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NICEATM Support of the Tox21 Program

The multiagency "Tox21" research initiative aims to improve regulatory hazard assessment of substances potentially harmful to humans and the environment. This research initiative uses in vitro high throughput screening (HTS) assays to evaluate the biological activity of compounds in a 10,000 compound library and to map the observed activities to toxicity pathways. One of the goals of the Tox21 effort is to incorporate in vitro assay data into models that can be used to predict toxic (or adverse) effects in humans resulting from chemical exposures.

NICEATM provides support to the Tox21 effort primarily through its computational toxicology group. Read more about NICEATM computational toxicology projects.

NICEATM Tox21 Projects
  • NTP and the U.S. Environmental Protection Agency (EPA) are investigating the use of zebrafish in Tox21 and the EPA ToxCast high throughput screening program 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. Current SEAZIT activities include a webinar series considering the application of informatics to improve data analysis of zebrafish studies and a manuscript in preparation that will summarize findings of a literature review and information gathering from expert researchers.
  • Using the collective results of 16 Tox21 and ToxCast estrogen receptor pathway-related assays, NICEATM developed and applied one-compartment steady-state or physiologically based pharmacokinetic models to quantitatively correlate in vitro and in vivo dosimetry for estrogen receptor reference chemicals. NICEATM scientists then developed a workflow to semi-automate the modeling process. The first version of this work was published in December 2014 in Applied In Vitro Toxicology. Application of an improved approach to a larger chemical set has been submitted to Environmental Health Perspectives. Most recently, a poster describing open-source workflows to conduct these analyses was presented at the 2017 SOT annual meeting (Chang et al. poster).
  • 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 (EDSP) 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 and is described in detail in a manuscript published in Environmental Science and Technology.
  • Using data from 11 Tox21 and and ToxCast assays, NICEATM built a model to predict androgen receptor (AR) pathway activity. This model has been evaluated using a list of reference chemicals developed from a systematic literature review of in vitro data from androgenic activity assays. The model and evaluation are described in a manuscript published in Chemical Research in Toxicology.
  • From these data and the AR pathway predictions, NICEATM also developed predictive quantitative structure-activity relationship (QSAR) models for androgen receptor binding and activity (see Zang et al. poster presented at the 2016 SOT annual meeting).
  • 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, and have been shown to be useful in identifying environmental chemical pVDCs.
    • Ellis-Hutchings R et al. 2017. Embryonic vascular disruption adverse outcomes: Linking HTS signatures with functional consequences. Reprod Toxicol 71:16-31.
    • McCollum CW et al. 2017. Identification of vascular disruptor compounds by analysis in zebrafish embryos and mouse embryonic endothelial cells. Reprod Toxicol 70:60-69.
    • Tal T et al. 2017. Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity. Reprod Toxicol 70:70-81.
  • 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. Toward good read-across practice (GRAP) guidance. ALTEX.
    • Zhu H et al. 2016. Supporting read-across using biological data. ALTEX.
HTS Assay Nominations

To achieve its aims, the Tox21 initiative needs assays that assess the effects of chemicals on targets encompassing all pathways relevant to toxicity. The Assays and Pathways Working Group of the Tox21 Consortium is accepting nominations for in vitro HTS toxicity assays, particularly assays that identify toxicity pathway markers for either initiating or downstream events. Assays that evaluate 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 pathways: estrogen, thyroid, adrenal, and androgen

Nominated assays will be assessed for their overall applicability to the Tox21 HTS program in terms of biological relevance, cost, and potential to be adapted to an HTS platform. Suitable assays 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 (96-well format or higher, with no obvious impediments to miniaturization to a 1536-well format)
  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 Chair, Assays and Pathways Working Group. 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, NTP Chair, Assays and Pathways Working Group, or Warren Casey, NICEATM Director.


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 the agency websites:

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.