Since 2009, the Tox21 project has screened similar to 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.

In response to a growing recognition over the past couple of decades that the field of toxicology needed to transition from a largely descriptive science to one based on the mechanisms of chemical effects in living systems, a partnership among several US government agencies (initially the Environmental Protection Agency, the National Toxicology Program and the NIH Chemical Genomics Center, later joined by the Food and Drug Administration) emerged in the early 21st century. This partnership, which was formally established in 2007 and came to be called Tox21, was based on the complementary expertise of the member agencies and was committed to advancing the science in a transparent manner, with open access to all methodologies and results, and with community involvement in science generation and analysis. The consortium represents a unique collaboration across the US federal government, and in its relatively short existence has had a significant impact on moving the field of toxicology to more mechanism-based in vitro approaches. This chapter reviews the history of the consortium, its major objectives and accomplishments, and identifies new strategic directions that are being formulated to address gaps in technology and knowledge.

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.

The evaluation and regulation of chemical substances have undergone a major overhaul in Canada, the USA and the European Union (EU) over the last decade. To facilitate increasing concerns over chemical safety, changes in regulations and strategic plans were introduced. Specifically, the Canadian parliament adopted a new amendment to the Canadian Environmental Protection Act (CEPA) in 1999, the US National Academy of Sciences published the 2007 NRC report TT21C, the US EPA developed the Strategic Plan in 2009, and the EU introduced new legislation in 2007 called Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH substances). A 2013 workshop held in Ottawa (Risk Sciences International) focused on regulatory issues and challenges faced by these nations. This review summarises many of the discussions held during the workshop and specifically, five challenges that Canada has encountered when assessing chemicals with limited data on Canada's Domestic Substances List (DSL).

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.

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 mu M) on HepG2 cells over a 72-hr exposure period. The HCI end points included p53, c-Jun, histone H2A. x, alpha-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 mu M) 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.

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

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.

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 β-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.

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.

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 proliferatoractivated 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.

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.

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.

The U.S. Environmental Protection Agency's ToxCast research program uses high throughput screening (HTS) for profiling bioactivity and predicting the toxicity of large numbers of chemicals. ToxCast Phase I tested 309 well-characterized chemicals in more than 500 assays for a wide range of molecular targets and cellular responses. Of the 309 environmental chemicals in Phase I, 256 were linked to high-quality rat multigeneration reproductive toxicity studies in the relational Toxicity Reference Database. Reproductive toxicants were defined here as having achieved a reproductive lowest-observed-adverse-effect level of less than 500 mg kg−1 day−1. Eight-six chemicals were identified as reproductive toxicants in the rat, and 68 of those had sufficient in vitro bioactivity to model. Each assay was assessed for univariate association with the identified reproductive toxicants. Significantly associated assays were linked to gene sets and used for the subsequent predictive modeling. Using linear discriminant analysis and fivefold cross-validation, a robust and stable predictive model was produced capable of identifying rodent reproductive toxicants with 77% ± 2% and 74% ± 5% (mean ± SEM) training and test cross-validation balanced accuracies, respectively. With a 21-chemical external validation set, the model was 76% accurate, further indicating the model's potential for prioritizing the many thousands of environmental chemicals with little to no hazard information. The biological features of the model include steroidal and nonsteroidal nuclear receptors, cytochrome P450 enzyme inhibition, G protein-coupled receptors, and cell signaling pathway readouts—mechanistic information suggesting additional targeted, integrated testing strategies and potential applications of in vitro HTS to risk assessment.

BACKGROUND:Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents.RESULTS:The effects of 309 environmental chemicals on human constitutive androstane receptors (CAR/NR1I3), pregnane X receptor (PXR/NR1I2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPAR/NR1C), liver X receptors (LXR/NR1H), retinoic X receptors (RXR/NR2B) and steroid receptors (SR/NR3) were determined using in vitro data. Hepatic histopathology, observed in rodents after two years of chronic treatment for 171 of the 309 chemicals, was summarized by a cancer lesion progression grade. Chemicals that caused proliferative liver lesions in both rat and mouse were generally more active for the human receptors, relative to the compounds that only affected one rodent species, and these changes were significant for PPAR (p0.001), PXR (p0.01) and CAR (p0.05). Though most chemicals exhibited receptor promiscuity, multivariate analysis clustered them into relatively few NR activity combinations. The human NR activity pattern of chemicals weakly associated with the severity of rodent liver cancer lesion progression (p0.05).CONCLUSIONS:The rodent carcinogens had higher in vitro potency for human NR relative to non-carcinogens. Structurally diverse chemicals with similar NR promiscuity patterns weakly associated with the severity of rodent liver cancer progression. While these results do not prove the role of NR activation in human liver cancer, they do have implications for nuclear receptor chemical biology and provide insights into putative toxicity pathways. More importantly, these findings suggest the utility of in vitro assays for stratifying environmental contaminants based on a combination of human bioactivity and rodent toxicity.

Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multiple cellular pathways. In this study, metabolic clearance and plasma protein binding were experimentally measured for 35 ToxCast phase I chemicals. The experimental data were used to parameterize a population-based in vitro-to-in vivo extrapolation model for estimating the human oral equivalent dose necessary to produce a steady-state in vivo concentration equivalent to in vitro AC(50) (concentration at 50% of maximum activity) and LEC (lowest effective concentration) values from the ToxCast data. For 23 of the 35 chemicals, the range of oral equivalent doses for up to 398 ToxCast assays was compared with chronic aggregate human oral exposure estimates in order to assess whether significant in vitro bioactivity occurred within the range of maximum expected human oral exposure. Only 2 of the 35 chemicals, triclosan and pyrithiobac-sodium, had overlapping oral equivalent doses and estimated human oral exposures. Ranking by the potencies of the AC(50) and LEC values, these two chemicals would not have been at the top of a prioritization list. Integrating both dosimetry and human exposure information with the high-throughput toxicity screening efforts provides a better basis for making informed decisions on chemical testing priorities and regulatory attention. Importantly, these tools are necessary to move beyond hazard rankings to estimates of possible in vivo responses based on in vitro screens.

More than a few environmental substances are also known to bind unintentionally to hormone receptors and can imitate or block endogenous endocrine signaling. Yet other chemicals are known to modulate hormone synthesis and secretion or to alter metabolism and clearance. Collectively these materials are now known as “endocrine disruptors.” This chapter provides examples of the mechanisms of endocrine function, which can be perturbed by chemical exposure and a few examples of pesticides that act via these mechanisms. However, it does not cover all aspects of their toxicity. Impacts of altered endocrine function on development and reproduction, particularly in experimental animal models, have been emphasized. Given the central role of the endocrine system in regulating homeostasis and controlling developmental processes, it is not surprising that interference with their normal action leads to alterations in either function or morphology. In light of the complexity of the endocrine system and the multiple points at which it can be perturbed by exogenous agents, it is not surprising that endocrine disruption may be caused by a number of different chemical classes and structures, including a number of pesticides. Some of these pesticides are pervasive and widely dispersed in the environment. Some are persistent and can be transported long distances, and others are rapidly degraded in the environment or the human body. However, even a brief exposure to pesticides that alter endocrine function can cause permanent effects if the exposure occurs during critical windows of reproductive development.