Tox21 Cross-partner Projects

Tox21 is a collaboration among groups within four U.S. federal organizations aimed at developing more efficient approaches to predict how chemicals may affect human health. Tox21 studies use assays that are run at higher throughput than traditional tests. Test approaches developed and data collected via this initiative may enable agencies to reduce reliance on animal data for assessing chemical safety.

To more broadly address evolving challenges in toxicology, Tox21 partners developed a new strategic and operational plan (Thomas et al. 2018) that expands the focus of the program’s research activities. New areas of focus are:

  • Develop alternative test systems that are predictive of human toxicity and dose response.
  • Address key technical limitations of current in vitro test systems.
  • Curate and characterize legacy in vivo toxicity studies.
  • Establish scientific confidence in integrated assay batteries and in vitro test systems.
  • Refine and deploy in vitro methods for characterizing pharmacokinetics and in vitro disposition.

The four groups participating in the Tox21 collaboration are ICCVAM members:

  • U.S. Environmental Protection Agency
  • U.S. Food and Drug Administration
  • Division of the National Toxicology Program (within NIEHS)
  • National Center for Advancing Translational Sciences (within the National Institutes of Health)
Tox21 projects and projects using Tox21 data are described below and throughout this report.
Predictive Modeling of Developmental Toxicity with Human Pluripotent Stem Cells

EPA, FDA, and NICEATM scientists and collaborators applied IVIVE to evaluate the impact of pharmacokinetic models on predicting relevant external exposure from in vitro developmental toxicity potential concentrations derived from an in vitro human iPSC-based assay. Previous work showed that the devTOX quickPredict assay ranked ranked the developmental toxicity potential of valproate analogues in a manner that was consistent with observed developmental toxicity potency in vivo. The IVIVE analysis in this project estimated equivalent administered doses that would result in maternal and fetal blood concentrations equivalent to the developmental toxicity potential and cytotoxic in vitro concentrations (Chang et al. 2022). The estimated equivalent administered doses were compared to published lowest effect levels from in vivo developmental toxicity studies. Preliminary results of this analysis showed that equivalent administered doses for the valproate analogues based on different pharmacokinetic and PBPK models were quantitatively similar to in vivo data from both rats and humans, where available. The impact of in vitro kinetics on equivalent administered dose estimates was chemical-dependent. Equivalent administered doses from this study were within the range of predicted doses from other in vitro and model organism data. This suggested that the devTOX quickPredict assay and IVIVE approaches can quantitatively assess a chemical’s developmental toxicity potential.

Incorporating Genetic Susceptibility into Developmental Neurotoxicity Screening via Population Diversity

The potential for neurotoxicity in children following exposure to environmental chemicals is of concern due to recent increases in the prevalence of neurological disorders such as attention deficit hyperactivity disorder and autism. Neurotoxicity risk for an individual can be influenced both by genetic background and by exposures to neurotoxic chemicals in the environment. To investigate the role of genetic diversity in susceptibility to neurotoxicity, scientists at NIEHS, EPA, and FDA are using a genetically diverse set of cells to evaluate a curated set of chemicals with neurotoxic potential. Neural progenitor cells were derived from a set of mice bred to maximize genetic diversity. An initial set of 200 genetically different cell lines from male and female mice was narrowed down to 135 cell lines, considered to be the minimum number of cell lines needed to quantitatively assess diversity in population responses. The panel of cell lines was exposed to 8 concentrations of a 12-chemical test set. The intracellular morphometry of each treated cell was visualized using a high-content imaging assay called cell painting, which uses six fluorescent dyes to quantitatively describe cell features such as cell membranes, mitochondria, DNA and RNA, cytoskeleton, and Golgi bodies (Bray et al. 2016). Data analysis is ongoing, and data will be used to derive PODs for chemical-induced intracellular morphometric endpoints and characterize the variability in these PODs across cell lines for each chemical. Data will also be analyzed to explore differences in toxicity mode of action that may differ across lines and therefore be genetically linked. These data will inform data-driven uncertainty factors that account for interindividual variability, allowing for adequate protection of genetically sensitive subpopulations. A poster describing the project (Harrill et al.) will be presented at the 2022 annual meeting of the Society of Toxicology, with a talk planned for an accepted symposium at the 2023 meeting. A paper describing the project is planned for submission in 2023.

Automation of Reference Chemical Generation

To use data generated by HTS initiatives such as Tox21 and ToxCast in regulatory applications, the assays and models built from the assays must be validated based on their performance against the biological targets they query. This requires developing sets of reference chemicals that consistently yield reproducible results when assayed for these biological targets. Furthermore, the development of reference chemical sets needs to be streamlined and rapid enough to manage the tens to hundreds of assays that can help inform regulatory toxicity endpoints. To address this need, scientists at EPA and NIEHS developed a process to identify reference chemicals that consistently produce positive or negative results when assayed in defined assays (Judson et al 2019). Work under this project conducted in partnership with NCATS seeks to automate the generation of reference chemical lists without the need for new experimental tests or upfront literature review. The targets being examined for this project are androgen receptor, estrogen receptor, glucocorticoid receptor, peroxisome proliferator-activated receptor gamma, progesterone receptor, retinoic acid receptors, thyroid hormone receptor, tumor protein P53, mitochondria toxicity, and cell stress pathways. The list of assays and reference chemicals generated through this process will be used to validate new assays or AOPs, which will improve chemical screening abilities when predicting toxicity. The project will be described in one or more papers to be submitted for publication in 2022 and 2023.

Retrofitting Existing Tox21 HTS Assays with Metabolic Capability

The HTS assays that have been run in the Tox21 testing program to date generally lack the metabolic activity found in living systems, which can potentially increase or decrease the toxicity of chemicals. As a result, HTS results may not accurately reflect in vivo activity. Scientists at EPA, NCATS, and NIEHS are using several approaches to address this problem: adding human or rat liver microsomes into the existing assays, transfecting cells with mRNAs encoding human metabolic enzymes, or using metabolically capable human HepaRG cells. Work in 2020 and 2021 (Ooka et al. 2022) focused on three types of assays for which data have already been generated: cellular stress-related assays, endocrine disruption assays, and CYP450 enzyme inhibition assays. The retrofitted assays were used to screen the Tox21 10K chemical library to identify chemicals that are either bioactivated or detoxified by metabolic activity.

Expansion of Pathway Coverage by Tox21 HTS Assays for Better Prediction of Adverse Drug Effects

To date, Tox21 HTS assays have focused primarily on selected nuclear receptor and stress response pathways. This relatively limited focus suggests that activity in other toxicity pathways has not been adequately assessed; it is likely that some unexplored pathways relate to unanticipated adverse drug effects. Therefore, expanding the coverage of biological responses by adding assays that probe under-represented pathways in the current Tox21 assay portfolio may improve the predictivity of Tox21 data. Scientists at FDA, NIEHS, and NCATS are systematically identifying these under-represented pathways in a data-driven approach and nominating assays for development and Tox21 chemical screening. The data generated will be used to build models for human toxicity prediction, focusing on common adverse drug effects such as drug-induced liver injury and cardiotoxicity. The initial survey identified targets and pathways of interest, including cytochrome P450 metabolic enzymes and G protein-coupled receptors. HTS assays were optimized and validated for the proposed new targets and pathways, and these assays will be used to screen the Tox21 10K chemical library in 2022. Models are being built that could use HTS assay data to predict human toxicity. Predictions from the models will be validated in relevant assays. A paper describing the project and the prediction models will be prepared in late 2022.

Profiling Activity of Acetylcholinesterase Inhibitors

Acetylcholinesterase inhibitors cause a variety of adverse effects in the nervous system. Some acetylcholinesterase inhibitors serve as drugs, while others are used as pesticides or found in natural products. Scientists at FDA, EPA, and NCATS developed acetylcholinesterase inhibition assays (Li et al. 2017, 2019). These assays were then used to screen the Tox21 10K chemical library to identify environmental chemicals that inhibit acetylcholinesterase activity (Li et al. 2021). The screening study, which included NIEHS scientists and other collaborators, identified both known and not previously reported acetylcholinesterase inhibitors, which were further characterized in lower-throughput cell-based assays, enzyme-based activity assays, and molecular docking studies. Specifically, this study identified 18 potential novel acetylcholinesterase inhibitors, most of them clinically approved drugs. Cmax values, which represent the highest concentration of a drug in the blood or target organ after a dose, were found from the literature for 14 drugs. These were compared with half-maximal inhibitory concentration (IC50) values generated from this study (Santillo and Xia, 2022). The ratio of IC50 and Cmax can be used to prioritize the compounds for further study. This represents a robust and reliable approach to evaluating large sets of environmental compounds for their acetylcholinesterase inhibitory activity.

Development of High-throughput Assays to Detect Potential Sensitizers or Irritants

Assessing the sensitization or irritation potential is a key element in the safety evaluation of topically applied or exposed chemicals and drugs. European legislation now mandates the use of alternative test methods for these compounds, instead of testing them on animals. Developing HTS assays for these compounds would provide a faster and cheaper alternative testing method than current in vitro assays. Scientists from NIEHS and NCATS collaborated to explore development of HTS approaches to identify potential irritants and sensitizers. Studies described in a 2020 publication (Wei et al. 2020) found that HTS-compatible 2D cellular and 3D tissue skin models could be combined with irritation-relevant activity endpoints to assess the irritation effects of topical-use compounds and identify potential hazards. To identify potential skin sensitizers, the Tox21 10K compound library was screened for potential sensitizers using the KeratinoSens assay. Substances identified as active were further tested using a high-throughput version of the direct peptide reactivity assay (Wei et al. 2021), an interleukin-8 assay, and the human cell line activation test. Analysis of these data are ongoing, and the results will be reported in a paper to be published in 2022.

Predictive Toxicology of the Retinoid Signaling Pathway

The developing child is vulnerable to genetic, pharmacological, or chemical disruption of the retinoid biochemical pathway, especially during early growth and differentiation of embryonic tissues. Susceptibility of this pathway to chemical disruption is an important regulatory concern for developmental and reproductive hazard identification. In this study, which involves all four Tox21 participating offices, Tox21 data are being mined and modeled to identify potential retinoid pathway disruptors. Results from a dozen Tox21 molecular targets mapping to retinoid pathway targets identified over 100 structurally diverse chemicals with relevant bioactivity. Computational tools and approaches are now being built to integrate these data with embryological knowledge and construct data-driven models for developmental hazard prediction. For example, a computational model that linked chemical disruption of the retinoic acid signaling pathway with fetal skeletal defects has been aligned with a provisional AOP framework for craniofacial, vertebral, and/or appendicular phenotypes (Knudsen et al. 2021; Pierro et al. submitted). Papers in preparation related to this project describe the use of these data as input for IVIVE models to predict relevant exposure levels and to develop candidate reference chemicals for in vitro retinoid pathway assays. Participants in the project contributed to a “Detailed Review Paper on the Retinoid System” issued by OECD in 2021, and described findings in a poster presented at the 2021 annual meeting of the Society of Toxicology (Pierro et al.), a platform presentation at the 2021 annual meeting of the Society for Birth Defects Research and Prevention (Pierro et al.), and a workshop session at the 11th World Congress on Alternatives and Animal Use in the Life Sciences (Knudsen et al. 2021).

Investigation of Environmental Determinants of Pubertal Timing in Girls

Over the past decade, there has been a worldwide trend toward earlier breast development in girls. The rapid pace of this trend suggests the involvement of environmental factors. While some studies (e.g., Bandera et al. 2011) have suggested a relationship between the presence of endocrine-disrupting chemicals in girls and pubertal timing, more evidence is needed to develop clear associations. Scientists at NIEHS and NCATS are examining the potential effects of endocrine-disrupting chemicals on important components of the biochemical pathway responsible for pubertal timing. Chemicals in the Tox21 10K collection are being tested in human cell-based assays that measure activation or inhibition of the gonadotropin-releasing hormone receptor and kisspeptin receptor. These receptors are expressed in the hypothalamus and are important players in the control of pubertal timing. Chemicals that exhibit activity will be tested further in cell-based assays or possibly in animals to confirm the biological relevance of the identified activity and determine the chemical’s mechanism of action.