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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

Data Characterization and Applications

  • NICEATM is mapping HTS assay endpoints to toxicological adverse outcomes. Assays have been mapped to adverse outcomes of regulatory interest such as “acute systemic toxicity” and “developmental toxicity,” by leveraging known modes of action and assay annotation information. This mapping can facilitate data interpretation and aid in the development of adverse outcome pathways. This work was 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. The resulting curated Tox21 and ToxCast HTS data minimize low-confidence hit calls, providing a processed version of the HTS data that is ready for any application. Factors considered in the curation include chemical purity, quality control data, concentration-response curve fits, and testing range. These data are available through the NICEATM Integrated Chemical Environment (ICE).
  • 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.
  • 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).

Developmental Toxicity

  • 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 (summarized in Planchart et al. 2016) 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 that focused on the application of informatics to improve zebrafish data analyses, and a paper that summarized findings of a literature review and information gathering from expert researchers (Hamm et al. 2018).
  • 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; Saili et al. 2019) that confirmed the model to be useful in identifying environmental chemical pVDCs.
  • NICEATM, NTP, Oak Ridge National Labs, and the U.S. Food and Drug Administration are collaborating to automate the process of identifying high-quality developmental toxicity studies in the published scientific literature. Preliminary models were trained using the uterotrophic database (Kleinstreuer et al. 2016) built for the EPA Endocrine Disruptor Screening Program, and leverage natural language processing and machine learning to identify papers that meet minimum criteria to be considered guideline-like studies (Herrmannova et al. 2018).

Endocrine Disruptors

  • 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).
  • NICEATM scientists collaborated with the EPA National Center for Computational Toxicology to develop 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. 2020) collaborations.
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 developmental or endocrine pathways are of particular interest to NICEATM. However, nominated assays must address targets not already screened in the Tox21 program. 

Nominated assays will be assessed for their overall applicability to the Tox21 HTS program in terms of biological relevance (i.e., human and environmental health effect), cost, and potential to be adapted to an HTS platform. 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:

  1. Relevance to the goals of the Tox21 Initiative
  2. 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.)
  3. Evaluation of preliminary assay performance using appropriate reference compounds
  4. Validation status of the assay
  5. Availability of complete and detailed protocols
  6. 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:

  • Name
  • Affiliation
  • 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.

For more information about nominating an assay, please contact Kristine Witt or Warren Casey, Acting Chief, NTP Biomolecular Screening Branch.


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.

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.