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Tox21 Research Phases

The Tox21 program aims to develop state-of-the-art test methods that will better predict how chemicals may affect humans and the environment. To that end, research for the Tox21 program has been divided into phases to provide structure toward this goal. Data from all phases of Tox21, along with full chemical characterization and assay protocol details, are being deposited into publicly available databases and can be analyzed using the Tox21 Toolbox.


Phase I

In the first phase of Tox21 research (2005–2010), the partners used the high-throughput screening (HTS) technologies of the National Human Genome Research Institute's National Chemical Genomics Center (NCGC, now a part of the National Center for Advancing Translational Sciences, NCATS). Researchers performed quantitative HTS (qHTS) testing.

NCGC screened approximately 2800 compounds in more than 75 qHTS assays. During this time, the Environmental Protection Agency’s National Center for Computational Toxicology also screened 309 unique compounds across more than 500 biochemical- and cell-based assays through its ToxCast™ program. These compounds were also tested in NTP's "WormTox" laboratory. In the WormTox laboratory, toxicity screens used the roundworm, Caenorhabditis elegans.


Phase II

In Phase II of Tox21 research (2011–2016), the partners tested a chemical library at NCATS. The chemical library is expanded from Phase I to greater than 10,000 compounds. An HTS robotics system is used, sponsored by NTP.

More than 200 databases of chemicals and drugs in the United States and abroad were analyzed to select the compounds for testing. This chemical library included industrial and consumer products, food additives, drugs, and mixtures.

Tox21 partners are establishing a full spectrum of cell-based assays to further define and characterize activities identified in the initial qHTS tests. The results will provide information to help evaluate biological processes that may lead to adverse health effects in humans.


Phase III

Phase III of Tox21 research (2014–present) aims to better represent human health and disease in a variety of ways. In Phase III, the Tox21 federal partners developed a document entitled "The US Federal Tox21 Program: A strategic and operational plan for continued leadership" that was recently published (Thomas et al., ALTEX 2018 Mar 8. DOI: 10.14573/altex.1803011.). In this document, five key Areas of Focus were identified for moving forward in Phase III, which were:

  • 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 in vitro test systems and integrated assay batteries
  • Refine and deploy in vitro methods for characterizing pharmacokinetics and in vitro disposition

One specific focus at NTP in Phase III is to increase the biological diversity of human cell lines used in the HTS assays. In addition, NTP plans to investigate three-dimensional organoid (or organ-like) model cultures and simple model organisms in screening efforts. These three-dimensional models will address chemical metabolism and assess the effects of chemicals in more complex biological systems.

Phase III research also adds medium to high-throughput transcriptomics assays. These will test for effects of substances on gene expression. Phase III screening assays will look at multiple endpoints and processes in order to better identify substances that may cause adverse human health effects.

During Phase III, Tox21 partners have identified sets of genes in human cells and tissues that respond to toxic chemicals (e.g., toxicogenomics). The resulting human S1500+ gene set comprises approximately 2750 genes that will be used to evaluate gene expression changes from substance exposures. Similar gene sets are being developed for screening cells and tissues from rats, mice, and zebrafish. The human S1500+ gene set should provide transcriptomic information on cellular responses. These cellular responses are independent of cell type or species. The gene set will also provide information on those cellular responses that are specific by organ and/or cell type.