NICEATM Support of the Tox21 Program
The goal of the multiagency "Tox21" research initiative is to research, develop, evaluate, and translate innovative test methods that will better predict how substances may affect humans and the environment. This research initiative uses in vitro HTS assays to evaluate the biological activity of compounds in a ~10,000 compound library and to relate observed activities to toxicological endpoints.
NICEATM provides support to the Tox21 effort primarily through its computational toxicology group. Read more about NICEATM computational toxicology projects.
NICEATM Tox21 Projects
- NICEATM is mapping HTS assay endpoints to toxicological adverse outcomes. Over 100 assays have been mapped to each of the adverse outcomes “acute systemic toxicity” and “developmental toxicity,” respectively. This mapping can facilitate prioritization of chemicals for testing and aid in the development of adverse outcome pathways. This work will be described in a poster presentation (Karmaus et al.) at the 2019 SOT annual meeting.
- NICEATM curated HTS data from Tox21 and the EPA ToxCast program to identify low-confidence calls. Factors considered in the curation include chemical quality control, concentration-response curve fits, and testing range. These data are available through the NICEATM Integrated Chemical Environment (ICE). ICE, launched in 2017, provides high-quality, curated data from NICEATM and its partners as well as other data resources and tools to support development of new approaches for assessing chemical safety. ICE also includes an Integrator for exploration and comparison of in vivo and in vitro data and workflows to further explore data relationship and make toxicity predictions.
- To better characterize four chemicals identified in Tox21 quantitative HTS assays as having farnesoid X receptor alpha agonist or antagonist activity, NICEATM and collaborators evaluated them using four experimental approaches. Experiments generally confirmed the Tox21 results, provided orthogonal data on protein-to-protein interactions and receptor docking, and translated those results to an in vivo system (larval medaka assay). The study, presented at the 2018 SOT meeting (Hamm et al.), demonstrates an approach to targeted evaluation of putative bioactivity derived from HTS data.
- NTP and EPA are investigating the use of zebrafish in the Tox21 and ToxCast programs as a screening tool for hazard identification. A 2014 Collaborative Workshop on Aquatic Models and 21st Century Toxicology highlighted the need for standardization of husbandry and testing protocols. These issues are being addressed by the NTP Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative supported by NICEATM. Subsequent SEAZIT activities included a webinar series considering the application of informatics to improve data analysis of zebrafish studies and a paper that summarized findings of a literature review and information gathering from expert researchers (Hamm et al. 2018).
- To investigate the utility of in vitro HTS data to predict acute lethality, NICEATM investigated the use of computational approaches to predict LD50 values and binary toxicity categories (toxic vs. nontoxic) with ToxCast and Tox21 data. These analyses confirmed that no single in vitro assay can currently predict acute systemic toxicity in rodents; further efforts to identify relationships between in vitro cytotoxicity and cell stress assays and acute systemic toxicity produced similar results. The analyses were presented (Strickland et al.) at the 2017 World Congress on Alternatives and Animal Use in the Life Sciences.
- To follow up on these results, NICEATM conducted an analysis using the random forest machine-learning method and data from ToxCast and Tox21 to determine whether a specific group of high-throughput assays could be identified that would be informative in predicting oral LD50 values. Results suggested that the approach might be able to distinguish toxic from nontoxic chemicals, especially if the nontoxic chemicals were conservatively defined.
- NICEATM developed and applied one-compartment steady-state or physiologically based pharmacokinetic models to data from a Tox21 assay to quantitatively correlate in vitro and in vivo dosimetry for estrogen receptor reference chemicals (Chang et al. 2014). Subsequent work focused on understanding the impact of various parameters, such as using free plasma concentration as a surrogate for total plasma concentration, and comparing multiple modeling approaches (Casey et al. 2018).
- In June 2015, EPA accepted a plan integrating validated ToxCast/Tox21 high throughput assays and an associated computational model as an alternative to three Tier 1 tests, one of which is the rodent uterotropic assay, used in its Endocrine Disruptor Screening Program to assess estrogenic activity. It is envisioned that the use of high-throughput assays and computational methods will accelerate the pace of screening, decrease costs, and reduce animal testing. The EPA plan was developed and validated by EPA and NICEATM scientists (Browne et al. 2015).
- Using data from 11 Tox21 and and ToxCast androgen receptor pathway-related assays, NICEATM built a model to predict AR pathway activity (Kleinstreuer et al. 2017). This model has been evaluated (Kleinstreuer et al. 2018) using a list of reference chemicals developed from a systematic literature review of in vitro data from androgenic activity assays (Browne et al. 2018).
- NICEATM scientists and collaborators developed predictive quantitative structure-activity relationship (QSAR) models for endocrine receptor binding and activity though the CERAPP (Collaborative Estrogen Receptor Activity Prediction Project; Mansouri et al. 2016) and CoMPARA (Collaborative Modeling Project for Androgen Receptor Activity; Mansouri et al. in preparation) collaborations
- NICEATM scientists collaborated with EPA to build an adverse outcome pathway for disruption of embryonic vascular development leading to adverse prenatal outcomes, and an associated predictive signature using ToxCast assays to identify putative vascular disruptor compounds (pVDCs). These predictions have been tested in a variety of functional vascular development assays using zebrafish and complex in vitro cell-based models (Ellis-Hutchings et al. 2017; McCollum et al. 2017; Tal et al. 2017) and confirmed the model to be useful in identifying environmental chemical pVDCs.
- The bioactivity-based read-across (BaBRA) analysis approach presented by NICEATM at the 2015 SOT Annual Meeting was used as a case study for the Read Across Workshop sponsored by the Center for Alternatives to Animal Testing in October 2015. NICEATM participated in the biological data working group and contributed to publications from the workshop (Ball et al. 2016; Zhu H et al. 2016).
HTS Assay Nominations
The Tox21 initiative seeks assays that assess the effects of chemicals on targets encompassing all pathways relevant to toxicity. The Tox21 Assay Evaluation and Screening Team accepts nominations for in vitro HTS assays that target toxicity markers for key initiating or downstream events. Assays that measure events associated with the following pathways are of greatest interest:
- Developmental pathways: Wnt, SONIC hedgehog (aka SHH), Delta-notch, TGF-beta, receptor tyrosine kinase (aka RTK) and retinoid signaling
- Endocrine-related pathways: steroidogenesis and estrogen, thyroid, adrenal, and androgen signaling
Nominated assays will be assessed for their overall applicability to the Tox21 HTS program in terms of biological relevance (i.e., human health effect), cost, and potential to be adapted to an HTS platform. Nominated assays must address targets not already screened in the Tox21 program. Assays judged to be suitable, based on these criteria, will then be optimized to the HTS platform, and validated for use in the program.
Nominations should consider the following general criteria:
- Relevance to the goals of the Tox21 Initiative
- High-throughput capability of the assay (homogeneous, can be run in 48 h or less, volume up to 6 uL/well, robust signal readout, etc.)
- Evaluation of preliminary assay performance using appropriate reference compounds
- Validation status of the assay
- Availability of complete and detailed protocols
- Efficiency and cost of the assay
Assay nominations should be submitted to Kristine Witt, NTP Co-Chair, Assay Evaluation and Screening Team. When submitting HTS assay nominations and protocol information, please provide the following contact information for the submitter:
- Mailing address
- Phone number
- Fax number
- Email address
- Sponsoring organization (as applicable)
NTP prefers electronically submitted nominations, but will accept nominations by the means that is most convenient for the submitter.
The Tox21 Consortium is a multiagency collaboration among
- The National Toxicology Program
- The NIH National Center for Advancing Translational Sciences
- The EPA National Center for Computational Toxicology
- The Food and Drug Administration
The Tox21 partner agencies work together to develop, validate, and translate innovative in vitro HTS methods to characterize the impact of chemicals on key steps in toxicity pathways.
Data collected in the Tox21 initiative will be used in the near term to prioritize uncharacterized compounds for regulatory testing using both traditional and novel test methods. The eventual goal of Tox21 is to use HTS methods to generate data that will allow risk assessors to more accurately predict the effects of uncharacterized substances on human health and the environment.
More information about partner agencies' activities in support of Tox21 can be found on these agency websites:
- National Toxicology Program
- National Center for Advancing Translational Sciences
- Environmental Protection Agency
NICEATM’s mission, in part, is to facilitate the development, validation, and regulatory acceptance of new and revised test methods while maintaining and promoting scientific quality. The strategies used to accomplish NICEATM’s mission can be leveraged to support the Tox21 initiative while promoting the reduction, refinement, and replacement of animal use for regulatory toxicity testing.