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Mixture toxicity literature is dominated by water pollutants, pesticides, environmental pollutants, insecticides, soil pollutants, and chemicals described as persistent, bioaccumulative, and toxic.

The sodium-iodide symporter (NIS) mediates the uptake of iodide into the thyroid. Inhibition of NIS function by xenobiotics has been demonstrated to suppress circulating thyroid hormones and perturb related physiological functions. Until recently, few environmental chemicals had been screened for NIS inhibition activity. We previously screened over 1000 chemicals from the ToxCast Phase II (ph1v2 and ph2) libraries using an in vitro radioactive iodide uptake (RAIU) with the hNIS-HEK293T cell line to identify NIS inhibitors. Here, we broaden the chemical space by expanding screening to include the ToxCast e1k library (804 unique chemicals) with initial screening for RAIU at 1 × 10–4 M. Then 209 chemicals demonstrating > 20% RAIU inhibition were further tested in multiple-concentration, parallel RAIU and cell viability assays. This identified 55 chemicals as active, noncytotoxic RAIU inhibitors. Further cytotoxicity-adjusted potency scoring (with NaClO4 having a reference score of 200) revealed five chemicals with moderate to strong RAIU inhibition (scored > 100). These data were combined with our previous PhII screening data to produce binary hit-calls for ~ 1800 unique chemicals (PhII + e1k) with and without cytotoxicity filtering. Results were analyzed with a ToxPrint chemotype-enrichment workflow to identify substructural features significantly enriched in the NIS inhibition hit-call space. We assessed the applicability of enriched PhII chemotypes to prospectively predict NIS inhibition in the e1k dataset. Chemotype enrichments derived for the combined ~ 1800 dataset also identified additional enriched features, as well as chemotypes affiliated with cytotoxicity. These enriched chemotypes provide important new information that can support future data interpretation, structure–activity relationship, chemical use, and regulation.

[Image: see text] 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.