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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

  • To use Tox21 data in regulatory applications, the assays and the models built from the assays must be validated based on their performance against the biological targets they query. This requires establishing sets of reference chemicals and a process for assessing and reporting the performance evaluation of an assay that provides consumers of the data the ability to interpret the appropriate context for use of the assay. Both of these activities also need to be streamlined and rapid enough to manage the tens to hundreds of assays that can help inform regulatory toxicity endpoints. NICEATM and EPA scientists developed a process to identify reference chemicals that consistently produced positive or negative results when assayed in defined assays (Judson et al. 2019). Current efforts are focused on using the data collected for identification of reference chemicals and establishing protocols to evaluate the performance of specific Tox21 and ToxCast assays.
  • NICEATM is mapping HTS assay endpoints to adverse toxicological outcomes. Assays have been mapped to developmental toxicity, acute toxicity, and carcinogenicity based on established modes of action from literature. The assay mappings are used in NICEATM's Integrated Chemical Environment (ICE) to facilitate targeted access to the Tox21 data in ICE Tools. This mapping draws on established ontologies and controlled vocabularies such as the NCI Metathesaurus and UMLS Metathesaurus concept IDs to promote the interoperability of ICE data with partners.
  • An update of ICE launched in March 2020 featured improvements to its in vitro to in vivo extrapolation (IVIVE) tool, which uses curated high-throughput screening data from Tox21 to predict an in vivo exposure that could produce an adverse effect. The IVIVE tool provides an interface to the EPA high-throughput toxicokinetics (httk) package and uses quantitative structure activity relationship predictions from the Open Structure-activity Property App (OPERA) to provide input parameters for over 800,000 chemicals. It also allows users to upload their own data to generate predictions.
  • 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 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.

Developmental Toxicity

  • FDA and NICEATM scientists applied IVIVE to evaluate the impact of pharmacokinetics and different modeling approaches on predicting relevant external exposure using data from an in vitro human induced pluripotent stem cell-based assay. The IVIVE analysis estimated equivalent administered doses that would result in maternal or fetal blood concentrations equivalent to the in vitro developmental toxicity and cytotoxic potential concentrations. Preliminary results of this analysis showed close agreement between equivalent administered doses and in vivo rat lowest effect levels for three valproate analogues. This suggested that the stem cell-based assay and IVIVE approaches can quantitatively predict in vivo developmental toxicity potential. An abstract describing this work (Chang et al.) was accepted for a presentation at the SOT 2020 annual meeting.
  • 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 FDA 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 (now part of the Center for Computational Toxicology and Exposure) 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.

Background

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 has been used to prioritize uncharacterized compounds for regulatory testing using both traditional and novel test methods. To more broadly address challenges in the field of toxicology, in 2018 Tox21 released a new strategic and operational plan (Thomas et al. 2018) that expands the focus of its research activities. New focus areas include expanding its portfolio of alternative test systems, addressing technical limitations of in vitro test systems, curating legacy in vivo toxicity testing data, establishing scientific confidence in in vitro test systems, and refining alternative methods for characterizing pharmacokinetics and in vitro assay disposition. 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.