforts.

On selecting a minimal set of in vitro assays to reliably determine estrogen agonist activity.

The US EPA is charged with screening chemicals for their ability to be endocrine disruptors through interaction with the estrogen, androgen and thyroid axes. The agency is exploring the use of high-throughput in vitro assays to use in the Endocrine Disruptor Screening Program (EDSP), potentially as replacements for lower-throughput in vitro and in vivo tests. The first replacement is an integrated computational and experimental model for estrogen receptor (ER) activity, to be used as an alternative to the EDSP Tier 1 in vitro ER binding and transactivation assays and the in vivo uterotrophic bioassay. The ER agonist model uses a set of 16 in vitro assays that incorporate multiple technologies and cell lines and probe multiple points in the ER pathway. Here, we demonstrate that subsets of assays with as few as 4 assays can predict the activity of all 1811 chemicals tested with accuracy equivalent to that of the full 16-assay model. The prediction accuracy against reference chemicals is higher than that of the full chemical set, partly because the larger set contains many chemicals that can cause a variety of types of assay interference There are multiple accurate assay subsets, allowing flexibility in the construction of a multiplexed assay battery. We also discuss the issue of challenging chemicals, i.e. those that can give false positive results in certain assays, and could hence be more problematic when only a few assays are used.

ToxCast Chemical Landscape: Paving the Road to 21st Century Toxicology.

The U.S. Environmental Protection Agency's (EPA) ToxCast program is testing a large library of Agency-relevant chemicals using in vitro high-throughput screening (HTS) approaches to support the development of improved toxicity prediction models. Launched in 2007, Phase I of the program screened 310 chemicals, mostly pesticides, across hundreds of ToxCast assay end points. In Phase II, the ToxCast library was expanded to 1878 chemicals, culminating in the public release of screening data at the end of 2013. Subsequent expansion in Phase III has resulted in more than 3800 chemicals actively undergoing ToxCast screening, 96% of which are also being screened in the multi-Agency Tox21 project. The chemical library unpinning these efforts plays a central role in defining the scope and potential application of ToxCast HTS results. The history of the phased construction of EPA's ToxCast library is reviewed, followed by a survey of the library contents from several different vantage points. CAS Registry Numbers are used to assess ToxCast library coverage of important toxicity, regulatory, and exposure inventories. Structure-based representations of ToxCast chemicals are then used to compute physicochemical properties, substructural features, and structural alerts for toxicity and biotransformation. Cheminformatics approaches using these varied representations are applied to defining the boundaries of HTS testability, evaluating chemical diversity, and comparing the ToxCast library to potential target application inventories, such as used in EPA's Endocrine Disruption Screening Program (EDSP). Through several examples, the ToxCast chemical library is demonstrated to provide comprehensive coverage of the knowledge domains and target inventories of potential interest to EPA. Furthermore, the varied representations and approaches presented here define local chemistry domains potentially worthy of further investigation (e.g., not currently covered in the testing library or defined by toxicity "alerts") to strategically support data mining and predictive toxicology modeling moving forward.

Development of a quantitative morphological assessment of toxicant-treated zebrafish larvae using brightfield imaging and high-content analysis.

One of the rate-limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time-consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high-content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V(TI) high-content image analysis platform. The basic visual assessment adds in-life parameters, and the high-content analysis assesses each individual larva for various features (total area, width, spine length, head-tail length, length-width ratio, perimeter-area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high-content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans-retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd.

Predictive endocrine testing in the 21st century using in vitro assays of estrogen receptor signaling responses.

Thousands of environmental chemicals are subject to regulatory review for their potential to be endocrine disruptors (ED). In vitro high-throughput screening (HTS) assays have emerged as a potential tool for prioritizing chemicals for ED-related whole-animal tests. In this study, 1814 chemicals including pesticide active and inert ingredients, industrial chemicals, food additives, and pharmaceuticals were evaluated in a panel of 13 in vitro HTS assays. The panel of in vitro assays interrogated multiple end points related to estrogen receptor (ER) signaling, namely binding, agonist, antagonist, and cell growth responses. The results from the in vitro assays were used to create an ER Interaction Score. For 36 reference chemicals, an ER Interaction Score >0 showed 100% sensitivity and 87.5% specificity for classifying potential ER activity. The magnitude of the ER Interaction Score was significantly related to the potency classification of the reference chemicals (p < 0.0001). ERα/ERß selectivity was also evaluated, but relatively few chemicals showed significant selectivity for a specific isoform. When applied to a broader set of chemicals with in vivo uterotrophic data, the ER Interaction Scores showed 91% sensitivity and 65% specificity. Overall, this study provides a novel method for combining in vitro concentration response data from multiple assays and, when applied to a large set of ER data, accurately predicted estrogenic responses and demonstrated its utility for chemical prioritization.

Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays.

Understanding potential health risks is a significant challenge due to the large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes 976 chemicals (including failed pharmaceuticals, alternative plasticizers, food additives, and pesticides) in Phases I and II of the U.S. EPA's ToxCast project across 331 cell-free enzymatic and ligand-binding high-throughput screening (HTS) assays. Half-maximal activity concentrations (AC50) were identified for 729 chemicals in 256 assays (7,135 chemical-assay pairs). Some of the most commonly affected assays were CYPs (CYP2C9 and CYP2C19), transporters (mitochondrial TSPO, norepinephrine, and dopaminergic), and GPCRs (aminergic). Heavy metals, surfactants, and dithiocarbamate fungicides showed promiscuous but distinctly different patterns of activity, whereas many of the pharmaceutical compounds showed promiscuous activity across GPCRs. Literature analysis confirmed >50% of the activities for the most potent chemical-assay pairs (54) but also revealed 10 missed interactions. Twenty-two chemicals with known estrogenic activity were correctly identified for the majority (77%), missing only the weaker interactions. In many cases, novel findings for previously unreported chemical-target combinations clustered with known chemical-target interactions. Results from this large inventory of chemical-biological interactions can inform read-across methods as well as link potential targets to molecular initiating events in adverse outcome pathways for diverse toxicities.

Real-time growth kinetics measuring hormone mimicry for ToxCast chemicals in T-47D human ductal carcinoma cells.

High-throughput screening (HTS) assays capable of profiling thousands of environmentally relevant chemicals for in vitro biological activity provide useful information on the potential for disrupting endocrine pathways. Disruption of the estrogen signaling pathway has been implicated in a variety of adverse health effects including impaired development, reproduction, and carcinogenesis. The estrogen-responsive human mammary ductal carcinoma cell line T-47D was exposed to 1815 ToxCast chemicals comprising pesticides, industrial chemicals, pharmaceuticals, personal care products, cosmetics, food ingredients, and other chemicals with known or suspected human exposure potential. Cell growth kinetics were evaluated using real-time cell electronic sensing. T-47D cells were exposed to eight concentrations (0.006-100 µM), and measurements of cellular impedance were repeatedly recorded for 105 h. Chemical effects were evaluated based on potency (concentration at which response occurs) and efficacy (extent of response). A linear growth response was observed in response to prototypical estrogen receptor agonists (17ß-estradiol, genistein, bisphenol A, nonylphenol, and 4-tert-octylphenol). Several compounds, including bisphenol A and genistein, induced cell growth comparable in efficacy to that of 17ß-estradiol, but with decreased potency. Progestins, androgens, and corticosteroids invoked a biphasic growth response indicative of changes in cell number or cell morphology. Results from this cell growth assay were compared with results from additional estrogen receptor (ER) binding and transactivation assays. Chemicals detected as active in both the cell growth and ER receptor binding assays demonstrated potencies highly correlated with two ER transactivation assays (r = 0.72; r = 0.70). While ER binding assays detected chemicals that were highly potent or efficacious in the T-47D cell growth and transactivation assays, the binding assays lacked sensitivity in detecting weakly active compounds. In conclusion, this cell-based assay rapidly detects chemical effects on T-47D growth and shows potential, in combination with other HTS assays, to detect environmentally relevant chemicals with potential estrogenic activity.