National Toxicology Program

Traditional toxicological testing is based largely on the use of laboratory animals. However, this approach suffers from low throughput, high cost, and difficulties inherent to inter-species extrapolation - making it of limited use in evaluating the very large number of chemicals with inadequate toxicological data.

The NTP recognized that the dramatic technological advances in molecular biology and computer science offered an opportunity to use in vitro biochemical- and cell-based assays and non-rodent animal models for toxicological testing. These assays allow for much higher throughput at a much reduced cost. In some assays, many thousands of chemicals can be tested simultaneously in days.

The goal is to move toxicology from a predominantly observational science at the level of disease-specific models to a predominantly predictive science focused upon a broad inclusion of target-specific, mechanism-based, biological observations.

The High Throughput Screening program, represents a new paradigm in toxicological testing. The HTS program approach to toxicological testing screens for mechanistic targets active within cellular pathways considered critical to adverse health effects such as carcinogenicity, reproductive and developmental toxicity, genotoxicity, neurotoxicity, and immunotoxicity in humans. The Biomolecular Screening Branch (BSB) administers the NTP's HTS program.

Goals of the HTS Program

• To prioritize substances for further in-depth toxicological evaluation
• To identify mechanisms of action for further investigation (e.g., disease-associated pathways)
  
• To develop predictive models for in vivo biological response (predictive toxicology)
  

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Partners in the New Strategy - The "Tox21" Community

What is "Tox21"?

With a 2008 five-year memorandum of understanding (MOU), the NTP entered a partnership with two other government organizations:

• National Human Genome Research Institute's NIH Chemical Genomics Center (NCGC)
• U.S. Environmental Protection Agency's National Center for Computational Toxicology (NCCT)

Together these partners (informally known as the "Tox21" community) will test a large number of compounds broadly characterizing and defining the chemical-biological space occupied by chemicals of toxicological concern. Each partner provides needed expertise to the coordinated "Tox21" effort. The NTP, EPA, and NCGC bring expertise in experimental toxicology, computational toxicology, and high-throughput technologies, respectively.

On July 19, 2010 a new five-year MOU was announced which was signed by the original three partners plus the U.S. Food and Drug Administration (FDA). The FDA brings to the partnership their experience in human diseases and in animal models of human disease. The FDA also has expertise in toxicity pathway analysis and computational toxicology. Their active participation is in recognition of the FDA commitment to developing new methods to evaluate the toxicity of the substances they regulate.

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Early "Tox21" Research - Phase I

What are quantitative High Throughput Screening (qHTS) Assays? The National Chemical Genomics Center's (NCGC) qHTS assays test chemicals at multiple concentrations - as many as 15 per chemical. The range of low to high concentrations allow concentration-response curves to be generated, which provide more information about and better prediction of toxicity for each chemical.

The NCGC uses a 1536 well microtiter plate format which allows for the testing of 1408 samples along with concurrent negative and positive controls per plate. Each plate represents a different concentration. Processing 100 of these plates per day, the HTS robots can test one million samples in one week. In the first phase of "Tox21" HTS research, the original three MOU partners utilized the high throughput technologies of the NCGC and performed quantitative HTS (qHTS) testing. The NCGC screened approximately 2800 compounds in more than 50 qHTS assays.

Concurrently, EPA's NCCT was screening 309 unique compounds across more than 500 biochemical- and cell-based assays in their ToxCast™ program. These compounds were also tested in NTP's "WormTox" lab, where toxicity screens using Caenorhabditis elegans have been developed.

The resulting data, along with full chemical characterization and assay protocol details, are being deposited into publicly accessible relational databases, such as the National Library of Medicine's PubChem, EPA's ACToR (Aggregated Computational Toxicology Resource), or NTP's CEBS (Chemical Effects in Biological Systems).

Individuals interested in nominating an assay or assays for consideration by the NTP can do so by completing and submitting an Assay Nomination form available on the NTP Nominations webpage.

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"Tox21" Research - Phase II

In the next phase, the "Tox21" partners will be testing an expanded library of greater than 10,000 compounds at the NCGC. This library is currently being constructed, with expectations that expanded testing at the NCGC will begin later in 2010.

In addition, approximately 700 of these compounds will be tested in Phase II of EPA's ToxCast™ program.

During Phase II, the NTP, EPA, FDA, and NCGC will establish a full spectrum of secondary and tertiary screening assays to further define and characterize activities identified in initial high throughput screens.

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Contact

For further information on NTP's High Throughput Screening program, contact:

Raymond Tice, Ph.D.
Chief, Biomolecular Screening Branch
NIEHS/NTP
P. O. Box 12233, MD K2-17
111 T. W. Alexander Drive
Durham, NC 27713
T: (919) 541-4482
F: (919) 541-0947
[Send Email]

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History of the High Throughput Screening Initiative

The NTP Vision for the 21st Century is to move toxicology from a predominantly observational science at the level of disease-specific models to a predominantly predictive science focused upon a broad inclusion of target-specific, mechanism-based, biological observations.

To implement the Vision, the NTP developed a Roadmap that places an increased emphasis on the use of alternative assays for targeting the key pathways, molecular events, or processes linked to disease or injury, and attempts to incorporate them into a research and testing framework. The Roadmap positions the NTP program to provide scientific data and the interpretation of those data for public health decision making. As a logical outgrowth of the Roadmap, NTP established a High Throughput Screening (HTS) program, representing a new paradigm in toxicological testing.

In 2007, the National Academy of Sciences published its report "Toxicity Testing in the 21st Century: A Vision and Strategy" that envisioned a not-so-distant future in which virtually all routine toxicity testing would be conducted in vitro in human cells or cell lines by evaluating perturbations of cellular responses in a suite of toxicity pathway assays using high throughput robotic assisted methodologies. In response to this report and in recognition that a coordinated program among multiple government organizations would be needed to achieve this vision, by building on existing expertise and overcoming the resource limitations of a single agency, the NTP entered into a partnership with two other government agencies.

Together with the NTP, the National Human Genome Research Institute's NIH Chemical Genomics Center (NCGC) and the U.S. Environmental Protection Agency's National Center for Computational Toxicology, located within the Office of Research and Development, agreed to test a large number of compounds broadly characterizing and defining the chemical-biological space occupied by chemicals of toxicological concern.

This partnership was formally announced in a five-year memorandum of understanding (MOU) released on February 14, 2008 (see February 2008 News Release), along with a publication authored by Drs. Francis Collins (NHGRI), George Gray (EPA) and John Bucher (NIEHS/NTP) describing how this partnership would transform environmental health protection.

On July 19, 2010, a new five-year MOU was announced that was signed by the original three partners plus the U.S. Food and Drug Administration (FDA). The FDA brings to the partnership their experience in human diseases and in animal models of human disease, as well as toxicity pathway analysis and computational toxicology, and their active participation is in recognition of the their commitment to developing new methods to evaluate the toxicity of the substances they regulate.

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What is "Tox21"?

"Tox21" is a collaboration between

• the National Institute of Environmental Health Sciences/National Toxicology Program
• the National Institutes of Health/National Chemical Genomics Center (NCGC)
• the Environmental Protection Agency
• and most recently the Food and Drug Administration.

The agencies work together to:

Research, develop, validate and translate innovative chemical testing methods that characterize toxicity pathways.

Research ways to use new tools to identify chemical induced biological activity mechanisms.

Prioritize which chemicals need more extensive toxicological evaluation.

Develop models that can be used to more effectively predict how chemicals will affect biological responses.

Identify chemicals, assays, informatic analyses, and targeted testing needed for the innovative testing methods.

Complete construction in 2010 of a library of more than 10,000 chemicals for quantitative High Throughput Screening (qHTS) at the NCGC.

Implement Phase II of EPA's ToxCast™ program, which will include the screening of a 700 compound subset of the 10,000 compound library in various mid- and high-throughput assays. ToxCast™ is an initiative launched in 2007 by EPA to revolutionize the agency's chemical toxicity evaluation procedures. ToxCast™ will use advances in computers, genomics, and cellular biology to speed up toxicity testing and enhance capacity to screen new compounds.

The data generated from the innovative chemical testing methods by the "Tox21" partnership will be provided to risk assessors to use when making decisions about protecting human health and environment.

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Publications:

Collins FS, Gray GM, Bucher JR. Toxicology. Transforming environmental health protection. Science. 2008 Feb 15;319(5865):906-7.

Huang R, Southall N, Cho MH, Xia M, Inglese J, Austin CP. Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening. Chem Res Toxicol. 2008 Mar;21(3):659-67.

Huang R, Southall N, Xia M, Cho, M-H, Jadhav A, Nguyen, D-T, Inglese J, Tice RR, Austin CP. Weighted Feature Significance (WFS): a simple, interpretable model of compound toxicity based on the statistical enrichment of structural features. Toxicological Sciences 2009; 112(2):385-93.

Kavlock RJ, Austin CP, Tice RR. Toxicity Testing in the 21st Century: Implications for Human Health Risk Assessment, Risk Analysis 2009; 29(4):485-487.

Parham F, Austin C, Southall N, Xia M, Tice R, Portier C. Dose-response modeling of high-throughput screening data. Journal of Biomolecular Screening 2009; 14(10):1216-27.

Xia M, Huang R, Witt KL, Southall N, Fostel J, Cho MH, Jadhav A, Smith CS, Inglese J, Portier CJ, Tice RR, Austin CP. Compound cytotoxicity profiling using quantitative high-throughput screening. Environ Health Perspect. 2008 Mar;116(3):284-91

Xia M, Huang R, Sun Y, Semenza GL, Aldred SF, Witt KL, Inglese J, Tice RR, Austin CP. Identification of Chemical Compounds that Induce 1 HIF-1α Activity. Toxicological Sciences 2009;112(1): 153163.