National Toxicology Program

U.S. Department of Health and Human Services

National Toxicology Program

PO Box 12233

111 TW Alexander Drive

Research Triangle Park, NC 27709

http://ntp.niehs.nih.gov

Printed on recycled paper NIH Publication No. 15-5970
National Toxicology Program

ANNUAL REPORT

for

Fiscal Year 2014

National Institute of Environmental Health Sciences 

National Institutes of Health

National Center for Toxicological Research

U.S. Food and Drug Administration

National Institute for Occupational Safety and Health

Centers for Disease Control and Prevention

August 2015

U.S. Department of Health and Human Services

National Toxicology Program

NIH Publication No. 15-5970

Table of Contents

Letter From the NIEHS and NTP Director.............................................................................................. 1

1. National Toxicology Program: Mission and Goals........................................................................ 3

A. Organizational Structure and Oversight ....................................................................................... 4

B. Training Programs ........................................................................................................................ 6

C. Advisory Boards and Committees ................................................................................................ 6

i. NTP Executive Committee .................................................................................................6

ii.	 NTP Board of Scientific Counselors ...................................................................................7

iii.	 Scientific Advisory Committee on Alternative Toxicological Methods .................................9

iv. Special Emphasis Panels.................................................................................................11

2. Funding ........................................................................................................................................... 13

A. Current and Projected Research Capacity ................................................................................. 13

B. Interagency Agreements............................................................................................................. 15

i. FDA/NCTR .......................................................................................................................15

ii. CDC/NIOSH .....................................................................................................................15

iii. CDC/DLS..........................................................................................................................15

iv. NIH/NCATS/DPI ...............................................................................................................16

3. NTP Highlighted Activities............................................................................................................. 17

A. West Virginia Chemical Spill....................................................................................................... 17

B. Systematic Review and Evidence Integration for Literature-Based Environmental 

Health Science Assessments ..................................................................................................... 18

C.	 Lung Tumors in Mice Induced By Whole-Life Inorganic Arsenic Exposure at

Human-Relevant Doses ............................................................................................................. 18

D. National Research Council Reports Endorse the Listings of Formaldehyde and 

Styrene in Report on Carcinogens ............................................................................................. 18

E. Nonneoplastic Lesion Atlas ........................................................................................................ 19

F. Crowdsourcing Human Variability Data ...................................................................................... 20

G.	 Mouse Methylome Project .......................................................................................................... 20

H. NTP Impact on Regulatory Agencies.......................................................................................... 21

I. Additional Activities..................................................................................................................... 23

4. Literature Analysis Activities ........................................................................................................ 24

A. Noncancer Health Effects .......................................................................................................... 24

B. Report on Carcinogens............................................................................................................... 26

5. Testing and Research .................................................................................................................... 29

A. Technical Reports ....................................................................................................................... 30

B. NTP Testing ................................................................................................................................ 32

i.	 Disposition, Metabolism, and Toxicokinetic Studies .........................................................32

ii. Genetic Toxicity ................................................................................................................33

iii. Organ System Toxicity......................................................................................................34

iv. Toxicology and Carcinogenicity Studies ...........................................................................36

v. Toxicogenomic Studies.....................................................................................................40

vi. Project Review Committee Approved...............................................................................43
C. NTP Research ............................................................................................................................ 44

i.	 Assessing Exposure to Substances in the Workplace .....................................................46

ii. Comprehensive Assessment of Occupationally-Relevant Exposures..............................51

iii. Immunotoxicology Research ............................................................................................52

iv. NTP Research in Partnership with NCTR ........................................................................54

6. Alternative Methods Development................................................................................................ 63

A. Tox21 .......................................................................................................................................... 63

B.	 NICEATM.................................................................................................................................... 69

i.	 Workshops........................................................................................................................70

ii.	 NICEATM Support of Tox21 .............................................................................................71

iii.	 Other NICEATM Activities.................................................................................................71

C.	 ICCVAM...................................................................................................................................... 72

i.	 ICCVAM Activities ............................................................................................................72

ii.	 Test Method Evaluation Activities .....................................................................................73

iii. International Validation Activities ......................................................................................73

7. Public Tools .................................................................................................................................... 75

A. Nonneoplastic Lesion Atlas ........................................................................................................ 75

B. Chemical Effects in Biological Systems (CEBS) Database ........................................................ 75

C. NTP Archives.............................................................................................................................. 76

D. The NCGC BioPlanet ................................................................................................................. 76

E.	 DrugMatrix and ToxFX................................................................................................................ 76

F.	 Meta Data Viewer....................................................................................................................... 77

8. Appendices ..................................................................................................................................... 78

A. Program Contact Information ..................................................................................................... 78

B. Frequently Used Abbreviations................................................................................................... 78

C. FY 2013 Bibliography ................................................................................................................. 80

i. Reports.............................................................................................................................80

ii. Journal Publications .........................................................................................................80

iii.	 Book Chapters..................................................................................................................97
Letter From the NIEHS and NTP Director 1

Letter From the NIEHS and NTP Director

In FY 2014, the National Toxicology Program (NTP) made significant

accomplishments in testing chemicals of public health concern and 

in developing and validating improved toxicological methods. NTP 

also successfully collaborated with other federal agencies to provide 

information about environmental and public health. 

NTP received a nomination from the Centers for Disease Control and 

Prevention Agency for Toxic Substances and Disease Registry to conduct 

toxicity studies on the predominant chemicals known to be involved in the

West Virginia chemical spill. In response, NTP began a research program 

aimed at producing results in a fairly short period of time that could be 

used for making decisions about the need for any follow-up studies. The

NTP studies are focused on acute exposures and the potential for longterm effects, such as genetic toxicity and reproductive and developmental 

toxicity, and short-term effects such as skin irritation and hypersensitivity.

The West Virginia research program includes regular communications with 

the public about study results through the NTP website. 

NTP also developed a framework to address environmental health questions by applying systematic review

approaches developed for clinical medicine to environmental health sciences. The seven-step framework,

published by the NTP Office of Health Assessment and Translation, is used to evaluate scientific literature and

reach conclusions in literature-based, noncancer health assessments. 

With its partners in the federal government, NTP continued to provide data to the public from the Tox21 

initiative through various databases, such as PubChem, ACTor, and CEBS. The Tox21 initiative aims to 

develop more efficient approaches to predict how chemicals may affect human health.

The National Research Council’s review of the Report on Carcinogens listings of styrene as reasonably 

anticipated to be a human carcinogen, and formaldehyde as known to be a human carcinogen, were

completed. The council supported and reconfirmed the listings based on reviews of updated literature.

In addition, NTP published numerous studies on substances of public health concern, including heavy metals, 

flame retardants, and widely used industrial chemicals.

I proudly present our FY 2014 accomplishments and ongoing efforts.

Linda S. Birnbaum, Ph.D., D.A.B.T., A.T.S.
2 National Toxicology Program Annual Report FY 2014
1. National Toxicology Program: Mission and Goals 3

1. National Toxicology Program: Mission and Goals

Safeguarding public health depends upon identifying the effects of 

substances that are in contact with people and their environment, 

and determining the levels of exposure at which they may become 

potentially hazardous. The Toxic Substances Control Act Chemical 

Substance Inventory of January 2015 (www.epa.gov/oppt/

existingchemicals/pubs/tscainventory/basic.html) lists more than 

84,000 chemicals as being available for sale and use in the U.S. 

New chemicals are continuously introduced into the U.S. market

each year. While the effects of many of these substances on human 

health are unknown, people may be exposed to them during their

manufacture, distribution, use, and disposal, or as pollutants in our 

air, water, and soil. 

The U.S. Department of Health, Education, and Welfare, now the 

U.S. Department of Health and Human Services (HHS), established 

NTP in 1978 to coordinate toxicology testing in the federal 

government. In carrying out its mission, NTP has several goals.

● Coordinate toxicology testing programs within the federal government.

● Strengthen the science base in toxicology.

● Develop and validate improved testing methods. 

● Provide information about potentially toxic chemicals to health agencies, regulatory agencies, research 

agencies, scientific communities, medical communities, and the public.

To protect public health, regulatory agencies make decisions based on scientific information. NTP plays a

critical role in providing scientific data, interpretation, and guidance in the appropriate uses of these data

to regulatory agencies and other health-related research groups. The American people and government 

agencies, at state and federal levels, rely on NTP to provide a strong scientific basis for making credible

decisions that will protect public health. In the past 37 years, NTP has studied and shared information on 

the health effects of more than 2,500 substances, including dietary supplements, industrial chemicals, and 

consumer products. 

In following government-wide efforts to increase access to the results of federally funded scientific

research, NTP maintains open communications and dialogue with federal and state agencies, industry, 

nongovernmental organizations, academia, and the public. The NTP website (http://ntp.niehs.nih.gov) 

provides the public with a variety of information, including Federal Register notices, status of and data from 

NTP studies, lists of NTP reports and journal publications, media releases, calendar of upcoming events, 

and the NTP Update newsletter. 

The public and other interested parties can stay abreast of NTP activities and events by subscribing to the NTP 

listserv, an email notification system (http://ntp.niehs.nih.gov/go/getnews). In addition, requests for information 

can be made through the Central Data Management office (CDM@niehs.nih.gov or 919-541-3419), or the 

Freedom of Information Act coordinator (www.niehs.nih.gov/about/od/ocpl/foia/contact).

NTP welcomes input on its programs and priorities. This input can be through response to formal requests for 

public comment in Federal Register notices, or informal submissions to the NTP Office of Liaison, Policy, and

Review (919-541-7539 or ntpinfo@niehs.nih.gov).

TO EVALUATE 

AGENTS OF PUBLIC 

HEALTH CONCERN 

BY DEVELOPING 

AND APPLYING THE 

TOOLS OF MODERN 

TOXICOLOGY AND 

MOLECULAR BIOLOGY

NTP MISSION:
4 National Toxicology Program Annual Report FY 2014

A. Organizational Structure and Oversight

Three agencies form the core for NTP (Figure 1): the National Institute for Occupational Safety and Health 

(NIOSH) of the Centers for Disease Control and Prevention; the U.S. Food and Drug Administration (FDA), 

primarily through the National Center for Toxicological Research (NCTR); and the National Institute of 

Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH).

NTP is located administratively at NIEHS, and Linda Birnbaum, Ph.D., serves as director of both NIEHS and 

NTP. John Bucher, Ph.D., is the NTP associate director and director of the NTP Division at NIEHS, herein 

referred to as NIEHS/NTP, which is the focal point for NTP activities. NIEHS and NTP utilize best research 

practices and embrace developments in technology to discover how the environment affects people, and 

seek to lead the field of environmental health sciences in innovation and the application of research to solve

health problems.

John Howard, M.D., is the director of NIOSH, and Elizabeth Whelan, Ph.D., chief of the Industrywide Studies

Branch of the Division of Surveillance, Hazard Evaluations, and Field Studies, manages the NTP program 

within NIOSH, herein referred to as NIOSH/NTP. Staff from three NIOSH divisions participate in NTP activities: 

the Division of Surveillance, Hazard Evaluations, and Field Studies, and the Division of Applied Research and 

Technology; Education and Information Division; and Health Effects Laboratory Division.

The mission of NIOSH is to generate new knowledge in the field of occupational safety and health, and to

transfer that knowledge into practice for the betterment of workers. NIOSH’s participation in NTP is consistent

with its mandate to protect workers’ health and safety under the Occupational Safety and Health Act, and the

Federal Mine Safety and Health Act.

William Slikker Jr., Ph.D., is the director of NCTR, and Paul Howard, Ph.D., associate director of the Office of

Scientific Coordination, manages the NTP program within NCTR, herein referred to as NCTR/NTP. NCTR staff

scientists, in partnership with researchers from elsewhere in FDA, other government agencies, academia, and 

industry, provide innovative technology, methods development, vital scientific training, and technical expertise.

NCTR conducts an array of studies that reflect the NTP mission and is critical in supporting FDA product

centers and their regulatory roles.

Figure 1. Organizational Structure of NTP

U.S. Assistant Secretary for Health

NTP Core Agencies Policy Oversight External and Science Oversight and Review

NTP Director

• CDC/NIOSH • NTP Executive Committee

• FDA/NCTR

• NIH/NIEHS

• Board of Scientific Counselors

• SACATM

• Special Emphasis Panels
1. National Toxicology Program: Mission and Goals 5

NIEHS/NTP Staff 

NIOSH Staff: Division of Applied Research and 

Technology and the Division of Surveillance, Hazard 

Evaluations, and Field Studies

NCTR/NTP Staff

NIOSH Staff: Health Effects Laboratory Division

NIOSH Staff: Education and Information Division

NCTR/NTP Staff
6 National Toxicology Program Annual Report FY 2014

B. Training Programs

NTP offers a limited number of postdoctoral training fellowships that prepare scientists for careers in 

pharmaceutical and chemical industries, regulatory agencies, and academia. Full details on opportunities, 

benefits, and applications can be found at http://www.niehs.nih.gov/careers/research/postdoc-training. 

The training program falls into six areas: applied toxicology and carcinogenesis, biomolecular screening 

and computational toxicology, health assessment and translation, laboratory animal medicine, systems 

and mechanistic toxicology, and toxicological pathology. In FY 2014, NTP staff mentored 19 postdoctoral 

fellows at NIEHS.

Table 1. NTP Training Program Postdoctoral Fellows in FY 2014

Training Program Fellow

Applied toxicology and carcinogenesis 

Natasha Catlin

Georgia Hinkley

Kristen Ryan

Brian Sayers

In Ok Surh

Sheetal Thakur

Biomolecular screening and computational toxicology

Rachel Goldsmith

Jui-Hua Hsieh

Sreenivasa Ramaiahgari

Health assessment and translation Katie Pelch

Laboratory animal medicine Sheba Churchill

Systems and mechanistic toxicology

Xiaohua Gao

Ntube Ngalame

Ruben Orihuela Garcia

Rachel Person

Yuanyuan Xu

Toxicological pathology

Sachin Bhusari

Tanasa Osborne

Erin Quist

C. Advisory Boards and Committees

NTP receives policy guidance, science oversight, and peer review from formal external groups including 

the NTP Executive Committee, NTP Board of Scientific Counselors, and Scientific Advisory Committee on

Alternative Toxicological Methods. Ad hoc special emphasis panels also help guide NTP activities.

i. NTP Executive Committee

The NTP Executive Committee provides programmatic and policy oversight to the NTP director. The Executive 

Committee meets once or twice a year in closed forum. Members of this committee include the heads, or their

designees, from the following federal agencies:
1. National Toxicology Program: Mission and Goals 7

● U.S. Consumer Product Safety Commission (CPSC)

● U.S. Department of Defense (DOD)

● U.S. Environmental Protection Agency (EPA)

● U.S. Food and Drug Administration (FDA)

● National Cancer Institute (NCI)

● National Center for Environmental Health/Agency for Toxic Substances and Disease Registry (NCEH/

ATSDR)

● National Institute of Environmental Health Sciences (NIEHS)

● National Institute for Occupational Safety and Health (NIOSH)

● Occupational Safety and Health Administration (OSHA)

To enhance agency interactions, NTP uses agency points of contact, in lieu of formal committees, to 

streamline communication. Agency points of contact have a dedicated responsibility and time commitment; are 

knowledgeable about the NTP mission, programs, and their agency’s resources; and allow the most relevant

agency expertise to be brought to bear on NTP issues. 

ii. NTP Board of Scientific Counselors

The NTP Board of Scientific Counselors (BSC), a federally chartered advisory group, provides scientific

oversight to NTP on the scientific merit of its programs and activities. The HHS Secretary appoints members

to the BSC. The BSC can consist of up to 35 scientists, primarily from the public and private sectors, with 

scientific expertise relevant to NTP activities. The BSC charter and current roster are available at http://ntp.

niehs.nih.gov/go/164. Lori White, Ph.D., designated federal officer, manages the BSC. Table 2 provides the 

roster for FY 2014.

BSC members and NTP staff at the June 2014 BSC meeting
8 National Toxicology Program Annual Report FY 2014

The BSC met twice in FY 2014 (http://ntp.niehs.nih.gov/go/9741). At the meeting on April 17-18, 2014, which 

was rescheduled from December 2013 due to the federal government shutdown, the BSC:

● Reviewed the NTP Laboratory and its chief, Michael Waalkes, Ph.D.

● Reviewed three draft Report on Carcinogens (RoC) concepts: (1) selected viruses: Epstein-Barr virus, 

human immunodeficiency virus type 1, human T-cell lymphotrophic virus type 1, Kaposi sarcomaassociated herpes virus, and Merkel cell polyoma virus; (2) goldenseal root powder; and (3) cobalt.

● Heard a report on the RoC peer-review meeting on ortho-toluidine, and pentachlorophenol and byproducts of its synthesis, held in December 2013. 

● Reviewed two draft Office of Health Assessment and Translation (OHAT) concepts, Adverse Health

Effects Associated With Occupational Exposure to Cancer Chemotherapy Agents, and Pregnancy 

Outcomes Associated With Traffic-Related Air Pollution.

● Heard a report on the draft NTP Technical Reports peer review of vinylidene chloride, cobalt metal, 

glycidamide, and tetrabromobisphenol A, held in October 2013. 

● Was updated by the NIEHS and NTP director, and NTP associate director, on NTP progress since the 

June 2013 meeting.

The second BSC meeting was held June 17-18, 2014. During this meeting, the BSC: 

● Reviewed four NTP research concepts on biphenol S, triclocarban, alkylbenzenes, and xylenes.

● Reviewed two OHAT concepts, NIEHS-EPA Collaborative Project to Improve Characterization of 

Personal Care Product and Home Exposures, and Inflammation-Based Atherosclerosis Associated

With Environmental Exposures.

● Heard about the progress on Tox21, and The S1500 Genes High Throughput Transcriptomics Project 

from the Biomolecular Screening Branch. 

● Was updated on the current work on the adverse outcome pathways by the NTP Interagency Center

for the Evaluation of Alternative Toxicological Methods (NICEATM).

● Heard from the NIEHS and NTP director, and NTP associate director, on NTP progress since the April 

2014 meeting.

Table 2. NTP Board of Scientific Counselors Membership Roster FY 2014

Name and Title Affiliation Term Ends

Milton L. Brown, M.D., Ph.D.

Director

Drug Discovery Program

Georgetown University 

Medical Center

Washington, D.C.

06/30/17

Robert E. Chapin, Ph.D.

Laboratory Director

Pfizer

Groton, Connecticut 06/30/15

George B. Corcoran, Ph.D., A.T.S.

Chair and Professor 

Department of Pharmaceutical Sciences

Eugene Applebaum College of Pharmacy and Health Sciences

Wayne State University

Detroit, Michigan 06/30/16

David C. Dorman, D.V.M., Ph.D.

Professor

College of Veterinary Medicine

North Carolina State University

Raleigh, North Carolina 06/30/15
1. National Toxicology Program: Mission and Goals 9

Name and Title Affiliation Term Ends

Mary Beth Genter, Ph.D.

Associate Professor

Department of Environmental Health

University of Cincinnati

Goshan, Ohio 06/30/17

Jack R. Harkema, D.V.M., Ph.D., D.A.C.V.P.

Distinguished Professor

Department of Pathobiology and Diagnostic Investigation

Michigan State University

East Lansing, Michigan 06/30/15

Dale Hattis, Ph.D.

Research Professor

George Perkins Marsh Institute

Clark University

Worcester, Massachusetts 06/30/15

Steven Markowitz, M.D., Dr.P.H.

Professor and Director

Center for the Biology of Natural Systems

Queens College

City University of New York

Flushing, New York

06/30/17

Lisa A. Peterson, Ph.D., Chair of the BSC

Professor

Division of Environmental Health Sciences and Masonic Center

School of Public Health

University of Minnesota

Minneapolis, Minnesota 06/30/16

Sonya Sobrian, Ph.D.

Associate Professor

Department of Pharmacology

Howard University

Washington, D.C. 06/30/15

Iris G. Udasin, M.D.

Professor

Department of Environmental and Occupational Medicine

Robert Wood Johnson Medical School

University of Medicine and Dentistry

of New Jersey

Piscataway, New Jersey 

06/30/16

iii. Scientific Advisory Committee on Alternative Toxicological Methods

The Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) is a federally chartered

advisory committee established January 9, 2002, in response to the Interagency Coordinating Committee 

on the Validation of Alternative Methods (ICCVAM) Authorization Act of 2000 (42 U.S.C. 285l-3(d)). SACATM

advises ICCVAM, the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods

(NICEATM), and the director of NIEHS and NTP regarding statutorily mandated duties of ICCVAM and

activities of NICEATM (see pages 69 and 72). SACATM provides advice on priorities and activities related

to the development, validation, scientific review, regulatory acceptance, implementation, and national and

international harmonization of new, revised, and alternative toxicological test methods. The SACATM charter

and current roster are available at http://ntp.niehs.nih.gov/go/167. Table 3 provides the roster for FY 2014. 

SACATM typically meets once a year and members serve rotating terms of up to four years. Lori White, Ph.D.,

designated federal officer, manages SACATM.

SACATM met once during FY 2014 on September 16, 2014, at NIEHS (http://ntp.niehs.nih.gov/go/8202). 

At the meeting, ICCVAM and NICEATM updated SACATM on the new goals for ICCVAM and on activities

by NICEATM and the International Cooperation on Alternative Test Methods (ICATM). ICCVAM members

presented an overview of ICCVAM activities related to oral and dermal testing, animal reductions in Leptospira

vaccine testing, and skin sensitization testing. SACATM heard updates on new ICCVAM communication

activities and reports for four meetings: Aquatic Models and 21st Century Toxicology, Murine Histamine

Sensitization Test, Adverse Outcome Pathways, and ICCVAM Public Forum. SACATM provided input on the

reports from four federal agencies, including the U.S. Department of the Interior, FDA, EPA, and NIEHS.
10 National Toxicology Program Annual Report FY 2014

Table 3. NTP SACATM Membership Roster FY 2014

Name and Title Affiliation Term Ends

Lauren E. Black, Ph.D.

Senior Scientific Advisor

Navigators Services

Charles River Laboratories

Reno, Nevada 11/30/16

Tracie E. Bunton, D.V.M., Ph.D.

Consultant

Eicarte LLC

Gettysburg, Pennsylvania 06/30/15

Joy Cavagnaro, Ph.D., D.A.B.T., R.A.C., A.T.S., R.A.P.S.

President and Founder

Access BIO LC

Boyce, Virginia 11/30/14

Joan M. Chapdelaine, Ph.D.

Consultant Scott Township, Pennsylvania 06/30/15

Mark G. Evans, D.V.M., Ph.D., A.C.V.P.

Research Fellow

La Jolla Laboratories 

Pfizer

San Diego, California 06/30/15

Michael D. Kastello, D.V.M., Ph.D.

Vice President and Global Head

Animal Research and Welfare

Disposition, Safety and Animal Research

Sanofi

Bridgewater, New Jersey 11/30/16

Safdar A. Khan, D.V.M., M.S., Ph.D., D.A.B.V.T.

Senior Toxicologist

Senior Director of Toxicology Research

ASPCA Animal Poison Control Center

Urbana, Illinois 11/30/16

Steven M. Niemi, D.V.M. (chair)

Director

Center for Comparative Medicine

Massachusetts General Hospital

Charlestown, Massachusetts 06/30/13

Members of SACATM, ICCVAM, and ICATM, and staff from NIEHS and NTP at the September 

2014 SACATM meeting 
1. National Toxicology Program: Mission and Goals 11

Name and Title Affiliation Term Ends

Ricardo Ochoa, D.V.M., Ph.D., A.C.V.P.

President and Principal

Pre-Clinical Safety, Inc.

Niantic, Connecticut 11/30/14

Catherine E. Willett, Ph.D.

Director

Regulatory Toxicology, Risk Assessment and Alternatives

The Humane Society of the United 

States

Gaithersburg, Maryland

11/30/17

Daniel M. Wilson, Ph.D., D.A.B.T.

Mammalian Toxicology Consultant

Toxicology and Environmental Research and Consulting

The Dow Chemical Company

Midland, Michigan 11/30/14

Wei Xu, Ph.D.

Associate Professor

Department of Oncology

McArdle Laboratory for Cancer Research

University of Wisconsin at Madison

Madison, Wisconsin 11/30/17

iv. Special Emphasis Panels

NTP uses ad hoc scientific panels, referred to as special emphasis panels, to provide independent

scientific peer review and advice on targeted issues, such as agents of public health concern, new and

revised toxicological test methods, and other issues. These panels help ensure transparent, unbiased, and 

scientifically rigorous input to the NTP for its use in making credible decisions about human health hazards,

setting research and testing priorities, and evaluating test methods for toxicity screening. 

NTP Technical Report Peer-Review Panels

NTP Technical Reports (TRs) are published results of long-term studies, generally two-year rodent toxicology 

and carcinogenesis studies. NTP convenes external scientific panels to peer review draft TRs at public

meetings held at NIEHS. All reviews provide the opportunity for public comment. For each TR, the panel 

is charged with peer reviewing the scientific and technical elements and presentation of the study, and

determining whether the study’s experimental design and conduct support the NTP conclusions regarding the 

carcinogenic activity of the substance tested. There were two TR meetings in FY 2014. 

NTP convened a meeting on October 29, 2013 to peer review the draft TRs on cobalt metal, glycidamide, 

tetrabromobisphenol A, and vinylidene chloride. The peer-review panel included individuals with expertise 

in carcinogenesis, neurotoxicology, pathology, inhalation toxicology, reproductive toxicology, and molecular 

toxicology. Lori White, Ph.D., served as designated federal officer for the peer-review meeting.

NTP convened a second meeting on May 22, 2014, to peer review the draft TRs for bromodichloroacetic

acid, CIMSTAR 3800, green tea extract, and indole-3-carbinol. The panel consisted of experts in

biochemical metabolism, genetic toxicology, molecular mechanisms of toxicity, pathology, drug development, 

reproductive toxicology, and preclinical toxicology. Yun Xie, Ph.D., served as designated federal officer for

the peer-review meeting.

Both meetings were open to the public with time scheduled for oral public comment. The charge to both panels 

were to (1) review and evaluate the scientific and technical elements of the study and its presentation; and

(2) determine whether the study’s experimental design, conduct, and findings support the NTP’s conclusions

regarding the carcinogenic activity and toxicity of the substance tested. The panels for both meetings agreed 

with the NTP conclusions in all draft TRs. Additional information about review panel meetings is available at 

http://ntp.niehs.nih.gov/go/36051.
12 National Toxicology Program Annual Report FY 2014

NTP Monograph Peer-Review Panels

Monographs are publications on a single, detailed specific topic. There were no monograph review meetings

in FY 2014. Additional information about NTP monograph peer-review meetings is available at http://ntp.niehs.

nih.gov/go/36639. 

Report on Carcinogens Peer-Review Panels

Report on Carcinogens (RoC) monographs are prepared for each candidate substance selected for review. 

NTP follows an established, four-part process for preparation of the RoC. More information can be found at

http://ntp.niehs.nih.gov/go/rocprocess and on page 26. In FY 2014, NTP convened two external scientific

panels to peer review draft RoC monographs on three candidate substances. The meetings, held at NIEHS, 

were open to the public with time scheduled for oral public comment. Draft RoC monographs, consisting of a 

cancer evaluation component and a substance profile, were prepared for each substance. For each candidate

substance, the panel was charged with commenting on whether the draft cancer evaluation component was 

technically correct and clearly stated, whether NTP objectively presented and assessed the scientific evidence,

and whether the scientific evidence was adequate for applying the listing criteria. For each draft substance

profile, the panel was charged with commenting on whether the scientific justification presented supported the

NTP preliminary policy decision on the RoC listing status. 

On December 12-13, 2013, NTP convened a panel to peer review the draft RoC monographs for orthotoluidine, and pentachlorophenol and by-products of its synthesis. The panel voted on each draft level of 

evidence for carcinogenicity determination, based on the available scientific evidence in experimental animals

and human cancer studies, and whether the information cited in each draft substance profile supported

NTP’s preliminary listing recommendation for the substances in the RoC. The ortho-toluidine review covered 

the chemical properties and human exposure, cancer studies in experimental animals, metabolism and 

mechanistic data, human cancer studies, an overall cancer evaluation, and the draft substance profile. The

pentachlorophenol and by-products of its synthesis review covered the chemical properties and human 

exposure, human cancer studies, studies in experimental animals, supporting mechanistic evidence, an overall 

cancer evaluation, and the draft substance profile.

On August 12, 2014, NTP convened a panel to peer review the draft RoC monograph on trichloroethylene. The 

panel voted on the draft level of evidence for carcinogenicity determination, based on the available scientific

evidence from human cancer studies for kidney cancer, non-Hodgkin lymphoma, and liver cancer, and whether

the information cited in the draft substance profile supported the NTP’s preliminary listing recommendation

for trichloroethylene in the RoC. The review covered human cancer studies; toxicokinetic, toxicological, and

mechanistic studies; an overall cancer evaluation; and the draft substance profile for trichloroethylene.

Lori White, Ph.D., served as designated federal officer for both peer-review meetings. After each meeting, input

from the panels was considered in finalizing the monographs. Additional information about these meetings and

other RoC monograph peer-review meetings is available at http://ntp.niehs.nih.gov/go/38854.
2. Funding 13

2. Funding

A. Current and Projected Research Capacity

NTP relies on voluntary allocations from the program’s three core agencies, NIEHS, FDA/NCTR, and NIOSH, 

to support its activities. These allocations are specified after annual appropriations have been determined. As

shown in Figure 2, the total NTP budget for FY 2014 was $127.5 million.

Figure 2: Past, Current, and Projected Budget

NTP conducts its research studies through contract laboratories, in-house at the three core agencies, or 

through interagency agreements with other agencies (see page 15). In FY 2014, NIEHS funded 35 contracts 

(see Table 4), and held three workshops (see page 70), four special emphasis panel peer-review meetings 

(see page 11), and three scientific advisory meetings (see pages 7 and 9) for NTP.
14 National Toxicology Program Annual Report FY 2014

Table 4. NIEHS Contracts That Supported NTP Activities in FY 2014

Description Contractor

Absorption, Distribution, Metabolism, and Excretion

Lovelace Biomedical 

Research Triangle Institute

Analysis of Bisphenol A in Zebrafish Oregon State University

Analysis support for SEARCH project Johns Hopkins University

Analytical Chemistry Services

Battelle Memorial

Midwest Research Institute

Research Triangle Institute

Archives and Specimen Repository Experimental Pathology Laboratories

Bioinformatics Methylation Project Murdock Research Institute

Chemical Sample Analysis for SEARCH Project University of North Carolina at Chapel Hill

Evaluate Toxicity Following Early Life Exposure Southern Research Institute

Evaluation of Alternative Toxicological Methods ILS Inc.

Evaluation of the Toxicity of Selected Chemicals Battelle Memorial

Genetic Toxicity Testing Support Services Integrated Laboratory Systems

Immunotoxicity Burleson Research Technologies

In Life Data Collection and Management System INSTEM, LSS

NTP Computer and User Support Vistronix Inc.

NTP Information Systems Support Scimetrika

NTP Statistical and Computer Support SRA International

NTP Technical Reports Preparation Support Services Biotechnical Sciences Inc.

OHAT Literature-Based Evaluations ICF Inc.

Pathology Support

Charles River Laboratories International Inc.

Integrated Laboratory Systems

Pathology Support and Quality Assessment Experimental Pathology Laboratories

Production of B6C3F1 Mice Taconic Farms

Productive Assessments by Continuous Breeding Research Triangle Institute

Provision for Animals and Specialized Services Charles River, Jax, Taconic

Quality Assessment Oversight of NTP Toxicology Data System Validation Labscience

Quality Assessment Support, Audits and Inspections Dynamac

Research on Inhalation Toxicology of Environmental Chemicals Alion Science & Technology

Support for GeneCo Databases Thomson Reuters

Support for Preparation of the Report on Carcinogens Integrated Laboratory Systems

Toxicologic and Carcinogenic Potential of Test Agents Battelle Memorial

Toxicological and Carcinogenic Potential of Chemicals Battelle Memorial

Zebrafish Immune Function and Disease Resistance Assays North Carolina State University
2. Funding 15

B. Interagency Agreements

In FY 2014, NIEHS provided support for NTP activities through interagency agreements (IAGs) with other 

federal agencies. 

i. FDA/NCTR

In December 1992, NIEHS and FDA established a formal IAG, where NTP would support toxicology studies 

on FDA-regulated agents nominated to NTP, and conduct the studies at NCTR. These studies are designed to 

provide FDA and other regulatory agencies with hazard identification and dose-response data to support risk

assessment and risk management decisions that could affect public health.

This IAG has supported studies on endocrine active agents, dietary supplements, food contaminants, AIDS 

therapeutics, pediatric medicines, electromagnetic radiation, cosmetics, and nanoscale materials. Studies in 

these areas have produced about 16 published NTP Technical Reports and over 200 peer-reviewed journal 

publications. Some of the data from the IAG-supported studies have led to an increased understanding of the 

pharmacokinetics, mechanism of action, or dose-response of substances. Other data have led to refinement of

risk assessment models.

ii. CDC/NIOSH

NIEHS/NTP has two IAGs with NIOSH. One IAG was established in the early 1990s in general response to 

increased efforts by NTP to study noncancer endpoints. NIOSH and NTP have conducted studies to assess 

the potential toxicity of exposures to substances such as fungi, mycotoxins, volatile organics, lead, latex, 

nickel, isocyanates, and beryllium. Studies included workers that are exposed to mixtures of chemicals, such

as miners, farmers, health care workers, autoworkers, and firefighters. There have also been a number of

studies examining how genetic variability in immune-inflammatory-antioxidant responses contributes to the

development and severity of inflammatory and allergic disease in people of different occupations.

The second IAG involves multiple projects. NIEHS/NTP and NIOSH worked to establish methodologies to

assess complex mixtures, such as asphalt fume, welding fume, and tungsten fibers. NIOSH and NIEHS/

NTP are jointly supporting two large initiatives that evaluate emerging issues in nanotechnology. One 

project focuses on identifying workplaces engaged in the development, production, and use of engineered

nanomaterials, and determining the potential for worker exposure to selected engineered nanoparticles. A

second facet focuses on evaluating potential toxicity from workplace exposures to engineered nanomaterials.

A study with similar purpose and design is evaluating occupational exposure to bisphenol A. 

Many studies performed under these IAGs are published in the peer-reviewed literature and have been used

for hazard identification, and regulatory and intervention purposes.

iii. CDC/DLS

NIEHS/NTP and CDC/Division of Laboratory Sciences (DLS) are participating in a pilot study to characterize 

exposure profiles in a subset of 50 Danish women, who will be enrolled in a larger study of 500 women.

The pilot study is designed to assess whether exposure to several common endocrine-disrupting chemicals 

is related to reproductive health problems, such as infertility, risk of miscarriage, and low birth weight, or

childhood obesity and potentially other health outcomes in the children. Endocrine-disrupting chemicals from 

the organotins and phthalates classes will be evaluated. Blood and urine samples will be collected three 

times from each woman, once prior to pregnancy and twice during pregnancy. This collaboration between 
16 National Toxicology Program Annual Report FY 2014

NIEHS/NTP and CDC/DLS facilitates common goals in understanding individual exposure profiles for multiple

chemicals in woman of reproductive age.

iv. NIH/NCATS/DPI

This IAG supports ongoing and anticipated studies conducted at the National Center for Advancing 

Translational Sciences (NCATS)/Division of Pre-Clinical Innovation (DPI), to evaluate high throughput and 

high content screening assays in support of Tox21. Tox21 is an ongoing collaboration among federal agencies 

to characterize the potential toxicity of chemicals by using cells and isolated molecular targets instead of 

laboratory animals. The collaboration between NIEHS/NTP and NCATS/DPI will produce data for substances 

that lack needed toxicological information. Data from these assays can be used to prioritize substances for

further studies, including toxicological evaluation, mechanisms of actions investigation, and development of 

predictive modeling for biological response. The use of the assays should greatly increase the number of 

substances tested and decrease the cost of testing.
3. NTP Highlighted Activities 17

3. NTP Highlighted Activities

Figure 3 displays the major events for FY 2014. Additional activities of importance from FY 2014 are 

noted below.

Figure 3: FY 2014 Highlights

ICCVAM	 Interagency Coordinating Committee for the Validation of Alternative Methods

NICEATM	 NTP Interagency Center for the Evaluation of Alternative Toxicological Methods

SACATM	 Scientific Advisory Committee on Alternative Toxicological Methods

NTP Technical 

Reports Peer-review 

Panel Meeting

See page 11

Report on 

Carcinogens 

Peer-review 

Panel Meeting

See page 12

Launch of the 

Nonneoplastic 

Lesion Atlas

See page 19

NTP at Society 

of Toxicology 

Meeting in

Phonenix, Arizona

See page 23

Board of Scientific

Counselors Meeting

See page 8

NICEATM Collaborative

Workshop on Aquatic

Models and 21st

Century Toxicology

See page 70

NTP Technical 

Reports Peer-review 

Panel Meeting

See page 11

NTP Satellite 

Symposium 

on Pathology 

Potpourri

See page 23

Board of Scientific

Counselors Meeting

See page 8

ICCVAM

Public Forum

See page 72

National Research 

Council Report 

Endorses the Listing 

of Styrene in the RoC

See page 18

West Virginia Chemical Spill 

Nomination by CDC/ATSDR

See page 17

Release of the OHAT 

Approach, A Systematic Review 

and Evidence Integration for 

Literature-based Environmental 

Health Science Assessments

See page 18

 OCT ‘13 NOV ‘13 DEC ‘13 JAN ’14 FEB ’14 MAR ’14 APR ’14 MAY ’14 JUNE ’14 JULY ’14 AUG ’14 SEPT ’14 

National Research Council 

Report Endorses the Listing 

of Formaldehyde in the RoC

See page 18

Report on 

Carcinogens 

Peer-review 

Panel Meeting

See page 12

NICEATM Workshop

on Adverse 

Outcome Pathways: 

From Research 

to Regulation

See page 70

NICEATM Workshop

on the Murine

Histamine Sensitization 

Test (HIST) for 

Safety Testing of 

Acellular Pertussis

See page 70

SACATM

Meeting

See page 9

A. West Virginia Chemical Spill

In January 2014, about 10,000 gallons of a liquid used for washing coal to remove impurities was spilled 

from a leaking tank into the Elk River in West Virginia. The Elk River is a municipal water source that

serves about 300,000 people in the Charleston area. The spilled material was primarily composed of 

4-methylcyclohexanemethanol (MCHM). Other chemicals, including dipropylene glycol phenyl ether and

propylene glycol phenyl ether, were present in lower amounts. Limited toxicity data are available for the 

constituents of the spilled liquid. In July 2014, the Agency for Toxic Substances and Disease Registry within the 

Centers for Disease Control and Prevention nominated chemicals involved in the spill to NTP for toxicological 

characterization. Shortly after, NTP planned and began short-term toxicology studies to provide information 

relevant to the potential exposures of Charleston residents to the chemicals. More information can be found on

the NTP West Virginia chemical spill Web page http://ntp.niehs.nih.gov/go/wvspill.
18 National Toxicology Program Annual Report FY 2014

B. Systematic Review and Evidence Integration for Literature-Based 

Environmental Health Science Assessments

The Office of Health Assessment and Translation (OHAT)

developed a systematic review framework, the OHAT Approach,

to address environmental health questions by applying review 

approaches developed for clinical medicine to the greater 

range of data relevant to environmental health sciences, such 

as human, animal, and mechanistic studies. These systematic 

review methodologies provide a structure that increases 

transparency in the process of collecting and synthesizing 

scientific evidence for literature-based evaluations.

The OHAT Approach outlines a seven-step framework for

evaluating the scientific literature and reaching conclusions in

NTP literature-based, noncancer health assessments. In FY 

2014, OHAT published the methodology (http://www.ncbi.nlm.nih.

gov/pubmed/24755067). OHAT also held a webinar to present 

lessons learned in applying the OHAT Approach to case studies 

as part of testing the framework during methods development

(http://ntp.niehs.nih.gov/go/41629).

C. Lung Tumors in Mice Induced By Whole-Life Inorganic Arsenic Exposure 

at Human-Relevant Doses

Arsenic is a natural element that is widely distributed in the Earth’s crust. People may be exposed to arsenic 

through water, air, food, and soil. Arsenic is present in the drinking water of millions of people worldwide, and

exposure to high levels of arsenic is linked to higher cancer risk. NIEHS/NTP scientists studied whole-life

inorganic arsenic exposure from drinking water in mice at concentrations relevant to humans.

Mice were given arsenic at 0, 50, 500, or 5,000 parts per billion in their drinking water three weeks before

breeding, and throughout pregnancy and lactation. Arsenic at these same concentrations was then given to the 

offspring after weaning and all through adulthood. The researchers examined the adult offspring for tumors. 

Over half of the male offspring developed significant increases in benign and malignant lung tumors at 50 parts

per billion and 500 parts per billion. Female offspring developed benign tumors at the lower concentrations. 

This study provides evidence that there is potential for adverse health effects from very low exposures to 

arsenic. The study published in the journal Archives of Toxicology is available at http://www.ncbi.nlm.nih.gov/

pubmed/25005685.

D. National Research Council Reports Endorse the Listings of Formaldehyde 

and Styrene in Report on Carcinogens

In FY 2014, reports from the National Research Council (NRC) of the National Academy of Sciences 

supported the listing of styrene and formaldehyde in the 12th Report on Carcinogens (RoC), which was 

published in 2011. Styrene is an industrial chemical used to make plastics, resins, and rubber. Formaldehyde

is a major industrial chemical used in building materials, chemical manufacturing, household products, and 

other industries.
3. NTP Highlighted Activities 19

Congress directed the U.S. Department of Health and Human Services (HHS) to arrange for the National 

Academy of Sciences to independently review the listings and substance profiles for formaldehyde

and styrene. The NRC convened expert committees to conduct scientific peer reviews of styrene and

formaldehyde, and to carry out an independent assessment of the scientific literature published on both

chemicals through 2013. 

In July 2014, the NRC committee for styrene endorsed the listing of styrene as reasonably anticipated to 

be a human carcinogen, and reached a similar conclusion based on their independent assessment. They 

also agreed with the NTP scientific justification to support the listing, including (1) limited evidence of the

carcinogenicity of styrene from studies in humans; (2) sufficient evidence of the carcinogenicity of styrene

from studies in experimental animals; and (3) convincing, relevant information that the chemical acts 

through mechanisms that would cause cancer in humans. More information can be found at http://www8.

nationalacademies.org/onpinews/newsitem.aspx?RecordID=18725.

In August 2014, the NRC committee for formaldehyde endorsed the listing of formaldehyde as known to be

a human carcinogen, and reached a similar conclusion based on their independent assessment. Similar to 

the NTP assessment, they concluded there was (1) sufficient evidence of carcinogenicity of formaldehyde

from studies in humans based on consistent findings of increased incidence of nasopharyngeal cancer,

sinonasal cancer, and myeloid leukemia for which chance, bias, and confounding factors can be ruled out with

reasonable confidence; (2) sufficient evidence of the carcinogenicity from studies from experimental animals;

and (3) convincing, relevant information that formaldehyde induces mechanistic events associated with the 

development of cancer in humans. More information can be found at http://www8.nationalacademies.org/

onpinews/newsitem.aspx?RecordID=18948.

E. Nonneoplastic Lesion Atlas

In FY 2014, NTP continued work on the NTP

Nonneoplastic Lesion Atlas, a Web-based resource that 

currently contains hundreds, and will eventually contain 

thousands, of high-quality images and guidelines for 

nonneoplastic lesions in experimental rodent models. 

While nonneoplastic lesions are not cancerous, 

nonneoplastic diseases, such as cardiovascular and 

pulmonary diseases, are a major cause of illness and 

death, and many are thought to have environmental 

causes. For example, forms of pulmonary fibrosis, a

disease that causes lung scarring, have been linked

to exposures to inorganic materials such as asbestos, 

vanadium, cobalt, nickel, beryllium, and sulfur dioxide;

and organic materials such as dust from cotton, grain, 

and wood. 

Diagnosing and recording nonneoplastic lesions can be challenging, and terminology and diagnostic strategies 

can vary among pathologists. The purpose of the NTP Nonneoplastic Lesion Atlas is to standardize the 

terminology, diagnostic strategy, and recording of nonneoplastic rodent lesions; improve the consistency; 

and facilitate database searches, comparisons between studies, and generation of historical control data 

for nonneoplastic lesions. The atlas was launched in FY 2014 and will be completed in FY 2015. For more 

information, see http://ntp.niehs.nih.gov/nnl.
20 National Toxicology Program Annual Report FY 2014

F. Crowdsourcing Human Variability Data

In FY 2013, NIEHS, NCATS, the University of North Carolina at Chapel Hill (UNC), Sage Bionetworks,

and DREAM teamed up to launch an innovative crowdsourced challenge, the NIEHS-NCATS-UNC

DREAM Toxicogenetics Challenge, that utilized data collected from the Tox21 1000 Genomes Toxicity

Screening Project. The NIEHS-NCATS-UNC team conducted the largest ever, population-based, in vitro 

cytotoxicity study to understand how genetic variation affects individual response to common environmental 

and pharmaceutical chemicals. The study evaluated the extent of cytotoxicity induced in 1,086 human 

lymphoblastoid cell lines by 179 pharmaceutical or environmental chemicals. The 1,086 cell lines represented 

nine distinct geographical populations with defined genetic heterogeneity.

The crowdsourced challenge asked participants to use data from the toxicogenetics project to meet two

subchallenges: (1) use the biological data to develop a model that accurately predicts individual responses to 

chemical exposure, and (2) use the data on chemical properties to develop a model that accurately predicts 

how a particular population will respond to certain types of chemicals.

The challenge launched in June 2013 and closed in September 2013. There were 99 submissions from 34 

teams for subchallenge 1, and 85 submissions from 24 teams for subchallenge 2. The top-scoring teams were 

announced at the November 2013 DREAM Conference in Toronto. The winning teams for both challenges

were from the Quantitative Biomedical Research Center at the University of Texas Southwestern Medical

Center in Dallas. The results of the challenge will be published in Nature Biotechnology.

G. Mouse Methylome Project

The methylome, a component of the epigenome, is one of the factors that may affect susceptibility to cancer 

and other chemical exposure-related diseases. The methylome is an individual’s genome-wide pattern of 

cytosine methylation, which is the addition of a methyl group to cytosine, one of the four major bases of DNA. 

Presently, there is no central mouse reference database for the methylome, which significantly handicaps an

understanding of the mouse model in toxicology and environmentally related diseases, and the designing and 

conducting of research to understand associated mechanisms. 

The Mouse Methylome Project is creating a high-resolution map of the mouse liver methylome from three

different mouse strains — two parental strains C57BL/6N and C3H/HeN, and their first generation hybrid

offspring B6C3F1/N. These strains show dramatically different incidences of spontaneous liver tumors, which 

often confound two-year toxicology and carcinogenesis studies. This variable incidence of liver cancer may 

be due, in part, to differences in the epigenetic machinery, and also in the sites and amounts of cytosine 

methylation in critical tumor suppressor genes and other regulatory regions of the genome that affect liver 

cancer susceptibility and its heritability across generations. 

NIEHS scientists collected liver samples, as well as samples of four other tissues, including brain, cardiac 

and skeletal muscle, brown and white fat, and epididymal sperm, from all mice, at the same age, to minimize

age as a confounding factor for gene expression and epigenetics. They prepared sequencing libraries of 

the liver samples and banked samples of the other four tissue types for future use. In FY 2014, scientists

completed characterization of the liver methylome by genomic sequencing and bisulfite treatment of DNA,

to map all methylated sites in the mouse genome. Ongoing bioinformatics analyses found relationships with 

gene expression, and differentially methylated regions related to gender, strain, and heritability. Analyses 

will continue in FY 2015, methylome and genomic data will be made publicly available, and a manuscript is 

currently in the works.
3. NTP Highlighted Activities 21

H. NTP Impact on Regulatory Agencies

Federal and state regulatory agencies use NTP study data and recommendations in considering the need to 

regulate and test specific chemicals to protect human health. Table 5 lists NTP data and recommendations 

used by other agencies in FY 2014.

Table 5. Use of NTP Study Data or Recommendations by Federal and State Regulatory 

Agencies in FY 2014

Notice Summary of Notice NTP Information Cited

California Office of Environmental Health Hazard Assessment

Chemical Listed 

Effective May 2, 2014

as Known to the State 

of California to Cause 

Cancer: N,N-dimethylp-toluidine

Effective May 2, 2014, the Office of

Environmental Health Hazard Assessment is 

adding N,N-dimethyl-p-toluidine to the list of 

chemicals known to the state to cause cancer

for purposes of Proposition 65. 

May 02, 2014 -- Proposition 65

http://oehha.ca.gov/prop65/prop65_

list/050214list.html

NTP. 2012. National Toxicology Program 

Technical Report on the Toxicology and 

Carcinogenesis Studies of N,N-Dimethyl-pToluidine (CAS No. 99-97-8) in F344/N Rats and 

B6C3F1/N Mice (Gavage Studies). NTP TR 579,

NIH Publication No. 12-5921. Research Triangle 

Park, North Carolina. http://ntp.niehs.nih.gov/

go/37162. 

Notice of Adoption of 

Reference Exposure 

Levels for Benzene

The Office of Environmental Health Hazard

Assessment is adopting new and revised 

Reference Exposure Levels for benzene. 

June 27, 2014 -- Proposition 65

http://www.oehha.ca.gov/air/chronic_rels/

BenzeneJune2014.html

NTP conducted a chronic two-year toxicity 

bioassay in F344 rats and B6C3F1 mice of 

benzene by gavage in corn oil. Doses were 0, 25, 

50, and 100 mg/kg-day for females and 0, 50,

100, and 200 mg/kg-day for males. Dose-related

lymphocytopenia and leukocytopenia were

observed in both species in all dosed groups. 

Mice exhibited lymphoid depletion of the thymus

and spleen and hyperplasia of the bone marrow.

NTP. 1986. National Toxicology Program 

Technical Report on the Toxicology and 

Carcinogenesis Studies of Benzene (CAS No. 

71-43-2) in F344/N Rats and B6C3F1 Mice

(Gavage Studies). NTP TR 289, NIH Publication 

No. 86-2545. Research Triangle Park, NC. http://

ntp.niehs.nih.gov/go/7460. 
22 National Toxicology Program Annual Report FY 2014

Notice Summary of Notice NTP Information Cited

Environmental Protection Agency

Final Rule: Control 

of Air Pollution from 

Motor Vehicles: Tier

3 Motor Vehicle

Emission and Fuel 

Standards

This action establishes more stringent vehicle 

emissions standards and will reduce the sulfur 

content of gasoline beginning in 2017, as part of 

a systems approach to addressing the impacts 

of motor vehicles and fuels on air quality and 

public health. The gasoline sulfur standard will 

make emission control systems more effective

for both existing and new vehicles, and enable 

more stringent vehicle emissions standards. 

The vehicle standards will reduce both tailpipe 

and evaporative emissions from vehicles, 

resulting in significant reductions in pollutants

and help state and local agencies in their efforts 

to attain and maintain health-based National 

Ambient Air Quality Standards. These vehicle 

standards are intended to harmonize with 

California’s Low Emission Vehicle program, thus 

creating a federal vehicle emissions program 

that will allow automakers to sell the same

vehicles in all 50 states. The vehicle standards 

will be implemented over the same timeframe 

as the greenhouse gas/fuel efficiency standards

for light-duty vehicles (promulgated by EPA and 

the National Highway Safety Administration in 

2012), as part of a comprehensive approach 

toward regulating emissions from motor 

vehicles. 

April 28, 2014 -- 79 FR 23414

http://www.gpo.gov/fdsys/pkg/FR-2014-04-28/

pdf/2014-06954.pdf

In the NTP Report on Carcinogens the following 

compounds have been listed as: (1) known

carcinogens to humans: benzene, 1,3-butadiene, 

formaldehyde; (2) possibly carcinogenic to 

humans: acetaldehyde; or (3) reasonably 

anticipated to be a human carcinogen: 

naphthalene.

http://ntp.niehs.nih.gov/go/roc13

Final Rule: 

Diflubenzuron;

Pesticide Tolerances

This regulation establishes tolerances for 

residues of diflubenzuron (N-[(4-chlorophenyl)

amino]carbonyl]-2,6-difluorobenzimide) in

or on fruit, citrus, group 10-10 and citrus, 

oil. Chemtura Corporation requested these 

tolerances under the Federal Food, Drug, and 

Cosmetic Act. 

January 31, 2014 -- 79 FR 5294

http://www.gpo.gov/fdsys/pkg/FR-2014-01-31/

html/2014-02064.htm

Para-chloroaniline hydrochloride, a plant 

metabolite of diflubenzuron, tested positive for

splenic tumors in male rats and hepatocellular 

adenomas/carcinomas in male mice.

NTP. 1989. National Toxicology Program 

Technical Report on the Toxicology and 

Carcinogenesis Studies of para-Chloroaniline 

Hydrochloride (CAS No. 20265-96-7) in F344/N 

Rats and B6C3F1 Mice (Gavage Studies). NTP

TR 351, NIH Publication No. 89-2806. Research 

Triangle Park, North Carolina. http://ntp.niehs.nih.

gov/go/6963. 

A complete listing of NTP studies used by federal and state regulatory agencies can be found at http://ntp.

niehs.nih.gov/go/regact.
3. NTP Highlighted Activities 23

I. Additional Activities

NTP participates in a number of meetings with stakeholders and the scientific community. At the 2014 annual

meeting of the Society of Toxicology (SOT) in Phoenix, staff from NTP and NIEHS participated in more than 30 

workshops, symposia, and platform sessions; education and information sessions; and poster sessions. The

full program, including all NTP and NIEHS activities, can be found at http://www.toxicology.org/. 

NTP also hosts symposiums and workshops, to discuss the state of the science and advance the field. For

example, the 2014 annual NTP Satellite Symposium, Pathology Potpourri, was held in Washington, D.C., June 

21, in advance of the 33rd annual meeting of the Society of Toxicologic Pathology. The goal of the annual NTP 

symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology 

community. The Proceedings of the 2014 National Toxicology Program Satellite Symposium was published in 

the journal Toxicologic Pathology, along with summaries of presentations, including diagnostic or nomenclature 

issues that were presented, and select images that were used for audience voting and discussion.

NTP also hosted three workshops in FY 2014 related to alternative methods development (see page 70).
24 National Toxicology Program Annual Report FY 2014

4. Literature Analysis Activities

NTP conducts literature analysis and review to examine the state of the science and assess if a substance has 

adverse health effects.

A. Noncancer Health Effects 

NTP has made a commitment to studying noncancer health effects. The Office of Health Assessment and

Translation (OHAT) within NIEHS/NTP conducts evaluations to assess the evidence that environmental 

chemicals, physical substances, or mixtures, collectively referred to as substances, cause adverse health 

effects. OHAT also provides opinions on whether these substances may be of concern, given what is known

about current human exposure levels. Assessments of potential adverse effects of environmental substances 

on reproduction or development, carried out by the Center for the Evaluation of Risks to Human Reproduction

from 1998 to 2010, are now undertaken by OHAT, as are workshops and state-of-the-science evaluations

to address issues of importance in the environmental health sciences. Assessments are published as NTP 

monographs. The OHAT evaluation process can be found at http://ntp.niehs.nih.gov/go/38138. Kristina Thayer, 

Ph.D., is director of OHAT.

As highlighted on page 18, in FY 2014, OHAT published the Systematic Review and Evidence Integration for 

Literature-Based Environmental Health Science Assessments, methodologies that provide a structure that 

increases transparency in the process of collecting and synthesizing scientific evidence for literature-based

evaluations (http://ntp.niehs.nih.gov/go/38673). Table 6 lists literature analysis projects that were initiated, 

ongoing, or completed in FY 2014.

Table 6. NTP Noncancer Health Effects Ongoing Projects in FY 2014

NTP Project 

[Study Scientist] Objective and/or Summary

Identifying research needs for assessing 

safe use of high intakes of folic acid; state

of the science evaluation (http://ntp.niehs.

nih.gov/go/38144) 

[Boyles]

NTP, in conjunction with the NIH Office of Dietary Supplements, is convening

an expert panel to identify research needs, based on consideration of the 

state of the science related to the safe use of high intakes of folic acid. The

benefit of supplemental folic acid for pregnant women to prevent neural tube

defects in their children is well established. At the same time, there is interest 

in understanding potential adverse health effects from high intakes of folic

acid. The expert panel will discuss the areas of consistency and inconsistency 

in the literature at a public meeting at NIH on May 11-12, 2015, in Bethesda,

Maryland. The NTP literature review and expert panel report will be published.

More information about the meeting can be found at http://ntp.niehs.nih.gov/

about/org/ntpexpertpanel/past/index.html. This project aims to identify research 

needs and inform the development of a research agenda for evaluating the safe 

use of high intakes of folic acid.
4. Literature Analysis Activities 25

NTP Project 

[Study Scientist] Objective and/or Summary

Evaluation of inflammation-based

atherosclerosis associated with 

environmental exposures (http://ntp.niehs.

nih.gov/go/721549) 

[Rooney]

There is growing evidence that the environment plays a role in a wide range 

of diseases that involve inflammation. The extent to which environmental

exposures ultimately lead to these adverse health effects through an 

inflammatory pathway remains unclear. This evaluation will examine the

evidence that environmental substances contribute to inflammation, which

ultimately leads to atherosclerosis, and identify biomarkers of the inflammation

involved. Atherosclerosis was selected for investigation because of the 

significant public health impact of the disease, and the well-established role for

inflammation in the disease process leading to atherosclerosis.

Draft case-study protocols to assess the 

systematic review and evidence integration 

framework (http://ntp.niehs.nih.gov/

pubhealth/hat/noms/index-2.html)

[Rooney]

Case studies are being undertaken to determine if additional refinement or

revision to the OHAT Approach, a systematic review framework for evaluating

scientific literature, might be needed. Two case studies are underway to

evaluate the evidence regarding the association of (1) bisphenol A (BPA) 

exposure with obesity; and (2) perfluorooctanoic acid or perfluorooctane

sulfonate exposure with immunotoxicity.

Exposure to chemicals in consumer 

products: an NIEHS and EPA collaborative 

methods demonstration and evaluation pilot 

study (http://ntp.niehs. nih.gov/go/721548)

[Taylor]

There is concern about the endocrine-disrupting potential of some chemicals 

found in personal care products and other consumer products. Given the 

large number of co-occurring chemicals in these products, new strategies and 

techniques need to be developed, and existing tools need to be evaluated, for 

their utility in assessing the extent of human exposure to tens of thousands 

of chemicals. NIEHS is collaborating with EPA to perform a small-scale, 

longitudinal pilot study, to evaluate the performance of existing survey, 

measurement, and modeling methods for assessing exposures to chemicals in 

a number of consumer product categories, including personal and child care, 

household cleaning, lawn and garden, home improvement, and food packaging

products. The pilot study addresses a number of research needs related to the 

measurement and modeling of human exposures. 

State of the science for transgenerational 

inheritance of health effects (http://ntp.

niehs.nih.gov/go/38159)

[Walker]

Transgenerational inheritance is the phenomenon in which an individual’s 

exposures have far-reaching consequences, affecting multiple generations 

removed from the original insult. The traditional belief suggests that negative 

effects of exposure to environmental chemicals are reset in each generation, 

such that subsequent generations are unaffected by the exposure history 

of their parents and grandparents. This state of the science evaluation will 

examine the robustness of the evidence for transgenerational inheritance of 

health effects associated with exposure to a wide range of stressors, such as 

environmental chemicals, drugs of abuse, nutrition and diet, pharmaceuticals, 

infectious agents, and stress in humans and animals.

Evaluation of children’s health and trafficrelated air pollution (http://ntp.niehs.nih.gov/

go/721550) 

[Howdeshell]

Research on traffic-related air pollution and children’s health has increased in

the past decade with improvements in air monitoring technology and exposure 

methodology. Traffic-related air pollution has been measured in many different

ways, including direct traffic measures, such as proximity or density of traffic;

environmental gases, such as ozone and nitrogen dioxide; particulate matter, 

including coarse particles (PM10) and fine particles (PM2.5); and select

components of traffic-related pollution, including benzene, diesel exhaust,

and polycyclic aromatic hydrocarbons (PAHs). This topic will be addressed in 

a series of evaluations that examine the evidence for an association between 

traffic-related air pollution and health outcomes impacting fetal outcome and

children, including gestational hypertension and neurological development and 

function in children. 
26 National Toxicology Program Annual Report FY 2014

NTP Project 

[Study Scientist] Objective and/or Summary

Evaluation of adverse health effects 

and occupational exposure to cancer 

chemotherapy agents (http://ntp.niehs.nih.

gov/go/721547) 

[Howdeshell]

Cancer chemotherapy agents are cytotoxic drugs, and many of these agents 

are known mutagens and developmental toxicants. Occupational exposure

to cancer chemotherapy agents may occur in various professions including 

medical, veterinary, and manufacturing. While improved handling procedures 

and engineering controls have reduced contamination, surface contamination 

persists in pharmacy and nursing areas of some hospital-based cancer 

centers. This evaluation will examine the evidence that occupational exposure 

to cancer chemotherapy is associated with adverse health effects. It will 

focus on nonreproductive health, as NIOSH recently published a review of 

occupational exposure to anti-neoplastic drugs and reproductive health effects. 

B. Report on Carcinogens

The Report on Carcinogens (RoC) is a congressionally mandated listing of substances that either are known to

be human carcinogens or may reasonably be anticipated to be human carcinogens, and to which a significant

number of persons residing in the U.S. are exposed [Section 301(b)(4) of the Public Health Service Act, 42

U.S.C. 241(b)(4)]. 

Each substance listed in the RoC has a profile, which contains the listing status, a summary of the cancer

studies supporting the listing status, information on human exposure, and federal regulations to reduce 

exposure. The RoC is a cumulative report that consists of substances reviewed in previous editions, as well 

as newly reviewed substances. NTP, with assistance from other federal health and regulatory agencies, 

prepares the RoC for the HHS Secretary. Preparation of the RoC is conducted by the Office of the RoC, under

the direction of Ruth Lunn, Dr.P.H. Contract support for preparation of the RoC in FY 2014 was provided by 

Integrated Laboratory Systems Inc. 

NTP follows a four-part process for preparing the RoC (http://ntp.niehs.nih.gov/go/rocprocess), using 

established listing criteria (http://ntp.niehs.nih.gov/go/15209) to evaluate substances for possible listing 

in the report. The cancer evaluation for each substance is captured in the draft RoC monograph, which 

summarizes, assesses, and integrates the scientific information, and applies the RoC listing criteria to the body

of knowledge to reach a preliminary listing decision. NTP selects nominations for RoC evaluation, referred to

as candidate substances, and conducts cancer hazard evaluations on those substances. On a periodic basis, 

NTP submits the proposed listings status of substances, whose cancer evaluations are completed, to the HHS 

Secretary for review, approval, and release to the public and congressional members.

In FY 2014, the Office of the RoC continued efforts towards finalizing the 13th RoC, which is planned for

release in early FY 2015 (http://ntp.niehs.nih.gov/go/roc13). The cumulative report will include the listings of 

four newly reviewed substances. 

Other RoC activities in FY 2014 were related to completing the cancer hazard evaluations of the listed 

substances, continuing ongoing evaluations of previously identified substances, and identifying new

substances for evaluation (see Table 7). In December 2013, NTP convened a panel of experts to peer 

review the draft monographs on ortho-toluidine and pentachlorophenol in a public forum (http://ntp.niehs.nih.

gov/go/38854). Following the April Board of Scientific Counselors (BSC) meeting, these monographs were

finalized and posted on the RoC Web page (http://ntp.niehs.nih.gov/go/721814).
4. Literature Analysis Activities 27

The Office of the RoC completed the draft monograph for trichloroethylene, a substance previously

identified for review. The cancer hazard evaluation portion of the monograph was informed by expert

presentations and discussions at a public webinar in March 2014 (http://ntp.niehs.nih.gov/go/tcewebinar). 

The presentations and discussions were focused on assessing the quality of the exposure and outcome 

assessments in the human cancer studies. A panel of experts peer reviewed the draft RoC monograph on 

August 12, 2014 (http://ntp.niehs.nih.gov/go/38854). The Office of the RoC will share information concerning

the peer review, such as the revised draft monograph (available at http://ntp.niehs.nih.gov/go/37899), with 

the BSC at their December 2014 meeting.

At the April 2014 BSC meeting, the Office of the RoC presented, for review, concept documents for seven

substances, including five viruses, cobalt, and goldenseal root powder. These were selected as candidate

substances and their cancer hazard evaluations have been initiated (for more information, see http://ntp.niehs.

nih.gov/go/37894). 

Table 7. Candidate Substances for the RoC 

Candidate Substance

CASRN 

[Study Scientist]

Primary Uses/Exposures RoC Review Status

Cobalt

7440-48-4

[Spencer]

A naturally occurring element that is present in different 

forms, such as a metal and salts. Cobalt metal and cobalt 

compounds are used in the production of metal alloys 

for a variety of commercial applications, as a pigment for 

dying pottery and colored glass, and in green energies.

BSC review of draft concept, 

April 2014.

Cancer hazard evaluation 

initiated.

Goldenseal root powder

[Spencer]

Member of the plant family Ranunculaceae. The root

is used as an alternative medicine to treat a variety of 

ailments. 

People are exposed to this botanical by ingesting one of 

over 150 products, including dietary supplements and 

herbal remedies that contain goldenseal root powder.

BSC review of draft concept, 

April 2014.

Shift Work at Night, Light

at Night, and Circadian 

Disruption

[Lunn]

Circadian disruption occurs when endogenous circadian 

rhythms, daily and predictable variations in biological, 

physiological, and behavioral processes, are out of phase 

with the external environment or with each other.

People, by virtue of the nature of their work, lifestyle

choices, or residence, are subjected to interruptions in 

the natural light-dark cycles, leading to the potential for

circadian disruption.

BSC review of draft concept, 

June 2013.

Cancer hazard evaluation 

initiated.

Trichloroethylene*

79-01-6 

[Lunn]

Halogenated alkene used primarily for degreasing metals.

Peer-review meeting August 12, 

2014.

Revised draft monograph posted 

on RoC website.
28 National Toxicology Program Annual Report FY 2014

Candidate Substance

CASRN 

[Study Scientist]

Primary Uses/Exposures RoC Review Status

5 Selected viruses:

Epstein-Barr virus (EBV)

Human immunodeficiency

virus type 1 (HIV)

Human T-cell lymphotrophic 

virus type 1 (HTLV-1)

Kaposi’s sarcoma-associated 

herpes virus (KSHV)

Merkel cell polyoma virus

(MCV)

[Jahnke]

EBV and KSHV: herpes viruses (enveloped; double 

stranded DNA genome). Exposure occurs through saliva.

HIV and HTLV-1: retroviruses (enveloped; single stranded 

RNA genome). Exposure occurs via breast feeding and 

perinatal, parenteral, and sexual transmission.

MCV: polyomavirus (nonenveloped, double stranded DNA

genome). It is not known how people are infected with the

virus.

BSC review of draft concept, 

April 2014.

Cancer hazard evaluation 

initiated.

* Currently listed as reasonably anticipated to be a human carcinogen in the RoC.
5. Testing and Research 29

5. Testing and Research

NTP maintains a balanced research and testing program that provides data addressing a wide variety 

of issues important to public health. NTP actively seeks to identify and select study chemicals and other

substances for which there is insufficient information to adequately evaluate potential human health hazards.

The NTP nomination process is open to the public (http://ntp.niehs.nih.gov/go/nom), and nominations can be 

submitted via the NTP website (http://ntp.niehs.nih.gov/go/27911). Agencies represented on the NTP Executive 

Committee also identify and forward nominations to NTP. The review and selection of nominations for study is 

a multistep process with input from NTP participating federal agencies, Board of Scientific Counselors (BSC)

(see page 7), and the public (see http://ntp.niehs.nih.gov/go/156 for details). To facilitate review of proposed 

research and testing projects by the BSC and the public, NTP scientists develop research concepts to outline 

the general elements of a program of study that address the specific issues that prompted the nomination of a

chemical or substance for testing. 

In June 2014, four research concepts were reviewed by the BSC (see http://ntp.niehs.nih.gov/go/9741 for 

presentations and minutes). Table 8 below summarizes the rationale and objectives for each of these projects. 

The full research concepts can be found at http://ntp.niehs.nih.gov/go/41528.

Questions about the nomination, review, and selection process can be sent to Scott Masten, Ph.D., at

masten@niehs.nih.gov.

Table 8. Research and Testing Projects Initiated in FY 2014 

NTP Project

CASRN 

[Study Scientist]

Nomination Rationale and Project Aims

Bisphenol S

80-09-1

[Sutherland]

Bisphenol S (BPS) was nominated by EPA and NIEHS based on its potential endocrine activity, 

limited toxicological testing, and high probability for human exposure. BPS and some of its derivatives 

are currently used as an alternative to bisphenol A (BPA) in certain applications. The project aims to 

(1) characterize the dose-response effects of BPS; (2) assess metabolism of BPS and derivatives; (3) 

determine the need for chronic toxicology studies; and (4) compare and contrast BPS in vivo and in 

vitro data with other bisphenol analogues and derivatives.

Triclocarban

101-20-2

[Sutherland]

Triclocarban, an antimicrobial primarily found in bar soaps, was nominated by NIEHS for toxicological 

evaluation due to its widespread human exposure, potential endocrine activity, and limitations in 

publicly available toxicology data. The project includes phases to evaluate in vivo toxicity of oral 

administration, including a perinatal exposure window to assess the potential for reproductive and 

developmental toxicities, metabolism, toxicokinetics, and developmental immunotoxicology.
30 National Toxicology Program Annual Report FY 2014

NTP Project

CASRN 

[Study Scientist]

Nomination Rationale and Project Aims

C9 Alkylbenzenes

[Sayers]

C9 alkylbenzenes are a class of aromatic hydrocarbons that occur naturally in crude oil. Interest

in this class was spurred by NTP identification of a C9 alkybenzene, cumene, as a carcinogen in

rodents, and limitations in the current database that prevent developing safe exposure levels for all 

class members. The project aims to (1) identify suitable individual compounds or mixtures for testing; 

(2) evaluate the toxicity and carcinogenicity of up to two test agents; (3) conduct comparative shortterm inhalation toxicity studies on several class members; and (4) explore options for using in vitro 

assay systems to evaluate toxicity of these volatile chemicals.

Xylenes

1330-20-7

[Stout]

Xylenes are alkylated monoaromatic hydrocarbons produced in high volume and used as a solvent

in a variety of manmade solutions. Xylenes have been nominated for NTP testing on multiple

occasions, by different organizations, for a variety of endpoints. Although NTP has conducted chronic 

toxicity and carcinogenicity studies via oral gavage, significant data gaps remain. This project aims

to (1) obtain a high purity mixture of three xylene isomers, o-xylene, m-xylene, and p-xylene, free of 

ethylbenzene for use as a test agent; and (2) evaluate chronic toxicity, carcinogenicity, developmental 

toxicity, reproductive toxicity, and neurotoxicity of xylenes following whole body inhalation exposure.

A. Technical Reports

The results of NTP toxicology and carcinogenesis studies undergo rigorous peer review and are published 

in several NTP report series, including Technical Reports (TR), Toxicity reports (TOX), and Genetically

Modified Models reports (GMM). TRs present the results of NTP long-term, generally two-year, toxicology

and carcinogenicity studies. TOX reports are prepared for studies when the exposure period is short term,

generally up to 13 weeks. GMM reports present the results of substances evaluated by NTP in transgenic

mouse strains, such as p53+/- heterozygous or Tg.AC mice. Draft NTP reports undergo peer review by ad hoc 

scientific panels or by letter review. All peer reviewers are screened for conflict of interest prior to confirming

their service.

Abstracts and completed TR, TOX, and GMM reports are available on the NTP website (http://ntp.niehs.

nih.gov/go/reports) and are catalogued in PubMed. Study summaries for other types of studies, such as

immunotoxicity, developmental toxicity, and reproductive toxicity, are also available on the NTP study reports 

page on the website.

Table 9 lists NTP TRs published in FY 2014. NTP used established criteria to evaluate the findings (http://

ntp.niehs.nih.gov/go/baresults) and determine the strength of the evidence for conclusions regarding the 

carcinogenic activity of each substance. The conclusions for level of evidence of carcinogenic activity are 

included in the table. The final reports are available at http://ntp.niehs.nih.gov/. NTP anticipates one draft TR 

will undergo peer review in FY 2015, as shown in Table 10.
5. Testing and Research 31

Table 9. Technical Reports Published During FY 2014

Chemical

Technical 

Report 

Number/ 

CASRN

Use

Levels of Evidence of 

Carcinogenic Activity

Male 

Rats

Female 

Rats

Male 

Mice

Female 

Mice

3′-Azido-3′-

deoxythymidine

GMM-14

30516-87-1

Pyrimidine nucleoside 

analog with antiviral activity 

used in the treatment of 

AIDS (Merck 1989).

N/A N/A Clear 

evidence

Equivocal 

evidence

3′-Azido-3′-

deoxythymidine (AZT)

Lamivudine (3TC)

Nevirapine (NVP)

GMM-16

30516-87-1

134678-17-4

129618-40-2

Pyrimidine nucleoside 

analog with antiviral activity 

used in the treatment of 

AIDS (Merck 1989). Special

combination to study AIDS 

therapeutics.

N/A N/A

Clear 

evidence 

(ATZ)

No 

evidence 

(ATZ)

No 

evidence 

(3TC)

Equivocal 

evidence 

(3TC)

No 

evidence 

(NVP)

Equivocal 

evidence 

(NVP)

Clear 

evidence 

(AZT/3TC)

Equivocal 

evidence 

(AZT/3TC)

Clear 

evidence 

(AZT/3TC/

NVP)

Equivocal 

evidence 

(AZT/3TC/

NVP)

beta-Picoline

TR-580

108-99-6

Used as a solvent 

in the synthesis of 

pharmaceuticals, 

resins, dyes and rubber 

accelerators, and as a 

lab reagent. Also used 

as an intermediate in 

the manufacture of 

insecticides, waterproofing

agents, niacin, and 

niacinamide.

No 

evidence

Some 

evidence

Equivocal 

evidence

Clear 

evidence

Tetrabromobisphenol A

TR-587

79-94-7

Flame retardant for 

plastics, paper, and textiles.

Equivocal 

evidence

Clear 

evidence

Some 

evidence

No 

evidence
32 National Toxicology Program Annual Report FY 2014

Table 10. Technical Reports Expected to Undergo Peer Review in FY 2014

Chemical

Technical 

Report 

Number/ 

CASRN

Use

Pentabromodiphenyl ether 

mixture [DE-71 (technical

grade)]

TR-589

32534-81-9

DE-71, a pentabromodiphenyl ether mixture, was used in the past as an 

additive flame retardant, often in furniture materials, and the chemical can be

found in water, wildlife, and in humans, as well as in various food products, 

including meat, poultry, and fish.

B. NTP Testing

The NTP testing program evaluates substances for a variety of health-related effects, generally using rodent 

models for study. NTP staff develops protocols specifically designed to fully characterize a substance’s toxic

potential. For each agent studied, a study scientist designs a comprehensive testing strategy to address the 

identified research and testing needs, and a project review committee evaluates the strategy. The following

NIEHS/NTP branches are involved in the testing program: Biomolecular Screening Branch, led by Raymond 

Tice, Ph.D.; Cellular and Molecular Pathology Branch, led by Robert Sills, D.V.M., Ph.D.; NTP Laboratory, led

by Michael Waalkes, Ph.D., until his retirement in June, and now led by acting chief Michael Devito, Ph.D.;

Program Operations Branch, led by Michelle Hooth, Ph.D.; and Toxicology Branch, led by Paul Foster, Ph.D.

i. Disposition, Metabolism, and Toxicokinetic Studies

Complete dosimetry of a chemical or physical agent describes its absorption, distribution, metabolism, 

and excretion in the body in both humans and test animals at differing levels of exposure, over all ages, via 

several routes of exposure, and under varying genetic backgrounds. Data from NTP chemical disposition and

toxicokinetic studies are used in these studies. Substances evaluated during FY 2014 are listed in Table 11. 

More information can be found at http://ntp.niehs.nih.gov/go/sp. 

Table 11. Disposition, Metabolism and Toxicokinetic Studies During FY 2014

Chemical CASRN Species Route Study 

Scientist Status

Bisphenol A 80-05-7 Rats In vitro Delclos Ongoing

Bisphenol AF 1478-61-1 Mice, Rats Gavage

Intravenous

Sutherland Completed

N-Butylbenzenesulfonamide 3622-84-2 Mice, Rats Gavage

Intravenous

Rider Ongoing

2,2’-Dimorpholinodiethyl ether 6425-39-4 Mice, Rats

Dermal

Gavage

Intravenous

Irwin Completed
5. Testing and Research 33

Chemical CASRN Species Route Study 

Scientist Status

2-Ethylhexyl p-methoxycinnamate 5466-77-3 Mice, Rats

Dermal

Gavage

Intravenous

McIntyre Ongoing

Hydroquinone 123-31-9 Mice, Rats

Gavage

Intravenous

Topical application

Sutherland Ongoing

2-Hydroxy-4-methoxybenzophenone 131-57-7 Mice, Rats

Dermal

Gavage

Auerbach Completed

Hydroxyurea 127-07-1 Rats Gavage McIntyre Ongoing

L-beta-methylaminoalanine 15920-93-1 Mice, Rats Gavage

Intravenous Ryan Completed

Resveratrol 501-36-0 Mice, Rats Gavage

Intravenous

Germolec Ongoing

Tris(4-chlorophenyl)methane 27575-78-6 Mice, Rats Gavage

Intravenous

Surh Ongoing

Tris(4-chlorophenyl)methanol 3010-80-8 Mice, Rats Gavage

Intravenous

Catlin Ongoing

ii. Genetic Toxicity

Genetic toxicity test results are used to help interpret toxicity, carcinogenicity, or other in vivo test results, and 

provide a database for use in structure-activity analyses. Substances tested for genetic toxicity during FY 2014 

are listed in Table 12. More information can be found at http://ntp.niehs.nih.gov/go/gt. 
34 National Toxicology Program Annual Report FY 2014

Table 12. Genetic Toxicity Studies During FY 2014

Chemical CASRN Testing Battery Status

Aspergillus fumigatus mold N/A Salmonella Ongoing

3’-Azido-3’-deoxythymidine 30516-87-1 Salmonella Ongoing

3’-Azido-3’-deoxythymidine and 

2’,3’-Dideoxycytidine N/A Micronucleus Ongoing

Black cohosh 84776-26-1 Salmonella Ongoing

Bromodichloroacetic acid 71133-14-7 Salmonella Completed

N-Butyl glycidyl ether 2426-08-6 Micronucleus Ongoing

1-Butyl-1-methylpyrrolidinium chloride 479500-35-1 Micronucleus Initiated

N-Butyl-pyridinium chloride 1124-64-7 Micronucleus Initiated

Citral 5392-40-5 Micronucleus Ongoing

Clarithromycin 81103-11-9 Micronucleus Ongoing

Colchicine 64-86-8 Micronucleus Ongoing

Corn oil 8001-30-7 Micronucleus Initiated

Dimethylamine borane 74-94-2 Salmonella Completed

Echinacea purpurea extract 90028-20-9 Salmonella Initiated

N-Ethyl acetoacetamide 10138-46-2 Micronucleus Ongoing

4-Methylcyclohexanemethanol, crude N/A Micronucleus Initiated

2-Nitro-propanol 2902-96-7 Salmonella Ongoing

2-Nitro-2-ethyl-1,3-propanediol 597-09-1 Salmonella Ongoing

Perfluorodecanoic acid 335-76-2 Micronucleus Ongoing

Perfluorononanoic acid 375-95-1 Micronucleus Completed

Propylene glycol phenyl ether 770-35-4 Micronucleus Initiated

Sulfolane 126-33-0 Micronucleus Initiated

Tris(Chloropropyl)phosphate 13674-84-5 Salmonella Initiated

Vinpocetine 42971-09-5 Salmonella Initiated

Zinc carbonate, basic 5263-02-5 Micronucleus Ongoing

iii. Organ System Toxicity

NTP studies toxicity of environmental substances on organ systems for development, reproduction, and the 

immune system. Table 13 lists organ systems toxicity studies during FY 2014. More information can be found

at http://ntp.niehs.nih.gov/go/type. 
5. Testing and Research 35

Table 13. Neurotoxicity, Developmental Toxicity, and Reproductive Toxicity Studies During 

FY 2014

Chemical CASRN Species Route Study 

Scientist Testing Battery Status

Acrylamide 79-06-1 Rats Gavage Beland Neurotoxicology 

assessment Ongoing

Black cohosh 84776-26-1 Rats Gavage Blystone Continuous breeding Completed

Butyl paraben 94-26-8 Rats Feed Blystone Continuous breeding

Maternal transfer Completed

Diisobutyl phthalate 84-69-5 Rats Feed Blystone Continuous breeding Completed

Dimethylethanolamine 108-01-0 Rats Gavage McIntyre Conventional 

teratology Initiated

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats Feed Hansen Developmental toxicity Ongoing

4-Methylcyclohexanemethanol 34885-03-5 Rats Gavage Auerbach Conventional 

teratology Initiated

4-Methylimidazole 822-36-6 Rats Feed Behl Continuous breeding Completed

Vinpocetine 42971-09-5 Rats Gavage Catlin Teratology pilot 

studies Initiated

Immunotoxicity Testing

NTP immunotoxicity studies address adverse effects on the immune system that may result from exposure to 

environmental chemicals, biological materials, or therapeutic agents. The identification of substances that have

potential to cause injury to the immune system is of considerable public health significance, as alterations in

immune function can lead to increased incidence of hypersensitivity disorders, autoimmune disease, infectious 

disease, or neoplasia. There were no immunotoxicity studies initiated or ongoing during FY 2014. More

information can be found at http://ntp.niehs.nih.gov/go/9399. 

Modified One-Generation Reproduction Studies

Classical studies used to evaluate reproductive toxicity are of a multigenerational reproduction experimental 

design. NTP has modified this classical study design to better utilize the animals produced, and to reduce

animal use by improved experimental design and statistical power. Table 14 lists planned or ongoing modified

one-generation studies. More information can be found at http://ntp.niehs.nih.gov/go/MG. 
36 National Toxicology Program Annual Report FY 2014

Table 14. Modified One-generation Studies in FY 2014

Chemical CASRN Species Study 

Route Planned Cohorts Study Scientist Status

Bisphenol AF 1478-61-1 Rats Feed

Dose range-finding1

Maternal transfer2

Developmental Toxicity3

Fertility Assessment4

Subchronic toxicity5

F0 Generation6

Sutherland Completed

2-Ethylhexyl 

p-Methoxycinnamate 5466-77-3 Rats Feed

Developmental toxicity3

Fertility Assessment4

Subchronic toxicity5

F0 Generation6

McIntyre Completed

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats Feed

Developmental toxicity3 

Fertility Assessment4

F0 Generation6

McIntyre Completed

Perfluorooctane

sulfonate 1763-23-1 Rats Gavage Dose range-finding1 Blystone Completed

Perfluorooctanoic acid 335-67-1 Mice, Rats Gavage Dose range-finding1 Blystone Completed

Vinpocetine 42971-09-5 Rats Gavage Dose range-finding1 Catlin Initiated

Wyeth 14,643 (WY) 50892-23-4 Mice, Rats Gavage Dose range-finding1 Blystone Initiated

1 Dose range-finding: to find the ideal dose for toxicological studies. 2 Maternal transfer: to study the transfer of chemical from mother to offspring. 3

 Developmental toxicity: to study adverse developmental outcomes such as birth defects.

4

 Fertility assessment: to study adverse effects on fertility in males and females.

5

 Subchronic toxicity: 90-day study of adverse effects in the exposed rodent.

6

 F0 Generation: parental generation.

iv. Toxicology and Carcinogenicity Studies

NTP performs toxicity studies, in part, to provide dose-setting information for chronic studies, and address 

specific deficiencies in the toxicology database for the chemical. Toxicology and carcinogenicity studies

generally fall into two categories: prechronic toxicity studies, and two-year toxicology and carcinogenicity 

studies. Studies are generally conducted in rats and mice. Each study type is performed according to the 

Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical,

Biological, and Physical Agents in Laboratory Animals for the National Toxicology Program (January 2011). 

Additional information can be found at http://ntp.niehs.nih.gov/go/ba. 

Table 15, Table 16, and Table 17 list toxicity studies that were initiated, ongoing, or completed during FY 

2014. Chronic toxicology and carcinogenicity studies ongoing or completed in FY 2014 are listed in Table 18

and Table 19.
5. Testing and Research 37

Table 15. Prechronic Toxicology and Carcinogenicity Studies Initiated During FY 2014

Chemical CASRN Species Study Route Length Study Scientist

Black cohosh 84776-26-1 Mice Gavage 90 days Cora

Dimethylamine borane 74-94-2 Mice, Rats Dermal 2 weeks Germolec

Garcinia cambogia extract 90045-23-1 Mice, Rats Feed 14 days Rider

N,N-Dimethyl-p-toluidine 99-97-8 Rats Gavage 5 days Dunnick

N-Butylbenzenesulfonamide 3622-84-2 Mice Feed 14 days Rider

4-Methylcyclohexanemethanol 34885-03-5 Rats Gavage 5 days Auerbach

4-Methylcyclohexanemethanol,

crude N/A Rats Gavage 5 days Auerbach

Pentabromodiphenyl oxide 

(technical) (DE-71) 32534-81-9 Rats Gavage GD* 6 to PND** 21 Dunnick

Propylene glycol phenyl ether 770-35-4 Rats Gavage 5 days Auerbach

p-Toluidine 106-49-0 Rats Gavage 5 days Dunnick

2,2’,4,4’-Tetrabromodiphenyl 

ether (BDE-47) 5436-43-1 Rats Gavage

GD 6 to PND 

21, GD 6 to 

GD 20, GD 6 

to PND 21

Dunnick

Sulfolane 126-33-0 Guinea Pigs, 

Mice, Rats Gavage 28 days Blystone

Tungsten 7440-33-7 Rats Intratracheal 28 days Behl

Tungsten suboxide fibers N/A Rats Intratracheal 28 days Behl

Vanadyl sulfate 27774-13-6 Mice, Rats Water 14 days Sayers

* GD: gestational day

** PND: postnatal day

Table 16. Prechronic Toxicology and Carcinogenicity Studies Ongoing During FY 2014

Chemical CASRN Species Study Route Length Study Scientist

AZT/Drug combinations 

transplacental/neonatal study N/A Mice Gavage 28 days Beland

1-Butyl-3-methylimidazolium 

chloride 79917-90-1 Mice, Rats Water 90 days Ryan

1-Butyl-1-methylpyrrolidinium 

chloride 479500-35-1 Mice, Rats Water 90 days Ryan

tert-Butylphenyl diphenyl 

phosphate 56803-37-3 Rats Gavage 5 days Auerbach
38 National Toxicology Program Annual Report FY 2014

Chemical CASRN Species Study Route Length Study Scientist

N-Butylpyridinium chloride 1124-64-7 Mice, Rats Water 90 days Ryan

2-Ethylhexyl diphenyl phosphate 1241-94-7 Rats Gavage 5 days Auerbach

1-Ethyl-3-methylimidazolium 

chloride 65039-09-0 Mice, Rats Water 90 days Ryan

Isodecyl diphenyl phosphate 29761-21-5 Rats Gavage 5 days Auerbach

Isopropylated phenol phosphate 68937-41-7 Rats Gavage 5 days Auerbach

Melamine and cyanuric acid

combination N/A Rats Gavage 90 days Gamboa

Nanoscale silver 7440-22-4 Rats Gavage 13 weeks Boudreau

Pentabromodiphenyl oxide 

(technical) (DE-71) 32534-81-9 Rats Gavage GD* 6 to PND** 21 Dunnick

Tetrabromobisphenol A 79-94-7 Rats Gavage 13 weeks Dunnick

Triclosan 3380-34-5 Mice Dermal 90 days Fang

Tricresyl phosphate 1330-78-5 Rats Gavage 5 days Auerbach

Usnea lichen N/A Mice, Rats Feed 2 weeks Leakey

(+)-Usnic Acid 7562-61-0 Mice, Rats Feed 2 weeks,

90 days Leakey

* GD: gestational day

** PND: postnatal day

Table 17. Prechronic Toxicology and Carcinogenicity Studies Completed During FY 2014

Study CASRN Species Study Route Length Study Scientist

AZT+3TC+NVP combination N/A Mice Gavage 28 days Leakey

beta-Picoline 108-99-6 Mice, Rats Water 13 weeks Wyde

Cell phone radiation: CDMA* N/A Mice, Rats Whole body 

exposure

28 days, 

49 days Wyde

Cell phone radiation: GSM** N/A Mice, Rats Whole body 

exposure

28 days, 

49 days Wyde

Perfluorononanoic acid 375-95-1 Rats Gavage 28 days Blystone

Perfluorooctane sulfonate 1763-23-1 Rats Gavage 28 days Blystone

Perfluorooctanoic acid 335-67-1 Rats Gavage 28 days Blystone

Tetrabromobisphenol A 79-94-7 Mice, Rats Gavage 13 weeks Dunnick

Wyeth 14,643 50892-23-4 Rats Gavage 28 days Blystone

* CDMA: code division multiple access

** GSM: global system for mobile communication
5. Testing and Research 39

Table 18. Chronic Toxicity and Carcinogenicity Studies Ongoing During FY 2014

Study CASRN Species Study Route Length Study Scientist

Aging cohort study, mouse 

strains:

• 129S1/SvImJ

• B6C3F1 (Jackson)

• C3H/HeJ

• C57BL/6J (Jackson)

• CAST/EiJ (M. m. castaneus)

• NZO/HiLtJ

• PWK/PhJ

• WSB/EiJ (M. m. domesticus)

• A/J

• NOD. B10Sn-H2(b)/J

N/A Mice N/A 2 years Dunnick

Antimony trioxide 1309-64-4 Mice, Rats Inhalation 2 years Sayers

AZT/Drug combinations: 

Transplacental carcinogenesis 

study

N/A Mice In utero, 

Gavage 2 years Beland

Bisphenol A 80-05-7 Rats Gavage 2-years Delclos

Black cohosh 84776-26-1 Mice, Rats Gavage 2 years Blystone

2,3-Butanedione 431-03-8 Mice, Rats Inhalation 2 years Morgan

Cell phone radiation: CDMA* N/A Mice, Rats Whole body 

exposure 2 years Wyde

Cell phone radiation: GSM** N/A Mice, Rats Whole body 

exposure 2 years Wyde

Di(2-ethylhexyl) phthalate 117-81-7 Rats Feed 2 years Foster

Di(2-ethylhexyl) phthalate 117-81-7 Rats Feed Perinatal, 

2 years Foster

Dibutyl phthalate 84-74-2 Mice, Rats Feed 2 years Blystone

Furan 110-00-9 Rats Gavage 2 years Beland

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Mice, Rats Feed 2 years McIntyre

Insertional mutagenesis: 

Definitive vector study N/A Mice Intravenous 14 months Germolec

p-Chloro-a,a,a-trifluorotoluene 98-56-6 Mice, Rats Inhalation 2 years Stout

Pentabromodiphenyl oxide 

(technical) (DE-71) 32534-81-9 Mice, Rats Gavage 2 years Dunnick

Perfluorooctanoic acid 335-67-1 Rats Feed 2 years Blystone

Resveratrol 501-36-0 Mice, Rats Gavage 2 years Germolec

Sodium tungstate dihydrate 10213-10-2 Mice, Rats Water 2 years Behl

Triclosan 3380-34-5 Mice Dermal 2 years Fang

Trim VX N/A Mice, Rats Inhalation 2 years Morgan

Tris(Chloropropyl)phosphate 13674-84-5 Mice, Rats Feed 2 years Ryan
40 National Toxicology Program Annual Report FY 2014

Study CASRN Species Study Route Length Study Scientist

Zinc carbonate, basic 5263-02-5 Rats Feed 2 years Wyde

* CDMA: code division multiple access

** GSM: global system for mobile communication

Table 19. Chronic Toxicity and Carcinogenicity Studies Completed During FY 2014

Study CASRN Species Study 

Route Length Study Scientist

Bromodichloroacetic acid 71133-14-7 Mice, Rats Water 2 years DeVito

CIMSTAR 3800 N/A Mice, Rats Inhalation 2 years Morgan

Cobalt 7440-48-4 Mice, Rats Inhalation 2 years Behl

Glycidamide 5694-00-8 Mice, Rats Water 2 years Beland

Green tea extract N/A Mice, Rats Gavage 2 years Blystone

Indole-3-carbinol 700-06-1 Mice, Rats Gavage 2 years Wyde

Tetrabromobisphenol A 79-94-7 Mice, Rats Gavage 2 years Dunnick

Vinylidene chloride 75-35-4 Mice, Rats Inhalation 2 years Wyde

v. Toxicogenomic Studies

NTP is working to bring the latest toxicogenomics technology into its testing program to help revolutionize the

way studies are conducted. Toxicogenomics examines how the entire genetic structure, or genome, is involved 

in an organism’s response to environmental toxicants. Microarray, next-generation (NextGen) sequencing,

proteomics, and metabolomics are among the advanced technologies that NTP is using to study the way 

chemical exposures change the expression of genes, proteins, and metabolites in critical cells and tissues. 

Measuring genome-wide changes in affected tissues may be useful for identifying biomarkers of disease and

exposure to toxic substances, and for understanding individual genetic susceptibilities. Biomarkers, which can

be found in easily obtainable samples, such as blood and urine, could then be monitored in clinical studies. 

When biomarkers are validated, they can be repeatedly sampled during long-term NTP studies to determine

whether chemical exposures can be detected, or whether developing cancers provide a genetic signature.

NTP is interested in determining if analyzing the patterns of gene expression can provide indicators of toxicity 

at earlier time points and at lower doses than is possible with traditional toxicology parameters. Evaluating 

patterns of gene expression may provide more than just a link between genetics and morphology. It is also

expected to provide insights into the pathogenesis of the disease and how different rodent models respond to 

toxicants. Some of the FY 2014 toxicogenomic studies involved NextGen sequencing technologies that bring 

gene expression to a base-pair resolution of accuracy and increased sensitivity. In addition, metabolomics 

represents a promising area of study, as it can elucidate how chemicals affect metabolism within cells, relative 

to changes in gene expression. 

NTP is currently evaluating study conditions that may contribute to differential gene expression, such as animal 

and tissue variability, identifying best methods for tissue sampling, and establishing standards for conducting 

toxicogenomic studies under laboratory conditions. FY 2014 planned or ongoing NTP toxicogenomic studies 

are listed in Table 20. More information can be found at http://ntp.niehs.nih.gov/go/20358. 
5. Testing and Research 41

Table 20. Toxicogenomic Studies Planned or Ongoing in FY 2014

Chemical CASRN Species/ 

Cell Line

Study 

Route

Study Length/ Test 

Type (Platform)

Study 

Scientist

Aflatoxin B1 1162-65-8 Rats Feed

90 days/ Toxicity

NextGen sequencing

RNAseq (Illumina)

Merrick

Arsenite 7784-46-5 Human: 

Prostate

Cell 

culture

30 weeks/NextGen

sequencing 

DNAseq, RNAseq 

(Illumina)

Merrick

Black cohosh 84776-26-1 Mice Gavage 90 days/Toxicity 

Microarray (Affymetrix)

Cora

N,N-Dimethyl-p-toluidine 99-97-8 Rats Gavage

5 days, 90 days/ 

Toxicity

Microarray (Affymetrix)

Dunnick

Dong quai (Angelica sinensis) root 

extract 299184-76-2 Mice Gavage 90 days/Toxicity

Microarray (Affymetrix) McIntyre

Elk River spilled chemicals:

• Dipropylene glycol phenyl ether 

• 4-Methylcyclohexane methanol

(MCHM)

• 4-Methylcyclohexane methanol,

crude/mixture

• Propylene glycol phenyl ether 

51730-94-0

34885-03-5

N/A

770-35-4

Rats Gavage 5 days/Toxicity 

Microarray (Affymetrix)

Auerbach

Estrous cycle study in corn oil controls 8001-30-7 Rats Gavage 90 days 

Microarray (Affymetrix)

Merrick

Phosphate flame retardants:

• tert-Butylphenyl diphenyl phosphate 

• 2-Ethylhexyl diphenyl phosphate 

• Isodecyl diphenyl phosphate

• Isopropylated phenol phosphate

56803-37-3

1241-94-7

29761-21-5

68937-41-7

Rats Gavage

5 days/Toxicity

Microarray (Affymetrix)/

metabolomics

Auerbach
42 National Toxicology Program Annual Report FY 2014

Chemical CASRN Species/ 

Cell Line

Study 

Route

Study Length/ Test 

Type (Platform)

Study 

Scientist

Ginkgo biloba extract 90045-36-6 Rats Gavage 5 days/Toxicity

Microarray (Affymetrix)

Rider/ 

Auerbach

2-Hydroxy-4-

methoxybenzophenone

131-57-7 Rats Feed 90 days/Toxicity 

Microarray (Affymetrix)

Auerbach

Methyleugenol extract and Ginko biloba

extract

93-15-2

90045-36-6

Mice Gavage

2 years/Toxicity

NextGen sequencing 

Exome-Seq

RNASeq (Illumina)

Auerbach/

Merrick

Polycyclic aromatic compounds: 

• Acenaphthenequinone

• Benzo[b]fluoranthene

• Benzo(a)pyrene

• Dibenz[a,h]anthracene

• 9-Methylanthracene

• 1-Methylfluorene

• Perinaphthenone

• Phenanthrene

• Pyrene

82-86-0 

205-99-2

50-32-8

53-70-3

779-02-2

1730-37-6

548-39-0

85-01-8 

129-00-0 

Human: 

hepatocyte 

cell line

In vitro 

exposure

48 hour/Cytotoxicity 

and Gene expression

Rider/ 

Tokar

BDE Toxicogenomics Studies:

• 2,2’,4,4’,5-Pentabromodiphenyl ethers

• Pentabromodiphenyl oxide (technical) 

(DE-71) 

• 3,3,4,4,5-Pentachlorobiphenyl

• 2,2’4,4’-Tetrabromodiphenyl ether 

(DE-47)

5436-43-1

32534-81-9

57465-28-8

5436-43-1

Rats, Mice Gavage

GD* 6 through 3 weeks/

Toxicity

Microarray (Affymetrix)

Dunnick

Tetrabromobisphenol A 79-94-7 Rats Gavage

90 days/Toxicity

Microarray (uterus and

liver) (Affymetrix)

Dunnick/

Merrick

p-Toluidine 106-49-0 Rats Gavage 5 days, 90 days/Toxicity

Microarray (Affymetrix)

Dunnick

* GD: gestational day
5. Testing and Research 43

vi. Project Review Committee Approved

Table 21 lists studies that were approved by either the internal NIEHS/NTP protocol approval committee or the 

internal NIEHS/NTP project review committee, but were not started during FY 2014. 

Table 21. Protocol Approval Committee or Project Review Committee Approved Studies in 

FY 2014

Study CASRN Species Study 

Route Test Type Study Scientist

Aspergillus fumigatus N/A Mice Nose only 

inhalation Subchronic toxicity Germolec

Deoxynivalenol 51481-10-8 Rats Gavage Perinatal dose 

range-finding Germolec

Elk River chemical spill:

• 4-Methylcyclohexanemethanol

• 4- Methylcyclohexanemethanol, crude/

mixture

34885-03-5

N/A

Mice Dermal

Irritation and 

hypersensitivity 

responses

Auerbach/ 

Germolec

Ethylene glycol 2-ethylhexyl ether 1559-35-9 Rats, Mice

Gavage

Intravenous

Dermal

Absorption, 

distribution, 

metabolism, and 

elimination

Blystone/ 

Waidyanatha

Ethylene glycol 2-ethylhexyl ether 1559-35-9 Rats Gavage Dose range-finding Blystone

lsopropylated phenyl phosphate 68937-41-7 Rats Feed

Modified onegeneration 

Dose range-finding

Behl

4-Methylcyclohexanemethanol 34885-03-5 Rats Gavage Teratology dose 

range-finding Auerbach

Phenolic benzotriazole class:

• 3-(2H-Benzotriazol-2-yl)-

5-(1,1-dimethylethyl)-4-

hydroxybenzenepropanoic acid, octyl 

ester 

• 2-(2H-Benzotriazol-2-yl)-4-(1,1-

dimethylethyl)phenol 

• 2-(2H-Benzotriazol-2-yl)-4,6-bis(1,1-

dimethylpropyl)phenol 

• 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-

methyl-1-phenylethyl)phenol 

• Bumetrizole

• 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-

bis(1,1 dimethylethyl)phenol 

• Drometrizole

• Octrizole 

• Phenolic benzotriazole

84268-23-5

3147-76-0

25973-55-1

3864-99-1

70321-86-7

3896-11-5

2440-22-4

3147-75-9

10096-91-0

Rats Gavage Prechronic toxicity Blystone
44 National Toxicology Program Annual Report FY 2014

Study CASRN Species Study 

Route Test Type Study Scientist

alpha-Pinene 80-56-8 Rats, Mice Whole body 

inhalation

Chronic toxicity and 

carcinogenicity

Male reproductive

toxicity

Rider

Propylparaben 94-13-3 Rats Feed Dose range-finding Blystone

Simvastatln 79902-639 Rats Gavage Perinatal dose 

range-finding McIntyre

Sulfolane 126-33-0 Rats, Mice

Gavage

Intravenous

Dermal

Absorption, 

distribution, 

metabolism, and 

elimination

Blystone/ 

Waidyanatha

Triphenyl phosphate 115-86-6 Rats Feed

Modified onegeneration 

Dose range-finding

Behl

Vinpocetine 42971-09-5 Rats Gavage

Teratology

Perinatal dose 

range-finding

Catlin

C. NTP Research

In addition to testing activities, a variety of NTP research projects are underway at NIEHS, NCTR, and NIOSH. 

The following NIEHS/NTP branches are actively involved in NTP research activities: Biomolecular Screening 

Branch, led by Raymond Tice, Ph.D.; Cellular and Molecular Pathology Branch, led by Robert Sills, D.V.M.,

Ph.D.; NTP Laboratory, led by Michael Waalkes, Ph.D., until his retirement in June, and now led by acting

chief Michael Devito, Ph.D.; Program Operations Branch, led by Michelle Hooth, Ph.D.; and Toxicology Branch,

led by Paul Foster, Ph.D.

The NTP Laboratory, within NIEHS/NTP, conducts in-house, agent-specific, targeted research related to the

development and application of modern toxicology and molecular biology tools. These tools are used in the 

evaluation of specific substances of concern to NTP, issues of central importance to NTP programs, and

methods development to advance the NTP mission. The NTP Laboratory also focuses on the study of the 

developmental origins of adult diseases. Table 22 includes projects in the NTP laboratory in FY 2014.

Table 22. NIEHS/NTP NTP Laboratory Projects in FY 2014

NTP Labs Project 

[Study Scientist] Objective and/or Project Summary

Development of in vitro models of metal 

carcinogenesis 

[Tokar]

To develop in vitro cell transformation models with target relevant cells using 

arsenic and cadmium. 
5. Testing and Research 45

NTP Labs Project 

[Study Scientist] Objective and/or Project Summary

Epigenetics in malignant transformation

[Thayer]

To assess the epigenome of a series of isogenic cell lines transformed by 

genotoxic or epigenetic carcinogens, and perform gene-specific methylation

analyses. 

Formaldehyde in p53 knockout mice

[Morgan]

To define the role of formaldehyde inhalation in hematopoietic tumor induction.

Formaldehyde-induced transformation of 

human myeloid progenitor cells 

[Morgan]

To perform a proof of concept study of in vitro formaldehyde-induced 

malignant transformation in hematopoietic stem cells. Companion study to the 

p53 study mentioned above.

Incorporating metabolism into high 

throughput screening assays 

[DeVito]

To develop in vitro methods that incorporate xenobiotic metabolism.

Indium-tin-oxide and indium compounds

[Morgan] To perform various in vivo inhalation or in vitro toxicity studies.

Metalloestrogens and uterine/breast

response 

[Dixon, Fenton]

To retest the ability of reported metalloestrogens, like cadmium and arsenic, to

cause estrogen receptor stimulation in the uterus as a mode of action towards 

cancer development.

Method for assessing biological impact of

metal particle dissolution 

[Morgan]

(1) To develop an in vitro transwell method with metal particles and 

macrophages in one well and cells of interest, such as lung epithelium, in the 

other; and (2) define the ability of various types of macrophages to release

different metals from different particles. 

Methods in histopathology of mammary

gland development 

[Fenton]

To develop standardized methods to quantitatively assess chemical insult on 

mammary gland development.

Refinement of developmental

neurotoxicology methods 

[Harry]

To improve methods for various efforts including genetic signatures, stem 

cells, inflammation, behavior, and conditioning.

Role of microRNAs in malignant 

transformation 

[Tokar]

To study genes of interest involved in the epigenetics of malignant 

transformation, using in vitro human model systems of carcinogenesis. 

MicroRNAs are thought to be a key epigenetic or posttranscriptional gene

express control mechanism.

Stem cells in toxicology and carcinogenesis 

[Tokar]

To perform various in vitro studies on the role of stem cells and cancer stem 

cells in carcinogenesis and the developmental basis of adult disease. 

Toxicants and mammary gland development 

[Fenton]

To determine the effects of different toxicants, including atrazine, on mammary 

gland development in rats and mice. 

Using Caenorhabditis elegans (C. elegans) 

for screening mitochondrial toxicants

[Boyd]

To study the effects of mitochondrial toxicants in C. elegans.

Using C. elegans as screens for toxicity of 

flame retardants

[Boyd]

To study the effects of flame retardants in C. elegans.
46 National Toxicology Program Annual Report FY 2014

Genetic and epigenetic differences between individuals in the human population are proposed as major 

factors for individual susceptibility to environmental stressors. Environmental and drug safety assessments 

are currently conducted with a small number of commonly used animal models, which have limited genetic 

diversity and is insufficient to evaluate the influence of individual genetic differences on chemical and drug

toxicity. These models are of limited value in extrapolating results to human toxicity and disease, and this 

program works toward developing models that are more appropriate. The NTP Biomolecular Screening Branch

conducts in-house projects aimed at understanding individual susceptibility. Table 23 includes projects in the 

Biomolecular Screening Branch in FY 2014.

Table 23. NIEHS/NTP Biomolecular Screening Branch Projects in FY 2014

Host Susceptibility Project 

[Study Scientist] Objective and/or Project Summary

Mouse Methylome Project

[Merrick/Wade]

Male and female C57BL/6N mice were crossed with C3H/HeN mice. Five tissues, brain,

liver, cardiac and skeletal muscle, brown and white fat, and epididymal sperm, from

the first generation offspring were collected at the average age NTP starts mice in a

subchronic toxicity study, and flash-frozen for DNA/RNA isolation and liver sequencing.

Progress in FY 2014 involved computational analysis on sites of genomic methylation in 

relation to known genes and transcriptionally active regions in both parental strains and

the resulting first generation offspring. The relationship of methylation to gene expression

is being determined, and the genomic variation between mouse strains, genders, and F1 

offspring is being catalogued. The epigenomic landscape is being carefully described 

in these two mouse strains and offspring to help interpret the contribution of differences 

in DNA methylation to their differential susceptibility to malignancy. A manuscript is in 

preparation to be followed by public release of the methylome data.

i. Assessing Exposure to Substances in the Workplace

In following its mandate to protect 

workers’ health and safety, NIOSH

carries out research projects, for NTP, 

to assess the effects of exposure to 

substances through an interagency 

agreement with NIEHS (see page 15). 

Setting priorities in occupational 

toxicological research is based upon 

several sources of information that 

NIOSH develops and maintains. 

These include health hazard 

evaluations, industry-wide studies, 

gaps in knowledge identified while

developing criteria for recommended 

standards or criteria documents, 

current intelligence bulletins, hazard 

reviews and alerts, other technical 

reports, and information profiles on

chemical hazards. Table 24 lists 

NIOSH/NTP projects in FY 2014. 

NIOSH mobile lab for field studies
5. Testing and Research 47

Table 24. NIOSH/NTP Research Projects FY 2014

NIOSH/NTP Projects

[Study Scientist] Objective and/or Summary

Biomonitoring, Biomarker Development and Health Assessment

Reproductive health 

assessment of male 

workers

[Schrader]

To evaluate reproductive health hazards, using a health profile consisting of biomarkers for

assessing male fecundity. Current research involves a multiyear study with Emory University 

to evaluate the reproductive health of adult men accidently exposed to endocrine disruptors in 

utero through mothers living and working on family farms in Michigan.

Immunochemical 

biological monitoring for 

occupational exposure and 

disease 

[Striley]

To evaluate industrial chemicals that present a significant risk to workers, using biological

monitoring to assess exposure, by analyzing acute and latent metabolites in various biological 

media. The goal of this project is to develop low-cost, rapid immunochemical and analytical 

chemistry biomonitoring methods that will be used to identify exposures and evaluate potential 

interventions. Current efforts include exposure assessments of workers employed at gas and oil

drilling and servicing sites.

Ultraviolet native 

fluorescence-based

monitor for workplace

exposures 

[Snawder]

To develop and evaluate a readily adaptable, next generation, direct reading, personal monitor 

for use in measuring worker exposure to a wide variety of chemicals, including naphthalene and

components of asphalt fume. The development of personal monitors for volatile and semivolatile 

workplace chemicals will be helpful in rapidly assessing chemical exposure, resulting in more

realistic occupational exposure assessments, allowing for rapid interventions leading to reduced 

worker exposures, and aiding prevention of occupational illness and disease.

Workplace exposure,

inflammation, and

cardiovascular toxicity 

[Erdely]

To investigate the role of ultrafine and nanoparticle-induced cardiopulmonary inflammation,

and to identify specific markers of lung inflammation that directly correlate to systemic effects.

This laboratory-based research will evaluate novel molecular mechanisms involved in the link

between occupational exposures to ultrafine and nanosize particulates, and the development

of cardiovascular diseases. Planned studies will help identify potential risk factors, biomarkers,

and specific targets for prevention and therapeutic intervention of occupational-related

cardiovascular diseases.

Systematic assessment 

of multiwalled carbon 

nanotubes in pulmonary 

disease

[Qian]

To detect and identify novel biomarkers and molecular mechanisms of multiwalled carbon

nanotube-induced pulmonary diseases, including fibrosis and cancer, for early detection

and treatment interventions. Concerns over potential adverse pulmonary effects of airborne 

exposure to multiwalled carbon nanotubes have been raised due to their high aspect ratio 

(length and diameter), nanoscale diameter, fiber like-shape, durability, and biopersistence.

Increasing evidence has indicated potential pulmonary health hazards associated with 

pulmonary exposure to multiwalled carbon nanotubes, including inflammation, damage, fibrosis,

and potential carcinogenesis. The mechanisms underlying these adverse multiwalled carbon 

nanotube-induced pulmonary responses are not well understood. Currently, there is no available 

noninvasive screening test for early detection of lung fibrosis.

Industry-wide studies 

of workers exposed to

carbon nanotubes and 

nanofibers

[Schubauer-Berigan]

To collect exposure data from participating pilot-scale or full-scale manufacturers or users of 

single-walled or multiwalled carbon nanotubes and carbon nanofibers. A study of biomarkers for

early pulmonary, cardiovascular, and carcinogenic effect will be carried out among workers at

these facilities.

Mortality, cancer

incidence, and biomarker

studies 

[Ruder]

To elucidate exposure-outcome associations, especially dose-response relationships, for risk

assessment; to examine relationships between biomarkers of exposure, susceptibility, and

oncogene expression; and determine health effects.
48 National Toxicology Program Annual Report FY 2014

NIOSH/NTP Projects

[Study Scientist] Objective and/or Summary

Environmental Monitoring

Analytical research and 

development infrastructure

[Streicher]

To provide for administrative needs, and analytical instrumentation repair and maintenance, 

in support of Chemical Exposure and Monitoring Branch chemists conducting research on

sampling, and analytical methods development for workplace chemicals. New methods needed

to assess chemicals, such as BPA and manganese speciation, are being investigated as part 

of the NIOSH/NTP exposure assessment interagency agreement, and are developed in this 

project.

Diacetyl exposure 

assessment

[Streicher]

To develop and evaluate sampling and analytical methods for diacetyl and other higher 

molecular weight alpha dicarbonyl flavoring compounds, to enable accurate exposure

assessment and evaluation of the effectiveness of control technology. Two sampling and 

analytical methods are being investigated for measurement of specific flavoring compounds,

most notably diacetyl and 2,3-pentanedione, in airborne particles and bulk powders. Broader

gas chromatography-mass spectrometry method(s) will be developed for a range of compounds 

present in flavorings.

Chemical exposure 

monitoring with indoor 

positioning 

[Brown]

To investigate a direct reading exposure method that uses a personal photo ionization detector 

chemical monitor with telemetry and indoor positioning system, to provide remote monitoring of 

a worker’s exposure to volatile organic chemicals (VOCs) with position and time. The personal

monitor continuously samples and analyzes the workers’ air for VOCs, while recording their

position and time of exposure. Indoor positioning is accomplished using a radio transmitter 

attached to the personal monitor and receivers placed in the ceiling corners of the room. The 

positioning receivers communicate with each other and a remote laptop, using wireless local 

area network technology. The remote laptop calculates and visualizes the worker position and

exposure level. Once developed, this technology will be applied to analyze workplace exposures

to diacetyl.

Exposure Assessment

Exposure assessment for 

toxicologically important 

chemicals 

[Curwin]

To characterize workplace exposures to (1) welding fumes with emphasis on manganese; (2)

occupational exposure to BPA; (3) diacetyl; and (4) occupational exposure to carbon nanotubes 

and nanofibers. A new study will also investigate worker exposures to BPA. These chemicals

have been nominated by various groups to NTP. Goals of the studies include identifying 

industries, workplaces, uses, and users; determining occupational health relevance; estimating

number of workers exposed; and conducting exposure sampling.

Industry-Wide Studies 

Branch research, 

development, and planning 

[Whelan]

To support strategic planning and feasibility studies of high priority and emerging problems in 

occupational health. 

Nanotechnology field

evaluations 

[Geraci]

To obtain information from as many different facilities as possible, in the field, on the nature

of engineered nanomaterials, the processes involved in their manufacture and use, potential 

worker exposures, and work practices and control procedures used where nanomaterials are

produced or used. As toxicology studies identify the biologic hazards of nanomaterials, it is 

important to gain a better understanding of actual workplace exposures.
5. Testing and Research 49

NIOSH/NTP Projects

[Study Scientist] Objective and/or Summary

Immunotoxicity and Immunology

Immunotoxicological 

evaluation of occupational 

chemicals

[Anderson]

To identify occupational and environmental chemical hazards, and evaluate immune 

function and mechanisms associated with exposure. This research will contribute to better 

risk assessment and increased identification of immunological hazards encountered in the

workplace, which will ultimately establish occupational exposure limits.

Identification of

occupational allergens 

[Beezhold]

(1) To identify exposures to substances that can cause inflammatory or immune reactions

in certain work environments. These exposures are important causes of occupational lung

diseases, such as asthma and allergic alveolitis. (2) Develop improved techniques for the 

detection of such immune reactions before adverse clinical outcomes occur. (3) Develop 

improved techniques for the detection and identification of inciting occupational agents.

This project will involve the analyses of clinical samples, environmental bulk samples, and

environmental aerosol samples. Successful completion of these investigations should lead to 

the development of effective prevention strategies for occupational allergies and asthma.

Genetics

Immunotoxicity of 

subchronic fungal 

exposures

[Nayak]

To determine the pulmonary immunopathological outcomes of subchronic exposures to fungal 

spores in mice. The study will focus on fungal species nominated by the NTP to determine 

the health effects associated with long-term exposures. This research will aim to identify and 

characterize the underlying immunological mechanisms of allergic fungal diseases in lungs. 

Proposed studies will help to identify biomarkers and specific targets for diagnostics, prevention,

and therapeutic intervention of occupational-related pulmonary fungal diseases.

Immunomodulatory effects 

of triclosan on effector 

CD4 T cell development 

[Marshall]

To identify the cellular and molecular mechanisms behind the immune-modulating effects of 

the antibacterial chemical triclosan. This information will provide the basis for the evaluation of 

other nonsensitizing antimicrobial chemicals, to help identify potentially conserved mechanisms 

for contributing to allergic disease. This will help determine the need for evaluating these types 

of chemicals, which are encountered in the workplace, leading to better risk assessment and

establishment of occupational exposure limits.

Highly sensitive and 

practical biomarkers for

nanotoxicity 

[Joseph]

To develop, validate, and test highly sensitive and minimally invasive biomarkers for early

detection of pulmonary toxicity potentially associated with exposure to toxic nanomaterials, 

using a rat model. Techniques will be employed to develop transcriptomic signatures in blood 

as surrogate biomarkers for the pulmonary toxicity induced by inhalation exposure to specific

nanomaterials. Bioinformatic analysis of the global transcriptomics data will be conducted to 

gain insights into the molecular mechanisms underlying the pulmonary toxicity of nanomaterials. 

Determining the molecular mechanisms of pulmonary toxicity and developing highly sensitive 

and minimally invasive biomarkers for nanotoxicity have implications in monitoring workers

for their risk of developing adverse health effects potentially associated with exposure to toxic

nanomaterials.

Toxicological evaluation 

of pulmonary exposure to 

graphenes 

[Roberts]

(1) To characterize the pulmonary, cardiovascular, and neurological toxicity that may be 

associated with respiratory exposure to graphenes, using an in vivo tiered toxicity testing 

approach; and (2) determine potential mechanisms of pathology where it exists. The project will 

address whether respiratory exposure to graphene as a nanomaterial poses a risk for workrelated illness. This project is from the Nanotechnology Research Center at NIOSH.
50 National Toxicology Program Annual Report FY 2014

NIOSH/NTP Projects

[Study Scientist] Objective and/or Summary

Neurological risks

associated with 

workplace chemicals and

nanomaterials 

[Sriram]

To evaluate potential neurotoxicological effects associated with exposure to chemical agents, 

incidental nanoparticles, and engineered nanomaterials in a laboratory-based animal model. 

This includes neurological hazard identification and risk characterization, evaluating molecular

mechanisms of neurotoxicity, and identifying potential biomarkers of neurotoxicity. This project

will attempt to identify brain electroencephalographic changes by telemetry, to determine 

correlations with early stages of neuronal injury, in an effort to establish real-time biomonitoring 

programs. Findings from this study may contribute towards developing (1) novel biomarkers

for monitoring exposures, including real-time biomonitoring technologies; (2) prejob planning 

protocols, and hazard and risk assessment paradigms; and (3) occupational safety standards

for neurotoxic exposures to chemical agents, incidental nanoparticles, and engineered 

nanomaterials.

Mechanism of carbon

nanotube (CNT)-induced 

carcinogenesis and 

aneuploidy

[Sargent]

In vitro exposure of human cells to 1-4 nanometer diameter single-walled carbon nanotubes 

(SWCNT) resulted in fragmented mitotic spindle poles (centrosomes), multipolar mitotic 

spindles, and errors in chromosome number. By contrast, treatment with 10-20 nanometer 

diameter multiwalled carbon nanotubes (MWCNT) caused monopolar mitotic spindles,

suggesting that the diameter of the nanotube was important in the genotoxic response. The 

integration of SWCNTs and MWCNTs with the nucleus, the mitotic spindle microtubules,

DNA, and centrosome, as well as errors in chromosome number, suggested that CNTs were 

potentially carcinogenic. We then utilized a two-stage initiation/promotion protocol in a mouse 

model to determine whether inhaled MWCNTs act as a complete carcinogen or promote the

growth of cells with existing DNA damage. The results demonstrated that inhaled MWCNTs

were strong promoters of lung adenocarcinoma. We therefore proposed further studies using in 

vitro and in vivo approaches to (1) examine the dose response of CNT-induced tumor promotion; 

(2) further examine the role of CNT physical properties and diameter in the genotoxic effect 

using an in vitro model where human cells will be exposed to well-characterized 49 nanometer 

native and nitrogen doped MWCNT; and (3) determine if native and nitrogen-doped carbon

nanotubes will induce mitotic disruption in vivo using a transgenic mouse model with a green 

mitotic spindle pole.

Epigenetic changes in 

response to nanoparticle 

exposure 

[Ding]

To investigate potential pulmonary carcinogenesis in response to tungsten carbide cobalt 

(WC-Co) particle exposure using cell culture and animal models. Occupational exposure to 

hard metal dust is associated with an increased risk of lung cancer. Tungsten heavy alloys are

used broadly for radiation shielding in both medical equipment and oil well drilling industries, 

weights and counterbalances, boring bars and grinding quills, and tooling for die cast 

manufacturing. Nanograined WC-Co composites are exceptional surfacing materials used for 

hard coatings, decarburization, and metal spraying. Exposure to WC-Co has been shown to 

cause pulmonary disease and induce lung cancer. Our previous studies indicate that WC-Co 

nanoparticles generate more reactive oxygen species (ROS) than fine particles when incubated

with cells. Both fine and nanoparticles of WC-Co stimulate ROS generation and MAPKs AP-1

cascade. The underlining mechanisms of WC-Co-induced carcinogenesis have not been 

investigated. Mechanistic investigations, including gene mutation, activation of transcription

factors, and ROS generation, will be conducted to explain the events of WC-Co-induced tumor 

initiation, promotion, and progression. Determining the mechanisms involved in WC-Co-induced 

carcinogenesis, and elucidating target signaling pathways, could provide insights for the 

development of biomarkers and possible prevention strategies for WC-Co-induced diseases.
5. Testing and Research 51

NIOSH/NTP Projects

[Study Scientist] Objective and/or Summary

Assessment of specific

carbon nanotube-induced 

lung carcinogenesis 

[Rojanasakul]

Carbon nanotubes (CNTs) exhibit a fibrous morphology and biopersistence similar to asbestos,

a known human carcinogen. Pulmonary exposure to CNTs results in disposition into alveolar

and pleural areas, raising a major concern about human lung cancer and mesothelioma risks

associated with long-term CNT exposure. The objective of this project is to test the hypothesis 

that pulmonary exposure to CNTs targets susceptible lung cells, such as epithelial and 

mesothelial cells, and induces neoplastic transformation of the cells, leading to carcinogenesis. 

This project addresses several critical needs and challenges associated with the poorly 

known cancer hazard potential of nanomaterials exposure. The specific aims are to (1)

develop in vitro chronic exposure models, to evaluate the carcinogenic potential of CNTs and 

determine the effects of dose, exposure time, and physicochemical properties of CNTs on their 

carcinogenicity; (2) assess the tumorigenic potential of CNT-transformed cells in vivo; and (3) 

identify molecular signatures and gene targets of CNT-induced cell transformation as potential 

biomarkers, to predict CNT-induced lung cancer and mesothelioma. This project is from the

Nanotechnology Research Center at NIOSH.

Fracking: toxicological

effects of silica and diesel 

exhaust exposure 

[Fedan]

To investigate the toxicities of inhaled diesel exhaust, in combination with inhalation of fracking

sand dust, mimicking worker exposures during fracking operations. Effects on several organ

systems will be investigated using a battery of in vivo and in vitro animal model tests.

Pulmonary function and 

nanoparticle inhalation: in 

vivo and in vitro effects 

[Fedan]

To characterize multiwalled carbon nanotube effects on critical aspects of lung function in vivo 

and airway function in vitro, and provide metrics to enable risk assessment strategies.

Health effects of inhaled 

crude oil 

[Fedan]

To design and build an inhalation exposure system to deliver crude oil vapor to rats,and use 

the system to study the effects of crude oil vapor inhalation on the respiratory, cardiovascular, 

immune, and central nervous systems.

ii. Comprehensive Assessment of Occupationally-Relevant Exposures

NIEHS is coordinating an NTP effort with NIOSH to better understand worker exposures, identify occupational

health research gaps, and educate workers. The NIEHS and NIOSH interagency agreement supports these

projects. FY 2014 efforts listed in Table 25 address worker exposures to welding fumes, nanosized materials,

food flavorings, bisphenol A, indium compounds, and other industrial chemicals.
52 National Toxicology Program Annual Report FY 2014

Table 25. NIEHS and NIOSH Interagency Agreement on Occupationally Relevant 

Exposures in FY 2014

Study 

[Study Scientist] Objective and/or Rationale

Administrative support 

[Whelan]

To enable NIOSH scientists to (1) participate in review and oversight of NTP activities; 

and (2) attend NTP-related meetings in Research Triangle Park, North Carolina, and

Washington, D.C.

Occupational exposure assessment 

of welding fume with emphasis on 

manganese compounds

[Hanley]

(1) To identify industries, such as construction, shipbuilding, railroad, and 

manufacturing companies; and unions involved in welding operations, where the 

potential for substantial manganese exposure exists, for exposure assessments; (2) 

develop methods to identify manganese compounds and different valence states, 

based on selective solubility with various welding fumes matrices; and (3) characterize 

welding fume exposures based on welding-associated jobs, tasks, and processes.

Exposure assessment of diacetyl 

and other flavorings in food

production industries

[Curwin]

(1) To characterize workplace inhalation exposures to diacetyl in food production

industries that use food flavorings; (2) document high-exposure activities and

processes in the flavored food production industries; (3) identify work practices and

procedures that affect exposure; (4) document engineering controls; and (5) field test

novel techniques for both gravimetric and volatile sampling.

Exposure assessment of 

engineered nanoparticles

[Geraci]

(1) To identify workplaces engaged in the synthesis, manufacture, and use of

engineered nanomaterials; and (2) characterize workplace exposure to selected

engineered nanoparticles.

Durability of nanoscale cellulose 

fibers in artificial human lung fluids

[Stefaniak]

To investigate the in vitro durability of nanocellulose materials in artificial lung fluids.

Data generated from this study will be used to inform larger and more costly in vivo 

inhalation studies.

Occupational exposure to 

bisphenol A (BPA) in the U.S.

[Hines]

(1) To understand BPA use in industry, including processes, jobs, and tasks, with

potential BPA exposure; (2) develop air, hand wipe and surface wipe sampling, and 

analytical methods for BPA, using liquid chromatography-tandem mass spectrometry 

and liquid chromatography with ultraviolet detection; and (3) assess exposure to BPA 

among workers through air, hand wipe, and urine sample collection.

Industry-wide exposure 

assessment study of workers

exposed to carbon nanotubes and 

nanofibers

[Dahm]

(1) To establish sampling and analysis protocols for detection and quantification

of carbon nanotubes and nanofibers; (2) recruit companies and conduct exposure

assessments for carbon nanotubes and nanofibers in a representative sample of U.S.

workplaces; (3) document high exposure tasks and processes, as well as collect full

work shift, personal breathing zone samples; (4) refine exposure assessment methods,

which include lowering the detection limit for elemental carbon (NMAM 5040); and (5)

evaluate higher-flow, respirable cyclones to assess health-relevant exposures.

iii. Immunotoxicology Research

The NIEHS and NIOSH interagency agreement provides support of NTP hazard identification activities aimed

at preventing diseases or adverse effects caused by environmental exposure to chemical or physical agents. 

These cooperative studies continue to improve risk assessment, by measuring what constitutes an adverse

health effect on the immune system in humans. The studies, listed in Table 26, evaluate unique cohorts of 

individuals from professions associated with immune-mediated occupational diseases.
5. Testing and Research 53

Table 26. NIEHS and NIOSH Interagency Agreement on Immunotoxicology Studies, FY 

2014

Study 

[Study Scientist] Objective and/or Rationale

Identification and

characterization of crossreactive fungal biomarkers

[Green]

To develop species-specific monoclonal antibodies to recombinant fungal biomarker

antigens. The utility of these monoclonal antibodies will be important for the quantification

of species-specific biomarkers of fungal exposure, particularly to those fungi that are being

studied by NTP. In this project, Aspergillus terreus was initially used as a model fungal 

organism for the development of fungal-specific proteins using recombinant technologies.

Aspergillus terreus hemolysin was produced by recombinant technology, and a panel 

of species-specific monoclonal antibodies was also produced. The same recombinant

technologies were used to identify and characterize additional candidate fungal allergens. 

Recombinant Chaetomium globosum enolase (rec-CgEnol) has been cloned, expressed, 

purified, and confirmed using mass spectrometry. Three rec-CgEnol specific IgG1

monoclonal antibodies have been produced and characterized. Several manuscripts have 

been published that report these findings. The development of a sensitive immunoassay

that can be used in the serological detection of this biomarker is anticipated.

Toxicity of subchronic fungal 

exposures

[Green]

To characterize the hazards associated with personal exposure to common fungal 

contaminants in indoor and occupational environments; advance the understanding 

of toxicological and pulmonary immune responses associated with subchronic fungal 

exposures using a model that replicates human exposure; utilize an Accoustical Generator 

System and multi-animal nose-only exposure chamber to characterize Aspergillus fumigatus 

toxicological endpoints following subchronic dry fungal spore and particle exposures; and 

following completion of NTP Study #C08022, characterize subchronic toxicity and pulmonary 

immune responses to fungal contaminants nominated to the NTP, including Stachybotrys

chartarum (mycotoxin producing and nonproducing chemotypes), Aspergillus versicolor, 

and Alternaria alternata. In FY 2015, protocols will be submitted to NTP for the evaluation 

of Avicularia versicolor and a mycotoxin producing chemotype of Stachybotrys chartarum. 

Alternaria alternata, mixed fungal exposures, and other occupationally relevant fungi 

identified in concurrent NTP funded diversity studies will be evaluated in future fiscal years.

These toxicological studies will provide novel datasets that will be used to characterize the 

hazards that fungal exposure may represent to human health.

Identification and

characterization of fungal 

exposures 

[Green]

To investigate and characterize the diversity of fungal bioaerosols in the indoor built 

environment, using large-scale ribosomal RNA sequencing. In preliminary studies, 

various methods of extraction were compared and tested on occupational dust samples. 

A standardized extraction method was then used on 30 air and dust samples derived from 

homes participating in the project. Results from the analysis have been compiled and 

provide detailed insight into the diversity of previously overlooked fungal bioaerosol sources

in the indoor environment. Future studies, in collaboration with Assured Bio Inc., Columbia 

University, and NIOSH, are planned for FY 2015. These studies aim to further evaluate 

indoor and occupational environments, using large scale ribosomal RNA approaches 

from samples collected in contaminated and noncontaminated environments. In addition, 

fungal species richness identified in this analysis will be directly compared to commercially

available methods of fungal analysis, including the Environmental Relative Mold Index.

Alternative methods 

for chemical allergen 

identification and assessment:

use of immunochemical 

techniques for investigation of 

diisocyanate allergic diseases

[Siegel]

(1) To develop amine and thiol-based probe for kinetic assessment of chemical binding

(haptenation) with potential for standardization as a screen test for direct-acting chemical 

allergens; (2) assess the kinetic probe assay to predict the allergenicity, with chemical

modification of a known chemical allergen; and (3) use in-house developed monoclonal

antibodies against diisocyanate bound proteins to identify potential haptenation targets and 

study disposition after dermal exposure.
54 National Toxicology Program Annual Report FY 2014

Study 

[Study Scientist] Objective and/or Rationale

Analysis of mycotoxins in 

dust samples from a waterdamaged building

[Park]

The ultimate goal of the project is to examine potential roles of exposure to fungal toxins on 

occupants’ health in water-damaged buildings. To achieve the goal, we plan to (1) develop 

a cost-effective and robust method, using an ultra-performance liquid chromatographytandem mass spectrometry, for simultaneous analysis of multiple fungal toxins in 

environmental samples; (2) increase sensitivity and accuracy of the method by incorporating 

isotopically labeled (C13) mycotoxin internal standards to compensate extraction loss and 

matrix effects; (3) apply the method to examine stability of the mycotoxins in floor dust

stored in different temperature conditions for different lengths of time; and (4) quantify 

fungal toxins in floor dust samples that will be collected from a funded school study in

FY 2015, and from samples that have been collected from previous studies of waterdamaged buildings. Since these studies already have other exposure data, including fungal 

components, bacterial components, and occupants’ health, we will analyze them to examine 

the effect of exposure to fungal toxins on occupants’ health. 

iv. NTP Research in Partnership with NCTR

NCTR, in partnership with researchers from elsewhere in FDA, other government agencies, academia, and 

industry, provides innovative technology, methods development, vital scientific training, and technical expertise.

The unique scientific expertise of NCTR is critical in supporting FDA product centers and their regulatory roles.

FY 2014 studies, funded by NCTR voluntary allocations, are listed in Table 27. Table 28 lists FY 2014 projects 

that are funded through FDA and NTP interagency agreements.

Table 27. NCTR/NTP Projects in FY 2014

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Biochemical and Molecular Basis of Toxicology

An evaluation of the effect 

of vehicle cream on the 

photococarcinogenicity of retinyl 

palmitate in SKH-1 mice

[Boudreau]

(1) To determine the stability and homogeneity of retinyl palmitate in control cream. In the 

previous photococarcinogenicity study of retinyl palmitate, diisopropyl adipate was used 

as a carrier and filler for the retinoids. The stability and homogeneity of retinyl palmitate

has not been determined for a base cream that does not contain diisopropyl adipate. 

(2) Evaluate the photococarcinogenicity of retinyl palmitate when incorporated into the 

control cream and applied to the skin of SKH-1 mice in the absence and presence of

simulated solar light. (3) Determine the photococarcinogenicity of diisopropyl adipate 

as the filler ingredient in the control cream, in the absence and presence of simulated

solar light. In the previous study on retinoic acid and retinyl palmitate, the control 

cream induced skin tumors even in the absence of simulated solar light. The suspect

ingredient in the control cream was diisopropyl adipate, which appears as an ingredient 

in numerous topically applied skin products, including sunscreens.

Biologically based dose-response 

modeling for the thyroid axis in 

the fetus and neonate

[Fisher]

(1) To create biologically based dose-response models for the hypothalamic-pituitarythyroid axis in the developing rat and human as a function of iodide status; (2) interface 

the models with physiologically-based pharmacokinetic (PBPK) or toxicokinetic models

for thyroid active chemicals, to predicted conditions (iodide status and chemical 

exposure) for which brain thyroid hormone homeostasis cannot be maintained in 

the fetus and neonate; and (3) use the models to evaluate the possible influence of

population exposures to thyroid active chemicals on fetal and neonatal thyroid status as 

a function of iodide intake.
5. Testing and Research 55

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Physiologically-based 

pharmacokinetic (PBPK) models

for BPA

[Fisher]

(1) To create PBPK models for BPA in three species of adult, neonatal, pregnant (mother 

and fetus), and lactating (mother and neonate) laboratory animals (mouse, rat, and 

rhesus monkey). These PBPK models will be used to calculate internal measures of

dose for both aglycone (active) and conjugated (inactive) forms of BPA. (2) Human 

PBPK models for BPA (adult, child, pregnant mother and fetus, and lactating mother and 

infant) will be created using information obtained from the monkey, mouse, and rat, and

from the limited human information published in literature. The human suite of PBPK 

models will be used to extrapolate the internal doses of BPA associated with toxicity in 

laboratory animals to humans, and to extrapolate dosimetry from regions of observation 

to low levels of exposure to BPA, for which no experimental data exist. This simulation 

protocol will help reduce the uncertainty in the assessment of health risks posed by BPA

to human populations.

Relationship between liver 

epigenetic phenotype and 

susceptibility to nonalcoholic 

steatohepatitis-induced 

hepatocarcinogenesis in mice 

[Pogribny]

(1) To determine the role of epigenetic dysregulation in the etiology and pathogenesis 

of dietary nonalcoholic steatohepatitis-induced hepatocarcinogenesis in mice; (2) 

determine whether or not interstrain-specific susceptibility of mice to nonalcoholic

steatohepatitis-induced hepatocarcinogenesis is associated with differences in individual 

hepatic epigenetic phenotypes; (3) determine the role of epigenetic dysregulation in the 

etiology and pathogenesis of nonalcoholic steatohepatitis-induced hepatocarcinogenesis 

in mice induced by tamoxifen administration; and (4) determine whether or not aberrant 

epigenetic markers can be used as targets for prevention of nonalcoholic steatohepatitisinduced hepatocarcinogenesis in mice.

Sex differences in drug-induced 

QT prolongation and torsade 

de pointes: establishing an in 

vitro model for high throughput 

screening and risk assessment of

torsadogenic drugs

[Pang]

(1) To establish a model and positive control; and (2) evaluate the sensitivity and 

specificity of the model and test the possibility of high throughput screening and ranking

QT prolonging drugs for the risk of torsade de pointes.

The role of ATP binding 

cassette-drug transporters in 

chemoresistance in pancreatic 

cancer

[Pang]

(1) To compare various ATP binding cassette transporters’ expression in normal and 

pancreatic adenocarcinoma specimens and determine whether the expression of ATP 

binding cassette transporters is correlated with clinical aggressiveness of the tumor; 

(2) evaluate whether the single nucleotide polymorphisms in ATP binding cassette 

transporter genes are associated with the abnormal expression of the efflux pumps

and drug sensitivity; and (3) assess the epigenetic regulation of ATP binding cassette 

transporters in pancreatic cancer.

Mechanism of tumorigenic

pyrrolizidine alkaloids

and development of liquid 

chromatography-electrospray 

ionization/multistage mass 

spectrometry (LC-ESI/MS/MS(n))

methodology for detection and 

quantification of pyrrolizidine

alkaloids

[Fu]

(1) To validate the proposed mechanism by which pyrrolizidine alkaloids induce tumors

in rodents; (2) develop an LC-ESI/MS/MS(n) method for detection and quantification of

dehaloperoxidase-derived DNA adducts in rodents; (3) develop an LC-ESI/MS/MS(n)

method for detection and quantification of genotoxic pyrrolizidine alkaloids in herbal

plants and herbal dietary supplements; and (4) develop an LC-ESI/MS/MS(n) method for

detection and quantification of dehaloperoxidase-derived hemoglobin adducts in rodents.
56 National Toxicology Program Annual Report FY 2014

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

In vitro analysis of factors 

influencing CYP1A2 expression

as potential determinants of sex 

and interindividual variation: role 

of hormones and epigenetics

[Hammons]

(1) To determine the effect of cigarette smoke condensate or I3C on CYP1A2 expression

in selected liver and lung cell lines; (2) determine the effect of combining menthol with 

cigarette smoke condensate; (3) determine the effect of cigarette smoke condensate

(with and without menthol) or I3C on DNA methylation and histone modification in

CYP1A2 as epigenetic regulatory mechanisms in liver and lung cells; and (4) determine 

the effect of hormones, such as estrogen, testosterone, and growth hormone, on 

CYP1A2 expression as factors underlying sex differences in expression.

Epigenetics, DNA methylation, 

and obesity

[Varma]

To evaluate the effect of differences in DNA methylation and agouti signaling protein 

in the offspring of Avy/a dams crossed with a/a sires as a result of nutrient x gene 

interactions. These preliminary data will be used to select the appropriate diets for further 

studies on obesity and Type 2 diabetes.

Development of 3-D human skin

model for in vitro genotoxicity 

testing of chemical and physical 

agents

[Ding]

To evaluate the ability of human 3-D skin models to assess the genotoxicity of dermal

exposure to FDA-regulated products, including nanomaterials.

Dose-response genotoxicity of 

ethylmethane sulfonate in mice 

using the phosphatidylinositol 

glycan complementation group A 

(Pig-a) and transgenic gpt delta 

assays

[Cao]

(1) To use sensitive genotoxicity endpoints with low background frequencies to

increase the sensitivity of the assays for detecting low-dose effects; (2) measure 

genotoxicity using a design to detect the maximum responses; (3) measure the effects of 

ethylmethane sulfonate exposure in neonatal as well as adult animals; and (4) measure 

genotoxicity in the major target tissues for ethylmethane sulfonate carcinogenicity.

Neurotoxicology

Development of novel 

histochemical markers of

brain-vascular elements and 

their application for localizing 

neurotoxicant-induced 

pathologies

[Schmued]

(1) To develop and characterize novel markers for brain vascular elements and

investigate the effects of three different classes of neurotoxicants, including kainic acid,

an N-methyl-D-aspartate (NMDA) agonist, 3-nitropropionic acid, an inhibitor of metabolic

respiration, and metamphetamine, a dopamine agonist on vascular elements, such as 

perivascular pericytes, vascular lumen, and perivascular sheath; (2) characterize the 

response of certain vascular elements to neurotoxic insults; and (3) fluorescent and

bright field labeling at the vascular lumen.

Addendum: Development of 

novel histochemical markers

of brain-vascular elements 

and their application for 

localizing neurotoxicant-induced 

pathologies

[Schmued]

To characterize the respective pathologies at different time points following kainic acid

administration. 

Methylphenidate (ritalin) exposure

during pregnancy: assessment of 

neurotoxicity in offspring 

[Ferguson]

To determine the neurobehavioral toxicity associated with prenatal and early postnatal 

treatment with methylphenidate in rats using a range of behaviors at preweaning, 

adolescent, and adult life stages.
5. Testing and Research 57

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Developmental neurotoxicity 

assessment of N-methyl-Daspartate (NMDA) receptor

antagonists in zebrafish

[Kanungo]

(1) To study wildtype zebrafish embryos exposed to NMDA receptor antagonists (MK801, dextromethorphan, ketamine and sevoflurane), in order to assess their effects

on Rohon-Beard sensory neurons. The effects on the primary and secondary motor 

neurons and their axons will also be assessed using hb9:GFP transgenic embryos. 

Post-exposure, wash-out experiments will be pursed to determine the effects of 

these drugs on the nervous system. (2) Certain NMDA receptor antagonists, such as

ketamine, are known to modulate hormone levels in mammals (existing literature) and

zebrafish (unpublished data). Based on the importance of the neuroprotective nature

of neurosteroids, the study will determine estradiol-17’ levels in control and treated 

embryos. Changes in gene expression for the two CYP aromatases/estrogen synthases 

(brain aromatase cyp19a1b and gonadal aromatase cyp19a1a) will be quantified using

qPCR. (3) Phenotype-based cell signaling mechanisms (MAPK, etc.) and neuron

development-specific gene expression will be assessed. (4) Reversal of noted adverse

effects of these compounds on neurons will be attempted, particularly by treatment with 

acetyl L-carnitine. 

Methods development for toxicity

assays using the zebrafish

embryo as a model system: whole 

animal high throughput assays for 

chemical testing 

[Kanungo]

(1) To study the effect of methamphetamine on zebrafish embryos, especially related to

sensory and motor neuron development, using an established high throughput assay 

system to evaluate morphological and behavioral endpoints of toxicity and subtle organspecific toxicities; (2) determine if carbon nanotubes pass through the blood brain barrier

in zebrafish embryos, and, if so, if the nanomaterials generate reactive oxygen species,

deplete dopamine and its metabolites (dihydroxyphenylacetic acid and homovanillic 

acid), and alter markers of oxidative stress; and (3) determine the effect of nicotine on

zebrafish embryos, especially relating to sensory and motor neuron development and the

mechanism of action.

Development of a small, voxel, 

short echo time 1H magnetic 

resonance spectroscopy toolbox 

for biomarkers of neurotoxicity in

rats

[Liachenko]

(1) To establish a preclinical 1H-magnetic resonance spectroscopy platform toolbox 

for sensitive and fast measurements in small voxels; and (2) characterize acute effects 

of psychomodulators (amphetamine, ketamine) in normal rats using 1H-magnetic

resonance spectroscopy S (preclinical biomarker development).

Nanotoxicology

Does the durable nanoparticle 

bioaccumulation in macrophages 

increase susceptibility to bacterial 

infection?

[Khan]

To determine whether animals exposed to durable nanoparticles are more susceptible to 

Listeria infection, as measured by the severity of disease and the length of time needed 

to clear the infection.

Determination of cytotoxicity and 

genotoxicity of nanomaterials 

of interest to the FDA and 

mechanism of action

[Fu]

(1) To develop a set of cell-free and cell-based in vitro tests that can be used to rapidly 

identify nanomaterials of interest to FDA that elicit oxidative damage; and (2) determine 

if in the presence of nanometal materials, endogenous and dietary antioxidants can 

display pro-oxidative activity.

Development of methods 

for determining nanoparticle 

penetration or permeation into 

vaginal mucosal tissue

[Zhang]

(1) To develop and adapt methods to deliver nanoscale material into rat vaginal tract; 

and (2) develop the analytical methods to quantify vaginal mucosal penetration of select 

nanomaterials following intravaginal lavage. 
58 National Toxicology Program Annual Report FY 2014

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Evaluation of the applicability 

of in vivo micronucleus assays 

for assessing genotoxicity of 

engineered nanomaterials 

[Chen]

(1) To assess the genotoxicity of four types of nanoscale materials (carbon nanotubes, 

nanoscale titanium dioxide, nanoscale gold, and nanoscale silver) in three standard 

tests used for genotoxicity assessment by FDA (Salmonella Ames test, mouse 

lymphoma assay, and in vivo mouse micronucleus assay); and (2) evaluate the possible 

mechanisms of nanomaterial-induced genotoxicity using a transgenic mutation system, 

comet assay, and genomic analysis.

Do engineered silver 

nanomaterials, varying by size 

and coatings, behave differently 

than bulk silver in their ability to

induce genetic damage? 

[Chen]

To evaluate the genotoxicity of various sizes of engineered silver nanoparticles and bulk

silver in the Ames test, mouse lymphoma assay, and in vitro micronucleus assay.

Assessment of iron oxide 

nanoparticle-induced 

neurotoxicity in cell cultures and 

whole animal models 

[Binienda]

To determine if acute or chronic exposure of different sizes of iron oxide nanoparticles 

produce (1) specific changes in the mitochondrial function, cell death, and generation

of reactive oxygen species in different regions of rat and mice brain using in vivo 

microdialysis; (2) significant changes in neurotransmitter concentrations in different

regions of mice/rat brains using microdialysis; (3) alterations in the brain free fatty acid 

levels; (4) alterations in lipid peroxidation or changes in antioxidant enzyme activity 

(catalase, superoxide dismutase, glutathione peroxidase) and glutathione levels in mice 

and rat brains; and (5) selective patterns of deposition and damage in different regions of 

rat and mice brain, using in vivo magnetic resonance imaging.

Complement assays for the 

detection of immune-sensitizing 

activity of nanomaterials

[Leakey]

(1) To establish two complement assays for routine evaluation of immune-sensitizing 

activity of nanomaterials; (2) validate the assays using nanoparticles with known

immunoreactivity; and (3) determine the immune-sensitizing activity of novel 

nanomaterials.

Physiologically based 

pharmacokinetic (PBPK)

modeling of nanomedicine; 

building clinically relevant 

standards for FDA-regulated 

nanoparticulate drug products

[Leakey]

(1) To determine in vivo liposomal doxorubicin release kinetics in individual tissues and

blood stream by PBPK modeling; (2) establish quantitative physicochemical property 

(liposomal size and content of ammonium sulfate) biodistribution relationships of 

liposomal doxorubicin products by PBPK modeling; and (3) extrapolate the PBPK model 

to rats and humans. A whole-body PBPK model will be developed to describe and 

simulate the biodistribution of liposomal vesicles and doxorubicin.

Development and evaluation of 

exposure dosimetry methods 

to optimize the standard in 

vitro mammalian genotoxicity 

assays for assessing engineered 

nanomaterials 

[Chen]

(1) To evaluate whether the in vitro mammalian genotoxicity assay is suitable for 

assessing the genotoxicity of nanomaterials; (2) explore the possible mechanisms 

underlying genotoxicity of engineered nanomaterials by conducting genomic analysis; 

(3) identify potential improvements to the assay and general strategies for evaluating 

nanomaterials; and (4) examine whether the suitable methods and other experiences 

learned from the micronucleus assay are applicable to other genotoxicity tests, such as 

mouse lymphoma assay and in vivo micronucleus assay.

Assessment of size and shape 

dependent toxicity of silver 

nanoparticles as measured by 

changes in the permeability at the 

gastrointestinal surface 

[Khare]

(1) To investigate various cellular components involved in the uptake of nanoparticles in

intestine, their accumulation in various cell types, and the effect of nanoparticles on the 

intestinal microbiome, by determining the effect of nanomaterials on the permeability of 

intestinal epithelial cells in vitro and ileal mucosa ex vivo; and (2) measure the toxicity of 

silver nanoparticles as measured by changes in the expression of genes involved in the 

epithelial integrity of polarized epithelial cells and ileal mucosa.
5. Testing and Research 59

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Bioassay and Biomarker Development and Evaluation

Biomarkers of liver toxicity

[Mendrick]

(1) To discover biomarkers of hepatotoxicity in preclinical studies, which are predictive

of adverse effects in humans, for eventual evaluation of predictivity in rodent assays 

(preclinical studies); and (2) qualify the discovered biomarkers.

Development of cancer-relevant 

biomarkers for identification of

potential carcinogens: research 

to understand the normal 

background frequencies in rats

[Mckinzie]

To understand the distribution and range of spontaneous oncogene mutant frequencies 

in the major organs of rats and mice, to provide important basic information for the 

validation of these oncogene mutant frequencies as biomarkers of chemically induced

carcinogenesis.

Phosphatidylinositol glycan 

complementation group A (Pig-A) 

mutagenesis: an international 

validation study comparing 

Pig-A mutation in rats with other 

biomarkers of genetic toxicity

[Heflich]

(1) To generate data using a standardized protocol, which in combination with results 

from other investigators will be used to determine the sensitivity, specificity, and

portability of the rat red blood cell/reticulocyte Pig-A gene mutation assay; and (2) 

compare the Pig-A assay results with the in vivo comet and micronucleus formation 

assays, and hypoxanthine-guanine phosphoribosyltransferase lymphocyte gene 

mutation assays.

Mechanistic study on the

disruption of thyroid hormone 

homeostasis resulting from 

subchronic dermal exposure of 

triclosan to mice

[Fang]

To examine the mechanisms underlying the effects of triclosan on thyroid hormone 

homeostasis in female B6C3F1 mice.

Development of a high throughput 

assay for measuring in vivo 

mutation in an autosomal gene

[Dobrovolsky]

To develop a high throughput, in vivo, mutation model that detects mutations induced 

by a range of mechanisms including gene mutation, large deletions, and loss of 

heterozygocity. The basic properties and sensitivity of the model will be evaluated in 

experiments employing well characterized mutagens.

Development of methods for 

evaluating DNA damage using 

single cell gel electrophoresis 

(comet assay) in rodents 

[Manjanatha]

To evaluate and establish methods and conditions that enhance the sensitivity 

and reproducibility of the in vivo alkaline-comet assay for use in preclinical-hazard

identification and genotoxicity testing of food ingredients and chemicals for regulatory

purposes.

Development and application of a 

mitochondria-specific gene array

(Mitochip) for the investigation of

preclinical and clinical predictive 

biomarkers of toxicity

[Desai]

(1) To develop a MitoChip for various mammalian species, including rat, nonhuman

primate, and human; (2) investigate the mechanisms of drug toxicities and degenerative 

diseases associated with mitochondrial dysfunction in different mammalian species by 

conducting transcriptional profiling of mitochondria-related genes; and (3) characterize

species-specific transcriptional profiles to predict risk of drug toxicity or disease onset in

different mammalian species.

Development of the mouse 

embryonic stem cell test

[Hansen]

To gain hands-on experience and better characterize and indicate potentially beneficial

modifications to the assay. 
60 National Toxicology Program Annual Report FY 2014

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Identification of protein

biomarkers for neurotoxicity

assessments using a high 

throughput antibody microarray 

approach

[Gu]

(1) To examine proteomic changes at both the expression and phosphorylation levels 

using five established in vivo models of neurotoxicity; (2) identify common changes

in protein expression and phosphorylation status in these animal model systems; (3) 

confirm the observed alterations in protein expression and phosphorylation status by

means of other independent methods; and (4) apply the proteomic findings to a global

ischemic animal model to further validate the utility of protein biomarkers for use in

neurotoxicity assessments.

Evaluation of microRNAs in 

blood and urine for detection of 

chemical-induced carcinogenicity

[Chen]

(1) To determine miRNAs in blood and carcinogenic target tissues that respond to 

exposure of carcinogens, and the best time for sampling of their expression after 

treatment in rats; (2) determine miRNA profiles from the blood and target tissue samples

of rats treated with different mode-of-action carcinogens, such as alkylating agents,

aneugens, clatogens, and nongenotoxic carcinogens at the appropriate sampling time 

determined by objective 1; (3) determine the functions and pathways of the dysregulated 

miRNAs by the carcinogen treatments, and examine whether the changes in blood relate 

to those in the target tissues; and (4) establish specific miRNA biomarkers in blood for

assessing different types of carcinogens.

Establishment of embryonic 

stem cells as an in vitro model to 

explore developmental toxicity

[Inselman]

(1) To develop an in vitro culture system utilizing mouse mesodermal embryonic stem 

cells and human pluripotent stem cells; and (2) examine the mechanisms(s) responsible 

for embryotoxicity associated with selected known or suspect embryotoxins that affect

differentiation into osteoblasts.

Effect of fetal exposure to 

oxybenzone on reproductive 

organs of postnatal day 21 rats 

[Nakamura]

To determine if fetal exposure to oxybenzone influences the male (testes, steroid

biosynthesis) and female (ovary) reproductive organs of Harlan Sprague Dawley rats on 

postnatal day 21 by examining morphology of the reproductive organs and messenger 

RNA expression of genes related to the endocrine system.
5. Testing and Research 61

Table 28. NIEHS-NCTR Interagency Agreement Studies in FY 2014

Study 

CASRN 

[Principal Investigator]

Objective and/or Rationale

Food Contaminants

Two-year chronic toxicology study of BPA 

administered by gavage to NCTR Sprague 

Dawley rats from gestational day 6 until birth 

and directly to pups from postnatal day 1; 

continuous and stop-dose exposures

80-05-7 

[Delclos]

To characterize the long-term toxicity of orally administered BPA, including 

developmental exposure, in the NCTR Sprague Dawley rat over a broad 

dose range. In addition, animals generated in this study will be assigned to 

separate protocols for assessment of a range of molecular, morphological, 

and functional endpoints to determine if these endpoints are predictive of 

long-term toxic effects or reveal potential effects undetected by standard 

toxicological evaluations. 

Evaluation of molecular, morphological, 

and functional endpoints in NCTR Sprague 

Dawley rats treated with BPA administered 

by gavage from gestational day 6 until birth 

and directly to F1 pups from postnatal day 1; 

continuous and stop-dose (postnatal day 21) 

exposures 

80-05-7

[Delclos]

To evaluate a range of molecular, morphological, and functional endpoints 

in rats dosed orally with a wide range of BPA doses in a chronic toxicology 

study. The endpoints were selected based on reports from previous animal 

toxicology or human epidemiology studies suggesting that they are affected 

by BPA exposure. Assessments will be conducted at various ages (postnatal 

days 1, 21, and 90, as well as 6 and 12 months) and it will be determined 

if any effects observed are predictive of long-term effects evaluated in 

the companion chronic toxicology study, or if they reveal potential effects 

undetected by standard toxicological evaluations.

Neurobehavioral effects of BPA across age 

and sex 

80-05-7 

[Ferguson]

(1) To determine if orally administered BPA or ethinyl estradiol treatment 

during the perinatal period in Sprague Dawley rats affects anxiety, learning, 

and memory-related behaviors during juvenile and adult life, at three 

human-relevant BPA doses, and one dose of the reference estrogen; and 

(2) identify the molecular and morphological changes induced by perinatal 

BPA treatment or ethinyl estradiol in the hypothalamus and hippocampus, 

which are critical brain regions governing these behavioral responses. These 

studies use animals from the above study.

Assessment of the nephrotoxicity from the 

90-day combined exposure to melamine and 

cyanuric acid in F344 rats

108-78-1, 108-90-5

[Gamboa]

To investigate the nephrotoxic effects noted in F344 rats coexposed to 

melamine and cyanuric acid, and melamine cyanurate. The initial toxicity was 

reported in the kidneys of pets and children exposed to adulterated foods.

Two-year carcinogenicity bioassay of furan in 

Fischer 344 rats 

110-00-9

[Beland]

To determine the dose-response relationship for the carcinogenicity of furan 

in F344/N (NCTR) male rats in a two-year bioassay.

Dermal Toxicology Program

Two-year dermal carcinogenicity of triclosan 

in B6C3F1 mice 

3380-34-5

[Fang]

To determine the toxicity and carcinogenicity of topically applied triclosan in 

mice.
62 National Toxicology Program Annual Report FY 2014

Study 

CASRN 

[Principal Investigator]

Objective and/or Rationale

Nanoscale Material Program

Thirteen-week study to evaluate the

toxicology of silver nanoparticles in Sprague 

Dawley rats 

744-22-4

[Boudreau]

To determine the toxicity of nanoscale (10, 70, and 107 nanometeres) silver 

particles in a 13-week toxicity bioassay.

Reproductive and Developmental Toxicology Program

Effect of oxybenzone on fertility and early 

embryonic development in Sprague Dawley 

rats (Segment I) 

131-57-7

[Inselman]

(1) To examine the reproductive toxicity of oxybenzone in male and female 

rats, focusing specifically on fertility and early embryonic development to

implantation; and (2) compare the results of a typical Segment I, II, and III 

study design with results from a modified one-generation study conducted

simultaneously by NIEHS/NTP.

Effect of oxybenzone on embryo/fetal 

development in Sprague Dawley rats 

(Segment II)

131-57-7

[Inselman]

To determine the potential developmental toxicity of oxybenzone and 

compare the results of a typical Segment I, II, and III study design with results 

from a modified one-generation study conducted simultaneously by NIEHS/

NTP.

Effect of oxybenzone on prenatal and 

postnatal development in Sprague Dawley 

rats (Segment III)

131-57-7

[Inselman]

(1) To determine the potential toxicity of oxybenzone on prenatal and early 

postnatal development in male and female rats; and (2) compare the results 

of a typical Segment I, II, and III study design with results from a modified

one-generation study conducted simultaneously by NIEHS/NTP.
6. Alternative Methods Development 63

6. Alternative Methods Development

A. Tox21

In FY 2008, NTP established a high throughput screening initiative, representing a 

new paradigm in toxicological testing. On February 14, 2008, the Memorandum of

Understanding (MOU), High Throughput Screening, Toxicity Pathway Profiling, and

Biological Interpretation of Findings, was signed (see http://ntp.niehs.nih.gov/go/28213

for information about the MOU). Through this MOU, NIEHS/NTP has partnered

with the NIH Chemical Genomics Center (NCGC), and the EPA National Center for 

Computational Toxicology. The MOU was amended in 2010 to include FDA, whose

active participation is in recognition of its commitment to developing new methods 

to evaluate the toxicity of the substances it regulates. This interagency partnership is the basis for the Tox21 

program, and makes it possible to pool resources to overcome the resource limitations of a single agency,

build on existing expertise, and avoid the need to create a new administrative and support structure.

The Tox21 program was developed in response to the 2007 National Research Council report, Toxicity Testing 

in the 21st Century: A Vision and a Strategy. A central component of the MOU is exploration of quantitative

high throughput screening and quantitative high content screening (qHTS/qHCS) assays, and assays using 

phylogenetically lower animal species, such as fish and worms, as well as high throughput, gene expression,

analytical methods to evaluate mechanisms of toxicity. The ultimate goal of Tox21 is to provide the data 

generated by these new tools to risk assessors for use in protecting human health and the environment.

The goals of Tox21 and the NTP high throughput screening program are the same: prioritization of chemicals 

for further in-depth toxicological evaluation; identification of mechanisms of action for individual chemicals, as

well as chemical classes and structures; and, ultimately, predictive toxicology. The results of this collaborative 

effort should yield test methods for toxicity determination that are more scientifically and economically efficient,

inform adverse outcome pathway analyses, and provide models for risk assessment that are more biologically

and mechanistically based than current models. This approach should ultimately reduce or replace animals in 

regulatory testing, and is anticipated to occur in parallel with an enhancement of the ability to evaluate the large 

numbers of chemicals that currently lack adequate toxicological evaluation.

Phase I

In Tox21 Phase I, conducted from FY 2006 through FY 2010, a library of 2,870 compounds provided by 

NTP and EPA to NCGC was tested in 140 assays, representing 77 predominately cell-based qHTS assays. 

These assays broadly evaluated the ability of compounds to induce cytotoxicity; apoptosis; DNA damage; 

changes in methylation status; mitochondrial toxicity; upregulation of various stress response pathways, such 

as antioxidant, hypoxia, and heat shock, in a variety of cell types; or perturb nuclear receptor signaling in 12

different signaling pathways, including those involving the critically important estrogen and androgen receptors. 

Phase II

Based on the knowledge gained from Phase I, a screening approach for Tox21 Phase II was identified to

evaluate the biological effects of a 10,500-substance library (8,307 unique), informally known as the 10K

library, with approximately one-third of the total substances assembled by each Tox21 partner. The compounds 

cover a wide variety of classifications and include consumer products, food additives, chemicals found in

industrial processes, and human and veterinary drugs. A complete list of the compounds is publicly available 

at http://www.epa.gov/ncct/dsstox. Chemical analyses to determine the identity, purity, and stability of all 
64 National Toxicology Program Annual Report FY 2014

compounds in the library were initiated in FY 2012. The identity and purity results for the initial stock solutions

will be posted on the NCATS Tox21 server starting in FY 2015. An analysis of the stability of the compounds 

under the conditions of use will also occur in FY 2015. 

In FY 2011, screening of the 10K library began, as well as screening on a subset of approximately 700 of these 

compounds in Phase II of the EPA ToxCast Program (http://www.epa.gov/ncct/toxcast). The data from all 

these assays, along with full chemical characterizations and assay protocol details, are being deposited into 

publicly accessible, relational databases, such as the National Library of Medicine PubChem database (http://

pubchem.ncbi.nlm.nih.gov), the EPA Interactive Chemical Safety for Sustainability (iCSS) Dashboard (http://

actor.epa.gov/dashboard/), and the NTP Chemical Effects in Biological Systems (CEBS) database (http://www.

niehs.nih.gov/research/resources/databases/cebs). 

The 10K library contains 88 duplicate compounds, which are present on each compound plate, as well 

as approximately 2,100 compounds, which are present in the library in multiple copies and were obtained 

from different sources. This compound replication provides a means of evaluating interplate and intraplate 

variability, as well as cross-assay variability in compound activity, and monitors technical quality of a screen. 

The emphasis during Phase II is on (1) assays measuring nuclear receptor signaling in agonist and antagonist 

mode, including the aryl hydrocarbon receptor, androgen receptor (partial and full), estrogen receptor alpha 

(partial and full), farnesoid x receptor, glucocorticoid receptor (full), liver x receptor, peroxisome proliferatoractivated receptor delta and gamma, pregnane x receptor, retinoic acid receptor-related orphan receptor, 

retinoid x receptor, thyroid receptor, and vitamin D receptor; (2) induction of stress-response pathways, such as 

antioxidant, DNA damage, heat shock, and hypoxia; and (3) assays measuring specific toxicity endpoints, such

as mitochondrial membrane changes, caspase activation, aromatase inhibition, and upregulation of nuclear 

factor kappa beta, which is a transcription factor with an important role in the immune system.

To date, data from 88 qHTS assays measuring activity in 22 nuclear receptor agonist and antagonist assays, 

eight stress response pathway assays, and three autofluorescence detection assays were released to the

public through PubChem. For the majority of assays, cell viability was assessed using a complementary 

readout in the same wells as the main readout. In addition to the nuclear receptor and stress response assays, 

compound biological stability was assessed by screening the 10K library at 0, 2, 4, and 6 months of being 

maintained in use at room temperature in an assay measuring upregulation of p53. 

Tox21 Phase II is being conducted in a high-speed robotics screening facility opened at NCGC in FY 2011. It 

was built with funds provided by NIEHS/NTP. The robotics facility is used to screen the 10K compound library 

in triplicate (about 30,000 15-point concentration response curves per screen).

In FY 2011, the differential cytotoxicity response of cells from humans was evaluated by screening 1,089 

densely sequenced lymphoblastoid cell lines, representing nine racial groups, against 179 toxic chemicals. 

This study was designed to measure interindividual differences in sensitivity to environmental toxicants, based 

on genotype. The resulting data were analyzed and, in a collaboration with NCATS, the University of North 

Carolina (UNC), and Sage Bionetworks, portions of the data were used to conduct the first-ever crowdsourcing

challenge of its kind, the NIEHS-NCATS-UNC DREAM Toxicogenetics Challenge. For more details about the

challenge, see http://dreamchallenges.org. 

Phase III

Tox21 Phase III was initiated in FY 2013. The intent of this phase is to improve biological coverage and 

relevance of the screening activity by (1) focusing on more physiologically-relevant in vitro cell systems, such 

as human stem cell derived differentiated cell populations; (2) incorporating cell types, such as HepaRG, 
6. Alternative Methods Development 65

in 2-D and 3-D in vitro models that incorporate xenobiotic metabolism and allow for longer-term exposures; 

(3) increasing the characterization and use of computational models to predict metabolism and toxicity; (4) 

increasing the testing of compounds in alternative animal models like zebrafish and C. elegans; and (5) 

developing and implementing a high throughput transcriptomics platform for human, rat, mouse, zebrafish,

and C. elegans.

Table 29 describes the NTP Tox21 projects in FY 2014 that are being carried out by NIEHS/NTP staff. 

Table 29: NTP Tox21 Projects in FY 2014

NTP Project Title 

[Study Scientist] Objective and/or Summary

Assay Development

Developing a stable cell line to 

screen compounds that effect 

the estrogen-related receptor/

peroxisome proliferator-activated 

receptor coactivator pathway 

[Merrick]

To develop an assay that will detect compounds that interfere with the estrogen-related 

receptor/peroxisome proliferator-activated receptor gamma coactivator pathway, a 

critical pathway for metabolic homeostasis. Studies with human cells transiently infected 

with the appropriate reporter construct have been successful, and efforts are underway 

to produce a stable cell line expressing peroxisome proliferator-activated receptor 

gamma coactivator-1-alpha, with an expectation that the assay will be functional in FY 

2015.

High content screening with 

HepaRG cells 

[Ferguson]

To establish metabolically functional human HepaRG liver cells in 96-well or 384-well 

format to multiplex high content screening assays in collaboration with NCGC. In FY 

2014, the focus was on establishing a laboratory that could conduct high content studies 

using HepaRG cells. Studies with these cells will begin in FY 2015.

Testing for gene signatures and 

profiles in NTP archival tissues

project

[Merrick]

To determine if RNA can be extracted from fixed tissue blocks in the NTP Archives,

in order to measure gene signatures and develop chemically induced transcriptomics 

profiles. The potential usefulness of the NTP Archives is for measuring molecular

changes in different organs of the rat and mouse caused by chemical exposures. An 

effort on establishing a relational database that will allow identification and linkage of all

tissues in the NTP Archives is underway.

Stem cells projects

[Ferguson, Parham, Hsieh]

To screen for chemical toxicity in human or mouse stem cell lines (undifferentiated or 

differentiated) by qHTS at NCGC, or using lower throughput assays at NIEHS. Initially, 

the project is focused on fostering collaborations with stem cell technology providers 

and assessing control compounds and subsets of NTP chemicals using various assay 

approaches. Stem cell technology platforms and model systems shown to be useful for 

in vitro toxicology screening will be employed with larger sets of chemicals for hazard 

identification and chemical prioritization for toxicity testing. Data have been generated

on a library of 80 predominantly developmental neurotoxicants evaluated for effects on 

neurite outgrowth in a human stem cell derived neural cell population, cytotoxicity in 

different neural populations derived from human stem cells, and effects on the beating 

of human stem cell derived cardiomyocytyes. Dose-response analysis has been carried 

out on the resulting data from the assays and manuscripts are scheduled for submission 

in FY 2015.
66 National Toxicology Program Annual Report FY 2014

NTP Project Title 

[Study Scientist] Objective and/or Summary

Assay Interpretation

Validation of high throughput 

screening estrogen receptor (ER) 

qHTS transactivation assay (TA) 

[Casey-NICEATM]

(1) To compare the quality and accuracy of the qHTS assay that evaluates 

transcriptional activation of the ER in human ovarian cancer cells (BG1 ER 

transactivation assay [TA] assay) relative to the manual method that has been

validated for regulatory use by EPA and listed as a test guideline by the Organisation 

for Economic Co-operation and Development. The results of this evaluation indicate 

that the performance of the high throughput screening method is equivalent to the 

manual method. A manuscript detailing these results will be published in FY 2015. (2) 

To compare the BG1 ER TA assay (full-length receptor) with the assay that evaluates 

transcriptional activation of the estrogen receptor in human embryonal kidney cells

(HeK293 ER TA assay) that uses a transfected partial (ligand binding domain) receptor. 

Reproducible qHTS data were obtained in both assays, although there are differences 

in the percentages of compounds classified as active and inactive. Understanding

the factors contributing to differences in performance of these assays is critical to 

their regulatory acceptance and utilization. Once the high throughput screening data 

processing is complete and finalized (anticipated early FY 2015), results can be used

to assess and further develop quantitative structure-activity relationship models for 

estrogen receptor binding.

Data Analysis

Modeling mixtures of androgen

receptor- and estrogen receptoractive compounds screened in 

Tox21 qHTS assays

[Parham]

To determine (1) which mathematical models can best describe the toxicity of the 

mixtures of these compounds; and (2) whether the behavior of the mixtures can be 

predicted from the behavior of the individual components.

Analysis of Tox21 qHTS assay 

data 

[Hsieh]

To develop data analysis pipelines for Tox21 Phase II qHTS data to determine the 

activity of compounds in assays. The developed ranking/calling procedure takes into

account compound potency, efficacy, and data reproducibility. A manuscript describing

this pipeline was submitted for publication in FY 2014.

Prioritization of Tox21 compounds 

for genotoxicity

[Hsieh]

To develop a prioritization approach that includes compounds that show clear evidence 

of activity in the qHTS genotoxic assays and those that are weakly active compounds

based on chemical structure-activity relationship analysis. The resulting data are being 

compiled for a manuscript to be submitted in FY 2015.

Design of Tox21 data exploration 

graphical user interface

[Hsieh]

To develop two graphical user interfaces for viewing Tox21 data. One graphical user 

interface is to explore the concentration-response data in a line chart, and the second 

graphical user interface is to explore compound-similarity relationships in terms of their 

activities in Tox21 qHTS assays and their chemical structures. Prototype graphical user 

interfaces were developed during FY 2013 and made public in FY 2014.

Low-dose extrapolation for Tox21 

Phase I qHTS data

[Parham]

To determine points of departure for low-dose extrapolation by using signal-to-noise 

ratios and a benchmark-dose method. Data generated by this approach will be used to

help prioritize compounds for more extensive toxicological testing.

Sequencing quality control 

toxicogenomics project

[Auerbach]

To compare transcriptomic technologies (technologies that examine the RNA molecules 

in a cell population by microarray, high throughput screening, etc.) within the context 

of toxicogenomics. This is a multi-organization, international collaboration. The results 

were published in FY 2014 (http://www.ncbi.nlm.nih.gov/pubmed/25150838).
6. Alternative Methods Development 67

NTP Project Title 

[Study Scientist] Objective and/or Summary

Genomic signatures forecasting 

chemical carcinogenicity project

[Auerbach]

To develop transcriptomic signatures related to carcinogen treatment using NTP 

DrugMatrix data in combination with a machine-learning approach. Signatures are

being validated using an independent set of data from the Japanese Toxicogenomics 

Project. The results were published in FY 2014 (http://www.ncbi.nlm.nih.gov/

pubmed/25058030).

Unsupervised, data-driven 

analysis of Tox21 assay data 

project

[Auerbach]

To employ unsupervised data analysis methods (data organization based on patterns 

and performed by software) to identify chemicals that exhibit similar biological 

properties to well-characterized toxicants among the qHTS assays used to screen the 

10K library. The results are being used to help prioritize compounds for more extensive 

toxicological testing.

Next generation sequencing in 

toxicology project

[Merrick]

To develop bioinformatics pipelines for genomic and transcriptomic gene expression 

and mutational analysis on a genome-wide level, using next generation sequencing 

technologies to build signatures of toxicity and chemical exposure.

Development of a database related 

to skin sensitization

[Casey-NICEATM]

To develop the world’s largest public database for skin sensitization. The database,

which is publically available on the NICEATM website, serves as a valuable resource for

reference data to help develop alternative toxicological methods, including quantitative 

structure-activity relationship models that can be used to eliminate the use of animals. 

Such data are useful for evaluating in vitro results obtained in the Tox21 program. 

Collaborations are in progress with EPA and Cosmetics Europe to evaluate submitted 

studies and expand the database even further. 

Development of a reference 

database for estrogenic activity 

[Casey-NICEATM]

To support future validation of high throughput in vitro test methods and in silico models 

of estrogenic activity. NICEATM is creating a comprehensive database of high quality in

vivo data from over 1,000 scientific articles describing uterotrophic assay experiments

performed using over 1,500 different protocols. Analyses in progress include an 

evaluation of uterotrophic assay variability, comparison of ToxCast in vitro data to in vivo 

data, and comparison of uterotrophic data to published in vitro performance standards 

for the validation of in vitro estrogen receptor transactivation assays from ICCVAM.

These analyses will provide insights into the variability of uterotrophic data and the utility 

of in vitro assay data, including those from high throughput screening, for predicting in 

vivo responses.

In vitro to in vivo extrapolation 

using Tox21 data

[Casey-NICEATM]

To quantitatively correlate in vitro and in vivo dosimetry for estrogen receptor reference 

chemicals. Using data from a Tox21 estrogen receptor transactivation assay (BG1 ER 

TA), NICEATM developed and applied physiologically based pharmacokinetic models

to quantitatively correlate in vitro and in vivo dosimetry for estrogen receptor reference 

chemicals. The approach highlighted the importance of pharmacokinetic considerations

in assessing and ranking endocrine-active chemicals based on in vitro high throughput

screening assays.

Predicting skin sensitization with

ToxCast data

[Casey-NICEATM]

To build a cross-validated random forest model that predicts in vivo local lymph node 

assay (LLNA) results. NICEATM used ToxCast Phase II data and a balanced training

set of 60 chemicals with LLNA data to build a cross-validated random forest model 

that predicts LLNA results with 80 percent accuracy. The assays with highest variable 

importance included known adverse outcome pathway targets (e.g., Nrf2, T-cell

proliferation) and targets outside the current adverse outcome pathway (e.g., Coll III, 

PPAR, PXR, ER). These results were presented in a poster that won an Exemplary

Poster Award at the EPA ToxCast Data Summit on September 29-30, 2014, and may 

be used to develop efficient testing strategies that minimize animal use in regulatory

testing.
68 National Toxicology Program Annual Report FY 2014

NTP Project Title 

[Study Scientist] Objective and/or Summary

Evaluation of Tox21 data for 

predicting acute oral toxicity 

[Casey-NICEATM]

To determine the potential of high throughput screening data to reduce animal use for 

acute oral toxicity testing. NICEATM analyzed high throughtput screening data from

Tox21 and ToxCast for correlation and model fit to rat oral LD50 data, to determine

which tests or combinations of tests best characterized the rat oral toxicity data. The 

analysis suggested that combinations of in vitro assays and data from small model 

organisms, such as zebrafish, offer promise for predicting outcomes of rat acute oral

toxicity tests.

In silico prediction of metabolism 

project

[Ferguson]

To evaluate various in silico methods of predicting the extent of xenobiotic metabolism, 

identify metabolites, and prioritize chemicals in the Tox21 10K library. Computational 

methods will be used to bin the 10K library and develop subsets of chemicals likely

to be appreciably metabolized in humans. The results of these predictions are being 

summarized in a manuscript scheduled for submission in FY 2015.

Selection of a target set of genes 

for use in a high throughput 

transcriptomics screen

[Paules]

(1) To identify patterns of exposure-induced biological responses, in order to 

characterize toxicity and disease pathways; and (2) facilitate extrapolation of findings

from model species to humans. To accomplish this, an effort has been initiated to 

select a set of 1,500 genes to best capture and represent the full biological response 

to exposures and disease to be utilized in a high throughput transcriptomics screen. 

Criteria are being developed for the selection of the best target set of genes for this 

purpose. Efforts included a workshop in September 2013 to examine the utility and

feasibility identifying such a targeted set of genes for humans, rats, mouse, zebrafish,

and C. elegans. Expectations are that a draft human list will be released by mid FY 2015 

for comment by the scientific community.

Testing Projects

Epigenetic changes in chemical 

toxicity project

[Merrick]

To determine methylation patterns on a genome-wide basis and validation of selected 

CpG sites (regions of DNA where a cytosine nucleotide occurs next to a guanine 

nucleotide) altered by chemical exposure. Methylation of CpG sites can turn a gene

off while demethylation can cause transcriptional activation. A generalized approach 

for methylated DNA enrichment by MBD2 capture following bisulfite reduction and

sequencing is being developed.

Polycyclic aromatic hydrocarbon 

(PAH) project 

[Ferguson]

To evaluate approximately 20 PAHs considered relevant to human exposure in 

metabolism-competent HepaRG cells (derived from a human hepatic progenitor cell 

line), using multiplexed high content screening assays and gene expression platforms. 

Studies are in progress with completion expected in FY 2015.

Analysis of NTP’s 52 compounds 

in the EPA ToxCast Phase II 

program 

[Casey-NICEATM]

To screen 52 compounds in the EPA ToxCast program, based largely on immunological 

relevance. NICEATM will use in vitro chemical profiling data to identify predictive

signatures and adverse outcome pathways anchored to in vivo endpoints and toxicity 

pathways. Profiles in primary human cell systems will be examined. These analyses will

be used to enable chemical prioritization and hazard predictions. Collaborations have 

been established within the NIEHS inflammation faculty. A webinar is scheduled for FY

2015.
6. Alternative Methods Development 69

NTP Project Title 

[Study Scientist] Objective and/or Summary

NTP WormTox Laboratory Projects

Assay development

[Boyd]

To develop reproduction, growth, feeding, and locomotion assays that measure the 

effects of toxicant exposure on complex biological phenotypes, including development 

and neuron function. These assays have been developed in addition to an inducible 

gene expression fluorescence assay in transgenic C. elegans that measures stress 

pathway activation using high-content imaging system. Two mitochondrial toxicity 

assays are in development, including (1) an in vivo adenosine-5’-triphosphate assay, 

which provides real-time energetic status of the nematode; and (2) a fluorescence

dye-based mitochondrial membrane potential assay. Additionally, a low throughput C. 

elegans mitochondrial DNA damage and repair assay has been developed.

EPA’s ToxCast Phase II project

[Boyd]

(1) To determine the effects of the the EPA ToxCast Phase II 700-compound library 

on C. elegans development; and (2) compare the relative toxicities to those in higher 

organisms. The in vivo studies were completed in FY 2013, data analysis was 

completed in FY 2014, and publication is scheduled for FY 2015.

Ionic liquids project 

[Boyd]

(1) To describe the effects of approximatley 200 ionic liquids on C. elegans feeding, 

growth, and reproduction; and (2) compare the results in C. elegans to results in rodents 

and other toxicological models. Approximately 100 of these ionic liquids were screened 

in the growth assay in FY 2014, with more testing scheduled for FY 2015.

Flame retardants project

[Boyd]

(1) To describe the effects of eight flame retardants on C. elegans feeding, growth, and 

reproduction; and (2) compare the results in C. elegans to results in rodents. These 

studies were completed in FY 2014, with data analysis expected to be completed in FY 

2015.

Mitochondrial toxicants project

[Boyd]

To determine the effects of the mitochondrial toxicant subset from the Tox21 10K library 

on C. elegans growth and in vivo adenosine-5’-triphosphate levels and membrane 

potential. Compounds for testing were received in late FY 2014 with testing scheduled 

for FY 2015.

Fluorides project

[Boyd]

To compare the toxicities of three fluoride compounds commonly used in drinking water

treatment processes on C. elegans feeding, growth, and reproduction. The in vivo 

studies have been completed and the resulting manuscript was accepted for publication 

in FY 2014.

B. NICEATM

The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), an office

within NIEHS/NTP, supports the development and evaluation of new, revised, and alternative methods to 

identify potential hazards to human health and the environment, with a focus on replacing, reducing, or refining

animal use. NICEATM activities include the following:

● Conducting and publishing analyses and evaluations of data from new, revised, and alternative testing 

approaches. 

● Providing information to test method developers, regulators, and regulated industry, through their 

website and other communications, and by organizing workshops and symposia on topics of interest.

● Coordinating and providing logistical support for the Interagency Coordinating Committee on the 

Validation of Alternative Methods (ICCVAM) committee meetings, webinars, teleconferences, working

groups, and public forums (see ICCVAM Activities, page 72). 
70 National Toxicology Program Annual Report FY 2014

● Providing bioinformatics and computational toxicology support to NIEHS/NTP projects, especially 

those related to Tox21. 

Information on NICEATM activities is located at http://ntp.niehs.nih.gov/go/niceatm. The NICEATM website

includes resources for test method developers at http://ntp.niehs.nih.gov/go/resources. A complete list of 

articles on NICEATM activities published in scientific journals is available at http://ntp.niehs.nih.gov/go/niceatmpubs. Reports published by NICEATM are available at http://ntp.niehs.nih.gov/go/717297.

Warren Casey, Ph.D., is director of NICEATM. NICEATM received contract support during FY 2014 from

Integrated Laboratory Systems Inc.

i. Workshops

NICEATM and North Carolina State University

(NCSU) cosponsored the Collaborative Workshop

on Aquatic Models and 21st Century Toxicology, held

May 5–6, 2014, at NCSU. The workshop, which drew

nearly 150 scientists from the U.S., Canada, Europe, 

and Asia, provided an opportunity to consider the 

key role small fish and their embryos may play in

toxicity testing. More than 20 scientific talks were

given during the two-day workshop. Presentations

included descriptions of research studies and results, 

practical advice on the conduct of toxicity studies using small fish and fish embryos, and examples of use of

toxicity data derived from aquatic species in drug development and regulatory compliance. Information about 

the workshop is available at http://ntp.niehs.nih.gov/go/41308, and a workshop report is in preparation for

submission for publication in FY 2015.

NICEATM supported a workshop to consider implementation and regulatory acceptance of in vitro alternatives

to the murine histamine sensitization test for safety testing of acellular pertussis vaccines. The workshop was

a satellite meeting of the 9th World Congress on Alternatives and Animal Use in the Life Sciences on August 

24-28, 2014, in Prague. NICEATM is also providing support for an international workshop held in London in

March 2015, at which data from an ongoing collaborative study of in vitro alternatives to the murine histamine

sensitization test, and other recent advances in method development, will be reviewed.

Scientists from 21 countries, representing industry, academia, regulatory agencies, and special interest 

groups, attended the workshop, Adverse Outcome Pathways: From Research to Regulation. The meeting held

September 3-5, 2014, at NIH in Bethesda, Maryland, considered how the adverse outcome pathway (AOP)

concept could improve regulatory assessments of chemical toxicity. NICEATM cosponsored the workshop

with the Physicians Committee for Responsible Medicine. Plenary presentations were followed by breakout

sessions that considered AOP applications, development of new AOPs, and challenges to AOP adoption. 

The closing session summarized the breakout groups’ discussions and emphasized a need to maintain the

collaborations and momentum that were generated. Presentations and links to webcasts from the workshop

are available at http://ntp.niehs.nih.gov/go/41375. A report is being prepared for submission for publication in 

FY 2015.
6. Alternative Methods Development 71

ii. NICEATM Support of Tox21

In FY 2014, NICEATM conducted the following Tox21 projects. Full descriptions can be found in Table 29.

● Validation of high throughput screening estrogen receptor qHTS transactivation assay. 

● Development of a database related to skin sensitization.

● Development of a reference database for estrogenic activity.

● In vitro to in vivo extrapolation using Tox21 data.

● Predicting skin sensitization with ToxCast data.

● Evaluation of Tox21 data for predicting acute oral toxicity.

● Analysis of NTP’s 52 compounds in the EPA ToxCast Phase II program.

In addition to these projects, NICEATM built a mathematical model using data from nine Tox21 and ToxCast

assays to distinguish true androgen receptor pathway activity from technology-specific assay interference.

The results from the model were used to build a quantitative structure-activity relationship model to predict 

androgen receptor binding activity. NICEATM and the NTP Office of Health Assessment and Translation are

also collaborating on analyses of Tox21 data to determine potential links of environmental chemical exposures

with obesity.

iii. Other NICEATM Activities

NICEATM is collecting oral and dermal toxicity data to evaluate if acute oral toxicity data could be reliably used

to assign EPA acute dermal hazard classifications, potentially reducing the number of animals needed for

pesticide testing. EPA is providing NICEATM with acute oral toxicity data for both pesticide active ingredients

and formulations. NICEATM is reviewing the data for quality control purposes and using defined criteria

to evaluate the data’s reliability. NICEATM also formed a workgroup to focus on alternatives to acute oral

toxicity testing. Members represent federal agencies, nongovernmental organizations, international validation

organizations, and national and international animal welfare organizations.

NICEATM staff collaborated with scientists at Procter and Gamble to develop an approach to identify potential

skin sensitizers and characterize skin sensitization potency without conducting animal tests. The goal of the

collaboration is to develop a widely available, opensource version of a Procter and Gamble integrated testing 

strategy. The open-source, integrated testing strategy was described in a short communication in the journal 

ALTEX in 2014 (available at http://www.altex.ch/resources/Pirone_of_20140331.pdf). This strategy was 

recently revised, and the revision appears to have improved overall accuracy. Downloadable files to run the

analysis, along with documentation and sample data, are available at http://ntp.niehs.nih.gov/go/its.

NICEATM scientists worked with immunotoxicologists from NIEHS/NTP, EPA, academic institutions, and

international organizations to create a standardized ontology for in vivo immunotoxicity data. Using this 

ontology, NICEATM is working with EPA to create a comprehensive database consisting of high-quality data

from in vivo testing of potential immunotoxicants. This database will support the validation of high throughput, 

in vitro test methods for prediction of allergic contact dermatitis and other immunotoxic effects.

NICEATM is creating a comprehensive database of highquality, in vivo testing data to support future validation

of high throughput, in vitro test methods and in silico models of estrogenic activity. The database was compiled 

from over 1,000 articles from the scientific literature, describing uterotrophic assay experiments performed

using over 1,500 separate protocols. Analyses in progress using database data include (1) an evaluation 

of uterotrophic assay variability; (2) comparison of ToxCast data to in vivo data; and (3) comparison of 
72 National Toxicology Program Annual Report FY 2014

uterotrophic data for ICCVAM performance standards substances to data from an ICCVAM validation study

of in vitro estrogen receptor transactivation assays. These analyses will provide insights into the variability 

of uterotrophic data and allow for the evaluation of the utility of in vitro assay data, including those from high 

throughput screening, for predicting in vivo responses.

NICEATM is collaborating with the Physicians Committee for Responsible Medicine, EPA, and industry

scientists to organize a workgroup focused on inhalation toxicity. Specifically, the workgroup will review the

state of the science and determine technical needs for developing a dynamic flow, coculture system to reduce

or replace the use of animals for assessing the toxicity of inhaled nanomaterials.

NICEATM is participating in an expert working group, organized by the Center for Alternatives to Animal

Testing, to develop good cell culture practices for induced pluripotent stem cells.

Other ongoing projects by NICEATM scientists include (1) creating methods for bioactivity-based read-across,

using a proximity matrix that clusters chemicals based on their bioactivity patterns across all the ToxCast 

assays within the context of the entire chemical library; and (2) development of quantitative structure-property 

relationship models for prediction of chemical properties.

C. ICCVAM

The Interagency Coordinating Committee on the Validation 

of Alternative Methods (ICCVAM) is a permanent interagency

committee of NIEHS under NICEATM. The committee was formally

established by the ICCVAM Authorization Act of 2000 (42 U.S.C.

285l-3). Its purpose is to establish, wherever feasible, guidelines, 

recommendations, and regulations that promote the regulatory 

acceptance of new or revised scientifically valid toxicological tests that protect human and animal health and

the environment, while reducing, refining, or replacing animal tests and ensuring human safety and product

effectiveness (see http://iccvam.niehs.nih.gov/about/about_ICCVAM.htm).

ICCVAM is composed of representatives from 15 U.S. federal regulatory and research agencies that

generate or use toxicological and safety testing information. ICCVAM promotes the regulatory acceptance of

scientifically valid new test methods that replace, reduce, or refine animal use.

NICEATM administers ICCVAM and provides scientific support for ICCVAM activities. Warren Casey, Ph.D.,

serves as administrative director of ICCVAM. Information on ICCVAM activities is available at http://ntp.niehs.

nih.gov/go/iccvam. A complete list of articles on ICCVAM activities published in scientific journals is available at

http://ntp.niehs.nih.gov/go/niceatm-pubs.

i. ICCVAM Activities 

ICCVAM held eight teleconferences and one face-to-face meeting in FY 2014. NICEATM is also currently

providing support for three ad hoc ICCVAM working groups on ocular toxicity, skin sensitization, and

arsenic toxicity.

ICCVAM, with NICEATM support, held its first public forum on June 25, 2014, at NIH in Bethesda, Maryland.

This meeting provided an opportunity for public interaction with representatives from the 15 agencies 

represented on ICCVAM. ICCVAM members provided information about their agency’s activities relevant to

the development and use of alternative test methods. Groups interested in promoting alternative methods or 
6. Alternative Methods Development 73

reducing animal use in testing presented public comments. 

The agenda and presentations are available at http://ntp.

niehs.nih.gov/go/41491. 

ICCVAM is planning a Communities of Practice webinar on

in vitro to in vivo extrapolation in January 2015. Annually, 

ICCVAM plans to hold Communities of Practice webinars

and other events similar to the June 2014 public forum. 

NICEATM will provide support for this activity.

ICCVAM Member Agencies

CPSC

USDA

DOD

DOE

DOI

DOL

DOT

OSHA

EPA

HHS

CDC

FDA

NIH

ATSDR

NIOSH

NIEHS

NCI

NLM

OD

ii. Test Method Evaluation Activities

ICCVAM received no formal test method nominations or

submissions in FY 2014. ICCVAM is currently revising its

test method evaluation procedures, of which nominations 

and submissions are a key element. ICCVAM welcomes

submissions of innovative test methods, which may be 

acceptable for specific regulatory use, and for which

adequate validation studies have been completed. However, 

to maximize the potential for effective implementation of 

new test methods or approaches, ICCVAM will only conduct

evaluations and prepare recommendations on test method 

submissions proposed for regulatory uses that align with 

ICCVAM member agency needs and priorities. More

information on ICCVAM test method submissions is available at http://ntp.niehs.nih.gov/go/ICCVAM-submit. 

NICEATM and ICCVAM test method evaluation activities in FY 2014 are summarized in Table 30.

Table 30. Test Method Evaluation Activities in FY 2014

Test Method ICCVAM Recommendations/Agency Status

ICCVAM integrated decision strategy for

skin sensitization

ICCVAM is developing an integrated decision strategy based on an established

skin sensitization adverse outcome pathway using in vitro, in chemico, and in

silico information. NICEATM, on behalf of ICCVAM, issued a Federal Register

notice (78 FR 68076) requesting information about test methods, testing 

strategies, and other information relevant to identification of potential skin

sensitizers in support of this activity. 

iii. International Validation Activities

NICEATM and ICCVAM participate in international test method validation activities through the Organisation

for Economic Co-operation and Development (OECD), and collaborations with countries that are members 

of the International Cooperation on Alternative Test Methods (ICATM). ICATM collaborations address three

critical areas of cooperation, including test method validation studies, independent peer review of validation 

studies, and the development of formal recommendations on alternative testing methods. Members include the

European Union, Japan, Korea, Health Canada, and U.S.

In FY 2014, ICCVAM agencies commented on draft OECD documents through the U.S. National Coordinator

for the OECD Test Guidelines Programme, who is an ex officio member of ICCVAM. NICEATM and ICCVAM
74 National Toxicology Program Annual Report FY 2014

also participate in an OECD drafting group for a guidance document on integrated approaches to testing and 

assessment for skin sensitization.

Ongoing international validation studies that include participation of NICEATM or ICCVAM are listed in

Table 31. NICEATM and ICCVAM representatives attended ICATM coordination meetings in November 2013

and September 2014.

Table 31. Participation in International Validation Studies

Test Method Type of Test Lead 

Organization NICEATM-ICCVAM Involvement

Human cryopreserved HepaRG 

and cryopreserved hepatocytes 

cytochrome p450 induction test 

methods

Acute toxicity EURL ECVAM*

NICEATM and ICCVAM members served

on the validation management team and 

provided comments on study reports.

IL-8 in vitro test for assessing skin

sensitization potential 

Allergic contact 

dermatitis JaCVAM**

NICEATM members served on the validation

management team and provided comments 

on study design, chemical selection, and test 

method protocols.

Vitrigel-SST assay for assessing 

skin sensitization potential

Allergic contact 

dermatitis JaCVAM NICEATM members served on the validationmanagement team.

Vitrigel-EIT assay for eye irritation 

testing Ocular irritation JaCVAM NICEATM members served on the validationmanagement team.

Hand1-luc in vitro test for assessing 

reproductive toxicity potential 

Reproductive 

toxicity JaCVAM NICEATM members served on the validationmanagement team.

* European Union Reference Laboratory for Alternatives to Animal Testing, European Centre for the Validation of Alternative 

Methods

** Japanese Center for the Validation of Alternative Methods
7. Public Tools 75

7. Public Tools

NTP provides a variety of resources for public use. Those available in FY 2014 are described below. 

A. Nonneoplastic Lesion Atlas

The NTP Nonneoplastic Lesion Atlas is a Web-based resource currently containing hundreds, and will 

eventually contain thousands, of high-quality images and guidelines for nonneoplastic lesions in experimental 

rodent models. The purpose of the atlas is to standardize the terminology, diagnostic strategy, record of 

nonneoplastic rodent lesions, and improve the consistency of the NTP nonneoplastic lesion database. The 

atlas was launched in FY 2014 and will be completed in FY 2015. See page 19 for more information about the 

atlas or visit http://ntp.niehs.nih.gov/nnl/. 

B. Chemical Effects in Biological Systems (CEBS) Database

The CEBS database is a publicly accessible, 

NTP-integrated, data-management system 

that houses biological study data deemed of 

interest to toxicologists and environmental health 

scientists. The CEBS database was designed 

as a reference repository and accepts studies 

from many sources. Government, academic, and 

pharmaceutical laboratories contribute peerreviewed studies to the database. 

An important feature of the database is its 

flexibility. CEBS can house any type of biological

measurements from any type of study design. 

This flexibility allows data from different sources

to be integrated into one place to permit data 

mining and analysis. In addition, any one study can have different types of data associated with it, such as 

blood chemistry, histopathology, and microarray, and can all be viewed together by one study name. 

NIEHS/NTP continues to add legacy NTP data to CEBS. Phase II Tox21 data will also be publicly available 

through CEBS, as studies are released for reference and data mining. CEBS supports queries such as show 

me all test articles that cause a particular pathology or show me all test articles positive in a particular genetic 

toxicology assay, where test articles is the database term used to refer to substances being tested, such as 

chemicals or drugs.

CEBS captures the details of a study’s design and execution, as well as the biological responses of 

study subjects, such as animals and tissue culture cells. This enables the user to view the details of each 

study; search for particular studies or study subjects of interest, based on treatment, response, or other 

characteristics; and then either analyze the data within the CEBS database or download it for import into other 

tools. CEBS can be accessed at http://cebs.niehs.nih.gov.
76 National Toxicology Program Annual Report FY 2014

C. NTP Archives

The NTP Archives is an important NIEHS-supported resource that houses an unmatched collection of 

research specimens and supporting data from more than 2,000 studies, including toxicity, carcinogenicity, 

immunotoxicity, reproductive, and developmental studies. Currently, the Archives maintain 7.7 million 

histopathology slides; 4.8 million paraffin-embedded tissue blocks; 213,000 bags of formalin-fixed tissues and

organs; and 117,000 selected frozen tissue specimens. It also houses study data, including 4.4 million pages 

of paper records; 10.8 million pages of data on microfiche; 2.4 million pages of digital or electronic records

on CDs or DVDs; histological images, including over 38,000 kodachrome slides and 27,000 digital images in

a publically accessible, searchable, Web-based database; and educational and training materials on rodent 

toxicologic pathology on CDs and DVDs.

The NTP Archives provides researchers unique opportunities to characterize and compare diverse disease 

or disease processes that occur spontaneously or are chemically induced; examine collections of rare and 

unusual lesions; and explore observed pathologic responses at the cellular or molecular level to determine 

mechanisms of disease. There are ongoing efforts to organize the Archives, to make it more electronically

searchable for chemical exposure related to toxicity phenotype at the animal level, and to recommend 

protocols consistent with best practices for biobanking and archival storage. There are also ongoing efforts

to determine the usefulness of archival materials for various types of molecular analysis, including gene 

expression, epigenetics, and mutational analysis. The development of a querying system of archival materials 

from different organs and storage times is planned for the future to better determine the value in transcript 

profiling of the archived materials. In addition, a queryable database is in the planning stages to facilitate

integration of the frozen sample database, NTPFast, and formalin-fixed, paraffin-embedded tissues.

Additional information about the Archives can be found at http://ntp.niehs.nih.gov/ntp/pressctr/brochures/

ntparchives2014_508.pdf.

D. The NCGC BioPlanet

No one, comprehensive, uniform resource covers all known annotations of cellular pathways, and no single

platform allows integrated browsing, retrieval, and analysis of information from the many existing resources 

that do pathway mapping. Therefore, NCGC, with support from NIEHS/NTP (see page 63), built an integrated 

pathway resource to host information on approximately 2,000 human pathways from manually curated and 

publicly available resources. The NCGC BioPlanet complements this pathway warehouse by allowing easy 

browsing, visualization, and analysis of the universe of pathways. The BioPlanet is scheduled for public release 

in FY 2015.

E. DrugMatrix and ToxFX

NIEHS/NTP acquired DrugMatrix, a toxicogenomics reference database, informatics system, and frozen

tissue archive. The goal of DrugMatrix is to develop analysis tools and approaches, to allow an integrated

assessment of high throughput screening endpoints and associations with findings from traditional toxicology

and cancer models. This resource expands the ability of NTP to develop predictive models for toxicological 

effects based on gene signatures, provide additional tools for linking in vitro data to in vivo gene signatures and

disease outcomes, and provide additional tissue samples for NextGen-based investigations. DrugMatrix, and

its companion automated analysis tool ToxFX, were made accessible to the international scientific community

in early FY 2012 (https://ntp.niehs.nih.gov/drugmatrix and https://ntp.niehs.nih.gov/toxfx).
7. Public Tools 77

To date, more than 584 researchers have registered to use the DrugMatrix database. In addition, the data

and biological samples from DrugMatrix are a focal point in a number collaborations between NIEHS/NTP

scientists and research groups from NCTR, the City of Hope, Stanford University, Boston University, EPA, 

Health Canada, Abbott Laboratories, Eli Lilly and Company, SAS, Maastricht University, GeneData, GeneGo,

Ingenuity, Fondazione Bruno Kessler, Rush University Medical Center, Boku University Vienna, Loma Linda

University, University of Massachusetts, and University of North Dakota. Samples from the DrugMatrix

frozen tissue bank have been analyzed through the MicroArray Quality Control/Sequencing Quality Control

Consortium (http://www.fda.gov/ScienceResearch/BioinformaticsTools/MicroarrayQualityControlProject/

default.htm, http://www.nature.com/articles/sdata201421). In addition, the samples have been used in 

collaborations with researchers at Harvard University and NIEHS.

All of the RNA expression data from DrugMatrix have been integrated in NextBio, a database and analysis

tool, allowing analysis of the data within the context of all existing genomic and transcriptomic data. DrugMatrix

data have been made available through an easily accessible FTP portal (ftp://anonftp.niehs.nih.gov/drugmatrix) 

and the gene expression data are available in GEO and ArrayExpress databases. In addition, all of the in vitro 

screening data have been incorporated into the PubChem and ChEMBL databases. The clinical chemistry,

hematology, and histopathology results are in the process of being entered into the ChEMBL and PubChem

databases. Ultimately, the goal of these collaborations and data sharing is to use the DrugMatrix data to better

understand the molecular underpinnings of disease and toxicological pathology.

F. Meta Data Viewer

Staff within the Office of Health Assessment and Translation developed Meta Data Viewer, in collaboration

with SRA International, to be a user-friendly program for creating figures with multiple columns of

accompanying text, such as forest plots of epidemiology data or exposure-response arrays of toxicology data. 

The program allows users to quickly sort, group, and filter subsets of data from a larger database to look at

patterns of findings across a wide variety of studies or sets of results from a single study. Meta Data Viewer

is a public resource. Users are welcome to use the program and any associated data files provided for their

own purposes, including use in publications. Meta Data Viewer is available at http://ntp.niehs.nih.gov/go/

tools_metadataviewer. 
78 National Toxicology Program Annual Report FY 2014

8. Appendices

A. Program Contact Information

NTP Website: http://ntp.niehs.nih.gov

General Inquiries: NIEHS/NTP Central Data Management Office; CDM@niehs.nih.gov, 919-541-3419 

General Comments: Office of Liaison, Policy, and Review, NIEHS/NTP; ntpinfo@niehs.nih.gov, 919-541-7539

NIEHS Website: http://www.niehs.nih.gov

NCTR Website: http://www.fda.gov/AboutFDA/CentersOffices/OC/OfficeofScientificandMedicalPrograms/

NCTR/default.htm

NIOSH Website: http://www.cdc.gov/niosh

Staff Directory: http://directory.psc.gov/employee.htm

B. Frequently Used Abbreviations

3TC lamivudine

ATSDR Agency for Toxic Substances and Disease Registry

AZT 3’-azido-3’-deoxythymidine

BPA bisphenol A

BSC	 Board of Scientific Counselors

CASRN Chemical Abstracts Service Registry Number

CDC Centers for Disease Control and Prevention

CEBS Chemical Effects in Biological Systems 

CPSC U.S. Consumer Product Safety Commission

DOD U.S. Department of Defense

DOE U.S. Department of Energy

DOI U.S. Department of the Interior

DOL U.S. Department of Labor

DOT U.S. Department of Transportation

EPA U.S. Environmental Protection Agency

ER estrogen receptor 

FDA U.S. Food and Drug Administration

FY	 fiscal year

GMM	 genetically modified model

HHS U.S. Department of Health and Human Services

ICATM	 International Cooperation on Alternative Test Methods

ICCVAM	 Interagency Coordinating Committee on the Validation of Alternative Methods
8. Appendices 79

JaCVAM	 Japanese Center for the Validation of Alternative Methods

N/A not applicable

NCATS National Center for Advancing Translational Sciences

NCGC NIH Chemical Genomics Center

NCI National Cancer Institute

NCTR National Center for Toxicological Research

NICEATM	 NTP Interagency Center for the Evaluation of Alternative Toxicological Methods

NIEHS National Institute of Environmental Health Sciences

NIH National Institutes of Health

NIOSH National Institute for Occupational Safety and Health

NLM	 National Library of Medicine

NTP National Toxicology Program

NVP nevirapine 

OD	 Office of the Director, NIH

OHAT	 Office of Health Assessment and Translation

OSHA Occupational Safety and Health Administration

PBPK	 physiologically-based pharmacokinetic

qHTS quantitative high throughput screening

RoC Report on Carcinogens

SACATM	 Scientific Advisory Committee on Alternative Toxicological Methods

TR NTP Technical Report

USDA U.S. Department of Agriculture
80 National Toxicology Program Annual Report FY 2014

1 Funded by the NIEHS/NIOSH Interagency Agreement

2 Funded by NIOSH voluntary allocations to the NTP

3 Funded by the NIEHS/NCTR Interagency Agreement

4 Funded by NCTR voluntary allocations to the NTP

5 Funded by the NTP/NHGRI/EPA/FDA Interagency 

 Memorandum of Understanding

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

C. FY 2013 Bibliography

i. Reports

National Toxicology Program. 2013. National Toxicology Program Report on the Toxicology and Carcinogenicity Studies 

of 3’-Azido-3’-Deoxythymidine (AZT) (CASRN 30516-87-1) in Genetically Modified C3B6.129F1-Trp53tm1Brd N12

Haploinsufficient Mice (in utero and postnatal gavage studies). GMM-14, NIH Publication No. 14-5967. Research Triangle

Park, NC: National Toxicology Program.

National Toxicology Program. 2013. National Toxicology Program Report on the Toxicology and Carcinogenicity Study of 

Mixtures of 3′-Azido-3′-Deoxythymidine (AZT), Lamivudine (3TC), and Nevirapine (NVP) (CASRN 30516-87-1, 134678-

17-4, 129618-40-2) in Genetically Modified C3B6.129F1-Trp53tm1Brd N12 Haploinsufficient Mice (in utero and postnatal

gavage studies). GMM-16, NIH Publication No. 14-5973. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods. 2014.

Interagency Coordinating Committee on the Validation of Alternative Methods Biennial Progress Report 2012-2013.

Research Triangle Park, NC: National Institute of Environmental Health Sciences.

National Toxicology Program. 2014. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis 

Studies of beta-Picoline (CASRN 108-99-6) in F344/N Rats and B6C3F1/N Mice (drinking water studies). TR 580, NIH

Publication No. 14-5922. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2014. National Toxicology Program Technical Report on the Toxicology Studies of 

Tetrabromobisphenol A (CASRN 79-94-7) in F344/NTac Rats and B6C3F1/N Mice and Toxicology and Carcinogenesis

Studies of Tetrabromobisphenol A in Wistar Han [Crl:WI(Han)] Rats and B6C3F1/N Mice (gavage studies). TR 587, NIH

Publication No. 14-5929. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2014. Report on Carcinogens Monograph on Pentachlorophenol and By-Products of Its

Synthesis. NIH Publication No. 14-5953, ISSN 2331-267X. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2014. Report on Carcinogens Monograph on ortho-Toluidine. NIH Publication No. 14-5954,

ISSN 2331-267X. Research Triangle Park, NC: National Toxicology Program.

ii. Journal Publications

4 Aissa AF, Tryndyak V, de Conti A, Melnyk S, Gomes TDUH, Bianchi MLP, James SJ, Beland FA, Antunes LM,

Pogribny IP. 2014. Effect of methionine-deficient and methionine-supplemented diets on the hepatic one-carbon and

lipid metabolism in mice. Mol Nutr Food Res 58(7):1502-1512; doi:10.1002/mnfr.201300726 [Online 15 May 2014].

4 Ali SF. 2014. Porcine brain microvessel endothelial cells show pro-inflammatory response to the size and composition

of metallic nanoparticles. Drug Metab Rev 46(2):224-231; doi:10.3109/03602532.2013.873450 [Online 31 December

2013].

2 Anderson SE, Beezhold K, Lukomska E, Richardson J, Long C, Anderson K, Franko J, Meade BJ, Beezhold DH.

2014. Expression kinetics of miRNA involved in dermal toluene 2,4-diisocyanate sensitization. J Immunotoxicol

11(3):250-259; doi:10.3109/1547691X.2013.835891 [Online 25 September 2013].

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iii. Book Chapters

7 Auerbach SS, Paules RS. 2014. Application of in vivo genomics to the prediction of chemical-induced (hepato)

carcinogenesis. In: Toxicogenomics-Based Cellular Models: Alternatives to Animal Testing for Safety Assessment

(Kleinjans J, ed). Oxford, United Kingdom:Elsevier Inc., 15-33.

7 Ferguson SS, Bonzo J. 2014. In vitro approaches to study drug-drug interactions. In: Drug Metabolism Prediction

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7 Frawley R, Germolec D, van Loveren H. 2014. Use of toxicogenomics in immunotoxicology. In: Molecular

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27-66.

7 Harry GJ, McPherson CA. 2014. Microglia: neuroprotective and neurodestructive properties. In: Handbook of

Neurotoxicity (Kostrzewa RM, ed). New York:Springer, 109-132.

7 Harry GJ, Pont-Lezica L. 2014. Roles of microglia in developmental vascularization and myelination. In: Microglia in

Health and Disease (Tremblay M-E, Sierra A, ed). New York:Springer, 193-221.

7 Reed CE, Fenton SE. 2014. Risk factors as biomarkers of susceptibility in breast cancer. In: Biomarkers in Toxicology

(Gupta RC, ed). Oxford, United Kingdom:Elsevier Inc., 743-758.
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