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.

Accelerating the Pace of Chemical Risk Assessment.

Changes in chemical regulations worldwide have increased the demand for new data on chemical safety. New approach methodologies (NAMs) are defined broadly here as including in silico approaches and in chemico and in vitro assays, as well as the inclusion of information from the exposure of chemicals in the context of hazard [European Chemicals Agency, " New Approach Methodologies in Regulatory Science ", 2016]. NAMs for toxicity testing, including alternatives to animal testing approaches, have shown promise to provide a large amount of data to fill information gaps in both hazard and exposure. In order to increase experience with the new data and to advance the applications of NAM data to evaluate the safety of data-poor chemicals, demonstration case studies have to be developed to build confidence in their usability. Case studies can be used to explore the domains of applicability of the NAM data and identify areas that would benefit from further research, development, and application. To ensure that this science evolves with direct input from and engagement by risk managers and regulatory decision makers, a workshop was convened among senior leaders from international regulatory agencies to identify common barriers for using NAMs and to propose next steps to address them. Central to the workshop were a series of collaborative case studies designed to explore areas where the benefits of NAM data could be demonstrated. These included use of in vitro bioassays data in combination with exposure estimates to derive a quantitative assessment of risk, use of NAMs for updating chemical categorizations, and use of NAMs to increase understanding of exposure and human health toxicity of various chemicals. The case study approach proved effective in building collaborations and engagement with regulatory decision makers and to promote the importance of data and knowledge sharing among international regulatory agencies. The case studies will be continued to explore new ways of describing hazard (i.e., pathway perturbations as a measure of adversity) and new ways of describing risk (i.e., using NAMs to identify protective levels without necessarily being predictive of a specific hazard). Importantly, the case studies also highlighted the need for increased training and communication across the various communities including the risk assessors, regulators, stakeholders (e.g., industry, non-governmental organizations), and the general public. The development and application of NAMs will play an increasing role in filling important data gaps on the safety of chemicals, but confidence in NAMs will only come with learning by doing and sharing in the experience.

Chemical Risk Assessment: Traditional vs Public Health Perspectives.

Preventing adverse health effects of environmental chemical exposure is fundamental to protecting individual and public health. When done efficiently and properly, chemical risk assessment enables risk management actions that minimize the incidence and effects of environmentally induced diseases related to chemical exposure. However, traditional chemical risk assessment is faced with multiple challenges with respect to predicting and preventing disease in human populations, and epidemiological studies increasingly report observations of adverse health effects at exposure levels predicted from animal studies to be safe for humans. This discordance reinforces concerns about the adequacy of contemporary risk assessment practices for protecting public health. It is becoming clear that to protect public health more effectively, future risk assessments will need to use the full range of available data, draw on innovative methods to integrate diverse data streams, and consider health endpoints that also reflect the range of subtle effects and morbidities observed in human populations. Considering these factors, there is a need to reframe chemical risk assessment to be more clearly aligned with the public health goal of minimizing environmental exposures associated with disease.

Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis.

BACKGROUND: A recent review by the International Agency for Research on Cancer (IARC) updated the assessments of the > 100 agents classified as Group 1, carcinogenic to humans (IARC Monographs Volume 100, parts A-F). This exercise was complicated by the absence of a broadly accepted, systematic method for evaluating mechanistic data to support conclusions regarding human hazard from exposure to carcinogens. OBJECTIVES AND METHODS: IARC therefore convened two workshops in which an international Working Group of experts identified 10 key characteristics, one or more of which are commonly exhibited by established human carcinogens. DISCUSSION: These characteristics provide the basis for an objective approach to identifying and organizing results from pertinent mechanistic studies. The 10 characteristics are the abilities of an agent to 1) act as an electrophile either directly or after metabolic activation; 2) be genotoxic; 3) alter DNA repair or cause genomic instability; 4) induce epigenetic alterations; 5) induce oxidative stress; 6) induce chronic inflammation; 7) be immunosuppressive; 8) modulate receptor-mediated effects; 9) cause immortalization; and 10) alter cell proliferation, cell death, or nutrient supply. CONCLUSION: We describe the use of the 10 key characteristics to conduct a systematic literature search focused on relevant end points and construct a graphical representation of the identified mechanistic information. Next, we use benzene and polychlorinated biphenyls as examples to illustrate how this approach may work in practice. The approach described is similar in many respects to those currently being implemented by the U.S. EPA's Integrated Risk Information System Program and the U.S. National Toxicology Program. CITATION: Smith MT, Guyton KZ, Gibbons CF, Fritz JM, Portier CJ, Rusyn I, DeMarini DM, Caldwell JC, Kavlock RJ, Lambert P, Hecht SS, Bucher JR, Stewart BW, Baan R, Cogliano VJ, Straif K. 2016. Key characteristics of carcinogens as a basis for organizing data on mechanisms of carcinogenesis. Environ Health Perspect 124:713-721; http://dx.doi.org/10.1289/ehp.1509912.

Using ToxCast™ Data to Reconstruct Dynamic Cell State Trajectories and Estimate Toxicological Points of Departure.

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. CITATION: Shah I, Setzer RW, Jack J, Houck KA, Judson RS, Knudsen TB, Liu J, Martin MT, Reif DM, Richard AM, Thomas RS, Crofton KM, Dix DJ, Kavlock RJ. 2016. Using ToxCast™ data to reconstruct dynamic cell state trajectories and estimate toxicological points of departure. Environ Health Perspect 124:910-919; http://dx.doi.org/10.1289/ehp.1409029.

Lessons from Toxicology: Developing a 21st-Century Paradigm for Medical Research.

Biomedical developments in the 21st century provide an unprecedented opportunity to gain a dynamic systems-level and human-specific understanding of the causes and pathophysiologies of disease. This understanding is a vital need, in view of continuing failures in health research, drug discovery, and clinical translation. The full potential of advanced approaches may not be achieved within a 20th-century conceptual framework dominated by animal models. Novel technologies are being integrated into environmental health research and are also applicable to disease research, but these advances need a new medical research and drug discovery paradigm to gain maximal benefits. We suggest a new conceptual framework that repurposes the 21st-century transition underway in toxicology. Human disease should be conceived as resulting from integrated extrinsic and intrinsic causes, with research focused on modern human-specific models to understand disease pathways at multiple biological levels that are analogous to adverse outcome pathways in toxicology. Systems biology tools should be used to integrate and interpret data about disease causation and pathophysiology. Such an approach promises progress in overcoming the current roadblocks to understanding human disease and successful drug discovery and translation. A discourse should begin now to identify and consider the many challenges and questions that need to be solved.

Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway.

The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα ß-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ERα agonists and antagonists was evaluated using a set of 39 reference compounds with known ERα activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ERα binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ERα active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ERα signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.

Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms.

Addressing the safety aspects of drugs and environmental chemicals has historically been undertaken through animal testing. However, the quantity of chemicals in need of assessment and the challenges of species extrapolation require the development of alternative approaches. Our approach, the US Environmental Protection Agency's ToxCast program, utilizes a large suite of in vitro and model organism assays to interrogate important chemical libraries and computationally analyze bioactivity profiles. Here we evaluated one component of the ToxCast program, the use of primary human cell systems, by screening for chemicals that disrupt physiologically important pathways. Chemical-response signatures for 87 endpoints covering molecular functions relevant to toxic and therapeutic pathways were generated in eight cell systems for 641 environmental chemicals and 135 reference pharmaceuticals and failed drugs. Computational clustering of the profiling data provided insights into the polypharmacology and potential off-target effects for many chemicals that have limited or no toxicity information. The endpoints measured can be closely linked to in vivo outcomes, such as the upregulation of tissue factor in endothelial cell systems by compounds linked to the risk of thrombosis in vivo. Our results demonstrate that assaying complex biological pathways in primary human cells can identify potential chemical targets, toxicological liabilities and mechanisms useful for elucidating adverse outcome pathways.

Systems toxicology: from basic research to risk assessment.

Systems Toxicology is the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. Society demands increasingly close scrutiny of the potential health risks associated with exposure to chemicals present in our everyday life, leading to an increasing need for more predictive and accurate risk-assessment approaches. Developing such approaches requires a detailed mechanistic understanding of the ways in which xenobiotic substances perturb biological systems and lead to adverse outcomes. Thus, Systems Toxicology approaches offer modern strategies for gaining such mechanistic knowledge by combining advanced analytical and computational tools. Furthermore, Systems Toxicology is a means for the identification and application of biomarkers for improved safety assessments. In Systems Toxicology, quantitative systems-wide molecular changes in the context of an exposure are measured, and a causal chain of molecular events linking exposures with adverse outcomes (i.e., functional and apical end points) is deciphered. Mathematical models are then built to describe these processes in a quantitative manner. The integrated data analysis leads to the identification of how biological networks are perturbed by the exposure and enables the development of predictive mathematical models of toxicological processes. This perspective integrates current knowledge regarding bioanalytical approaches, computational analysis, and the potential for improved risk assessment.

The Tox21 robotic platform for the assessment of environmental chemicals--from vision to reality.

Since its establishment in 2008, the US Tox21 inter-agency collaboration has made great progress in developing and evaluating cellular models for the evaluation of environmental chemicals as a proof of principle. Currently, the program has entered its production phase (Tox21 Phase II) focusing initially on the areas of modulation of nuclear receptors and stress response pathways. During Tox21 Phase II, the set of chemicals to be tested has been expanded to nearly 10,000 (10K) compounds and a fully automated screening platform has been implemented. The Tox21 robotic system combined with informatics efforts is capable of screening and profiling the collection of 10K environmental chemicals in triplicate in a week. In this article, we describe the Tox21 screening process, compound library preparation, data processing, and robotic system validation.

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.

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.

Improving the human hazard characterization of chemicals: a Tox21 update.

BACKGROUND: In 2008, the National Institute of Environmental Health Sciences/National Toxicology Program, the U.S. Environmental Protection Agency's National Center for Computational Toxicology, and the National Human Genome Research Institute/National Institutes of Health Chemical Genomics Center entered into an agreement on "high throughput screening, toxicity pathway profiling, and biological interpretation of findings." In 2010, the U.S. Food and Drug Administration (FDA) joined the collaboration, known informally as Tox21. OBJECTIVES: The Tox21 partners agreed to develop a vision and devise an implementation strategy to shift the assessment of chemical hazards away from traditional experimental animal toxicology studies to one based on target-specific, mechanism-based, biological observations largely obtained using in vitro assays. DISCUSSION: Here we outline the efforts of the Tox21 partners up to the time the FDA joined the collaboration, describe the approaches taken to develop the science and technologies that are currently being used, assess the current status, and identify problems that could impede further progress as well as suggest approaches to address those problems. CONCLUSION: Tox21 faces some very difficult issues. However, we are making progress in integrating data from diverse technologies and end points into what is effectively a systems-biology approach to toxicology. This can be accomplished only when comprehensive knowledge is obtained with broad coverage of chemical and biological/toxicological space. The efforts thus far reflect the initial stage of an exceedingly complicated program, one that will likely take decades to fully achieve its goals. However, even at this stage, the information obtained has attracted the attention of the international scientific community, and we believe these efforts foretell the future of toxicology.

In vitro perturbations of targets in cancer hallmark processes predict rodent chemical carcinogenesis.

Thousands of untested chemicals in the environment require efficient characterization of carcinogenic potential in humans. A proposed solution is rapid testing of chemicals using in vitro high-throughput screening (HTS) assays for targets in pathways linked to disease processes to build models for priority setting and further testing. We describe a model for predicting rodent carcinogenicity based on HTS data from 292 chemicals tested in 672 assays mapping to 455 genes. All data come from the EPA ToxCast project. The model was trained on a subset of 232 chemicals with in vivo rodent carcinogenicity data in the Toxicity Reference Database (ToxRefDB). Individual HTS assays strongly associated with rodent cancers in ToxRefDB were linked to genes, pathways, and hallmark processes documented to be involved in tumor biology and cancer progression. Rodent liver cancer endpoints were linked to well-documented pathways such as peroxisome proliferator-activated receptor signaling and TP53 and novel targets such as PDE5A and PLAUR. Cancer hallmark genes associated with rodent thyroid tumors were found to be linked to human thyroid tumors and autoimmune thyroid disease. A model was developed in which these genes/pathways function as hypothetical enhancers or promoters of rat thyroid tumors, acting secondary to the key initiating event of thyroid hormone disruption. A simple scoring function was generated to identify chemicals with significant in vitro evidence that was predictive of in vivo carcinogenicity in different rat tissues and organs. This scoring function was applied to an external test set of 33 compounds with carcinogenicity classifications from the EPA's Office of Pesticide Programs and successfully (p = 0.024) differentiated between chemicals classified as "possible"/"probable"/"likely" carcinogens and those designated as "not likely" or with "evidence of noncarcinogenicity." This model represents a chemical carcinogenicity prioritization tool supporting targeted testing and functional validation of cancer pathways.

Economic benefits of using adaptive predictive models of reproductive toxicity in the context of a tiered testing program.

A predictive model of reproductive toxicity, as observed in rat multigeneration reproductive (MGR) studies, was previously developed using high throughput screening (HTS) data from 36 in vitro assays mapped to 8 genes or gene-sets from Phase I of USEPA ToxCast research program, the proof-of-concept phase in which 309 toxicologically well characterized chemicals were testing in over 500 HTS assays. The model predicted the effects on male and female reproductive function with a balanced accuracy of 80%. In a theoretical examination of the potential impact of the model, two case studies were derived representing different tiered testing scenarios to: 1) screen-out chemicals with low predicted probability of effect; and 2) screen-in chemicals with a high probability of causing adverse reproductive effects. We define 'testing cost efficiency' as the total cost divided by the number of positive chemicals expected in the definitive guideline toxicity study. This would approach $2.11 M under the current practice. Under case study 1, 22% of the chemicals were screened-out due to low predicted probability of adverse reproductive effect and a misclassification rate of 12%, yielding a test cost efficiency of $1.87 M. Under case study 2, 13% of chemicals were screened-in yielding a testing cost efficiency of $1.13 M per test-positive chemical. Applying the model would also double the total number of positives identified. It should be noted that the intention of the case studies is not to provide a definitive mechanism for screening-in or screening-out chemicals or account for the indirect costs of misclassification. The case studies demonstrate the customizability of the model as a tool in chemical testing decision-making. The predictive model of reproductive toxicity will continue to evolve as new assays become available to fill recognized biological gaps and will be combined with other predictive models, particularly models of developmental toxicity, to form an initial tier to an overarching integrated testing strategy.

Integration of dosimetry, exposure, and high-throughput screening data in chemical toxicity assessment.

High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, clearance, and exposure. Hepatic metabolic clearance and plasma protein binding were experimentally measured for 239 ToxCast Phase I chemicals. The experimental data were used in a population-based in vitro-to-in vivo extrapolation model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to in vitro AC(50) (concentration at 50% of maximum activity) or lowest effective concentration values across more than 500 in vitro assays. The estimated steady-state oral equivalent doses associated with the in vitro assays were compared with chronic aggregate human oral exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. A total of 18 (9.9%) chemicals for which human oral exposure estimates were available had oral equivalent doses at levels equal to or less than the highest estimated U.S. population exposures. Ranking the chemicals by nominal assay concentrations would have resulted in different chemicals being prioritized. The in vitro assay endpoints with oral equivalent doses lower than the human exposure estimates included cell growth kinetics, cytokine and cytochrome P450 expression, and cytochrome P450 inhibition. The incorporation of dosimetry and exposure provide necessary context for interpretation of in vitro toxicity screening data and are important considerations in determining chemical testing priorities.

Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data.

Environmental Protection Agency's ToxCast project is profiling the in vitro bioactivity of chemicals to assess pathway-level and cell-based signatures that correlate with observed in vivo toxicity. We hypothesized that developmental toxicity in guideline animal studies captured in the ToxRefDB database would correlate with cell-based and cell-free in vitro high-throughput screening (HTS) data to reveal meaningful mechanistic relationships and provide models identifying chemicals with the potential to cause developmental toxicity. To test this hypothesis, we built statistical associations based on HTS and in vivo developmental toxicity data from ToxRefDB. Univariate associations were used to filter HTS assays based on statistical correlation with distinct in vivo endpoint. This revealed 423 total associations with distinctly different patterns for rat (301 associations) and rabbit (122 associations) across multiple HTS assay platforms. From these associations, linear discriminant analysis with cross-validation was used to build the models. Species-specific models of predicted developmental toxicity revealed strong balanced accuracy (> 70%) and unique correlations between assay targets such as transforming growth factor beta, retinoic acid receptor, and G-protein-coupled receptor signaling in the rat and inflammatory signals, such as interleukins (IL) (IL1a and IL8) and chemokines (CCL2), in the rabbit. Species-specific toxicity endpoints were associated with one another through common Gene Ontology biological processes, such as cleft palate to urogenital defects through placenta and embryonic development. This work indicates the utility of HTS assays for developing pathway-level models predictive of developmental toxicity.

Developmental toxicity testing for safety assessment: new approaches and technologies.

The ILSI Health and Environmental Sciences Institute's Developmental and Reproductive Toxicology Technical Committee held a 2-day workshop entitled "Developmental Toxicology-New Directions" in April 2009. The fourth session of this workshop focused on new approaches and technologies for the assessment of developmental toxicology. This session provided an overview of the application of genomics technologies for developmental safety assessment, the use of mouse embryonic stem cells to capture data on developmental toxicity pathways, dynamical cell imaging of zebrafish embryos, the use of computation models of development pathways and systems, and finally, high-throughput in vitro approaches being utilized by the EPA ToxCast program. Issues discussed include the challenges of anchoring in vitro predictions to relevant in vivo endpoints and the need to validate pathway-based predictions with targeted studies in whole animals. Currently, there are 10,000 to 30,000 chemicals in world-wide commerce in need of hazard data for assessing potential health risks. The traditional animal study designs for assessing developmental toxicity cannot accommodate the evaluation of this large number of chemicals, requiring that alternative technologies be utilized. Though a daunting task, technologies are being developed and utilized to make that goal reachable.

Environmental impact on vascular development predicted by high-throughput screening.

BACKGROUND: Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High-throughput screening (HTS) in the U.S. Environmental Protection Agency (EPA) ToxCast™ project provides vast data on an expanding chemical library currently consisting of > 1,000 unique compounds across > 500 in vitro assays in phase I (complete) and Phase II (under way). This public data set can be used to evaluate concentration-dependent effects on many diverse biological targets and build predictive models of prototypical toxicity pathways that can aid decision making for assessments of human developmental health and disease. OBJECTIVE: We mined the ToxCast phase I data set to identify signatures for potential chemical disruption of blood vessel formation and remodeling. METHODS: ToxCast phase I screened 309 chemicals using 467 HTS assays across nine assay technology platforms. The assays measured direct interactions between chemicals and molecular targets (receptors, enzymes), as well as downstream effects on reporter gene activity or cellular consequences. We ranked the chemicals according to individual vascular bioactivity score and visualized the ranking using ToxPi (Toxicological Priority Index) profiles. RESULTS: Targets in inflammatory chemokine signaling, the vascular endothelial growth factor pathway, and the plasminogen-activating system were strongly perturbed by some chemicals, and we found positive correlations with developmental effects from the U.S. EPA ToxRefDB (Toxicological Reference Database) in vivo database containing prenatal rat and rabbit guideline studies. We observed distinctly different correlative patterns for chemicals with effects in rabbits versus rats, despite derivation of in vitro signatures based on human cells and cell-free biochemical targets, implying conservation but potentially differential contributions of developmental pathways among species. Follow-up analysis with antiangiogenic thalidomide analogs and additional in vitro vascular targets showed in vitro activity consistent with the most active environmental chemicals tested here. CONCLUSIONS: We predicted that blood vessel development is a target for environmental chemicals acting as putative vascular disruptor compounds (pVDCs) and identified potential species differences in sensitive vascular developmental pathways.