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. 14-5955
National Toxicology Program

ANNUAL REPORT

for

Fiscal Year 2013

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

July 2014

U.S. Department of Health and Human Services

National Toxicology Program

NIH Publication No. 14-5955
© Can Stock Photo Inc. / Lonely11
Table of Contents

Letter from the NIEHS/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.................................................................................................12

2. Funding ........................................................................................................................................... 14

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

A. Nonneoplastic Lesion Atlas ....................................................................................................... 17

B.	 Realignment of NICEATM and ICCVAM.................................................................................... 18

C. Chemotherapy Use During Pregnancy...................................................................................... 18

D. CLARITY-BPA Research Program............................................................................................. 18

E.	 Crowdsourcing Human Variability Data ..................................................................................... 19

F.	 Mouse Methylome Project......................................................................................................... 19

G. NTP Impact on Regulatory Agencies......................................................................................... 20

H. Additional Activities.................................................................................................................... 21

4. Literature Analysis Activities ........................................................................................................ 22

A. Noncancer Health Effects ......................................................................................................... 22

B. Report on Carcinogens.............................................................................................................. 23

5. Testing and Research .................................................................................................................... 25

A. Technical Reports ...................................................................................................................... 25

B. NTP Testing ............................................................................................................................... 28

i.	 Disposition, Metabolism, and Toxicokinetic Studies .........................................................29

ii. Genetic Toxicity ................................................................................................................30

iii. Organ System Toxicity......................................................................................................31

iv. Toxicology/Carcinogenicity Studies ..................................................................................36

v. Toxicogenomic Studies.....................................................................................................39

vi.	 Project Review Committee Approved...............................................................................42

C. NTP Research ........................................................................................................................... 42

i.	 Assessing Exposure to Substances in the Workplace .....................................................44

ii. Comprehensive Assessment of Occupationally-Relevant Exposures..............................48

iii. Immunotoxicology Research ............................................................................................49

iv. NTP Research in Partnership with NCTR ........................................................................51

6. Alternative Methods Development................................................................................................ 61

A. Tox21......................................................................................................................................... 61

B.	 ICCVAM..................................................................................................................................... 66

i.	 Workshops........................................................................................................................66
ii.	 Publications ......................................................................................................................67

iii.	 NICEATM Activities...........................................................................................................67

iv.	 Test Method Evaluation Activities .....................................................................................68

v.	 International Validation Activities ......................................................................................69

7. Public Tools .................................................................................................................................... 70

A. Nonneoplastic Lesion Atlas ....................................................................................................... 70

B.	 Chemical Effects in Biological Systems (CEBS) Database ....................................................... 70

C. NTP Archives............................................................................................................................. 71

D. The NCGC BioPlanet ................................................................................................................ 71

E.	 DrugMatrix and ToxFX............................................................................................................... 71

F.	 MetaDataViewer........................................................................................................................ 72

8. Appendices ..................................................................................................................................... 73

A. Program Contact Information .................................................................................................... 73

B.	 Frequently Used Abbreviations.................................................................................................. 73

C.	 FY 2013 Bibliography ................................................................................................................ 76

i. Reports.............................................................................................................................76

ii.	 Journal Publications .........................................................................................................76

iii. Book Chapters..................................................................................................................91
Letter from the NIEHS/NTP Director 1

Letter from the NIEHS and NTP Director

The National Toxicology Program (NTP) made great strides in conducting 

and coordinating toxicology testing and research, despite facing 

challenges like the budget sequestration in 2013. With its partners in the

federal government, NTP continued to lead the Tox21 initiative to develop 

more efficient approaches to predict how chemicals may affect human

health. NTP also distributed numerous reports on substances of public

health concern.

NTP published a monograph on chemotherapeutics use during pregnancy,

to serve as a resource for clinicians and their pregnant patients. Through 

five NTP Technical Reports, we disseminated information from studies

on the carcinogenic activity and toxicity of botanical dietary supplements,

an antiviral drug, and two substances used in industry. In addition, NTP

hosted an international workshop, and published three reports to facilitate

the development, validation, and regulatory acceptance of test methods 

that reduce, refine, or replace the use of animals in testing.

Through fiscal year (FY) 2013, the NTP Office of Health Assessment and Translation widely presented,

to agency partners and the public, their draft approach for systematic review and evidence integration for

literature-based health assessments. This draft approach describes how NTP will incorporate systematic

review methodologies in conducting literature-based analysis of published studies, to assess if a substance

may be of concern for human health. This new approach will increase transparency in how NTP conducts and

communicates literature-based assessments.

NTP continues to safeguard public health, by strengthening environmental health sciences, and working with

partners in the United States and abroad, to improve toxicology research and testing. I am proud to present our

achievements in FY 2013.

Linda S. Birnbaum, Ph.D., D.A.B.T., A.T.S.

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 2013 (http://www.epa.

gov/oppt/existingchemicals/pubs/tscainventory/basic.html) lists 

more than 83,000 chemicals as being available for sale and use in

the United States. New chemicals are continuously introduced into 

the U.S. market each year. According to the Board of Governors 

of the Federal Reserve System, chemical production in the 

United States increased 2.1-fold from 1972 to 2012 (http://www.

federalreserve.gov/datadownload/default.htm). While the effects

of many of these substances on human health are unknown,

people and our environment 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, as a focal point 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, regulatory, and research agencies,

scientific and 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 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, nongovernment 

groups, 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 and data of NTP studies, lists of reports and journal

publications, media releases, calendar of upcoming events, and the NTP Update quarterly 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 to the Central Data Management office (CDM@niehs.nih.gov or 919-541-3419), or through

Freedom of Information Act requests (http://www.niehs.nih.gov/about/od/ocpl/foia/contact/index.cfm).

As always, 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 Office of Liaison,

Policy, and Review, NIEHS/NTP (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 2013

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 its National Center for Toxicological Research (NCTR), and the National Institute of 

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

NTP is located administratively at NIEHS, and Linda Birnbaum, Ph.D., serves as director of 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 Gayle DeBord, Ph.D., associate director for science in the

Division of Applied Research and Technology, manages the NTP program within NIOSH, herein referred to 

as NIOSH/NTP. Staff from the following NIOSH divisions participate in NTP activities: Division of Surveillance, 

Hazard Evaluations, and Field Studies; 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 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 2013

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/

index.cfm. The training program falls into the following 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 2013, NTP staff mentored 20 

postdoctoral fellows at NIEHS.

Table 1. NTP Training Program Postdoctoral Fellows in FY 2013

Training Program Fellow

Applied toxicology and carcinogenesis 

Kristen Ryan

Brian Sayers

In Ok Surh

Sheetal Thakur

Biomolecular screening and computational toxicology

Rachel Goldsmith

Julie Hall

Jui-Hua Hsieh

Yang Sun

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

Michael Boyle

Michelle Cora

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 2013.

The BSC met twice in FY 2013 

(http://ntp.niehs.nih.gov/go/9741). 

At the meeting on Dec. 11, 

2012, the BSC reviewed a draft 

testing concept for polycyclic 

aromatic hydrocarbons. The

BSC was updated on the “NTP 

Approach for Systematic Review 

and Evidence Integration 

for Literature-based Health

Assessments,” and heard a 

report from the chair of the BSC 

working group that evaluated the 

draft approach. NTP provided 

updates on studies on bisphenol A

(BPA) and on NTP progress 

since the June 2012 meeting, 

from the NIEHS and NTP director 

and NTP associate director.

BSC members and NTP staff at the June 2013 BSC meeting
8 National Toxicology Program Annual Report FY 2013

The second BSC meeting was held June 25, 2013. The BSC voted unanimously to approve concepts for 

contracts regarding quality assessment support and global pathology support. NTP staff presented a report

on the Report on Carcinogens (RoC) peer-review meeting on 1-bromopropane and cumene, held March

2013. Office of Health Assessment and Translation (OHAT) staff briefed the BSC on the “NTP Monograph

on Developmental Effects and Pregnancy Outcomes Associated With Cancer Chemotherapy Use During

Pregnancy,” and on the revised approach on systematic review published for public comment in February

2013. The BSC reviewed a draft RoC concept on “Shift Work at Night, Light at Night, and Circadian

Disruption,” and heard about planned OHAT activities. The NIEHS and NTP director and NTP associate

director provided reports updating the BSC on NTP progress since the December 2012 meeting.

Table 2. NTP Board of Scientific Counselors Membership Roster FY 2013

Name and Title Affiliation Term Ends

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

David A. Eastmond, Ph.D. (chair, 2012)

Professor and Chair

Department of Cell Biology and Neuroscience

University of California

Riverside, California 12/27/12

Elaine M. Faustman, Ph.D.

Professor and Director

Institute for Risk Analysis and Risk Communication Department of 

Environmental and Occupational Health Sciences

University of Washington

Seattle, Washington 12/27/12

Miguel C. Fernandez, M.D., F.A.C.E.P., F.A.A.E.M., F.A.C.M.T., F.A.A.C.T.

Professor of Surgery

Division of Emergency Medicine

Director, South Texas Poison Center

University of Texas Health 

Science Center

San Antonio, Texas

12/27/13

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

Dana Loomis, Ph.D.

Professor and Chair

Department of Epidemiology

University of Nebraska

Medical Center

Omaha, Nebraska

12/27/12

Stephen W. Looney, Ph.D.

Professor

Department of Biostatistics

Department of Oral Health and Diagnostic Science

Georgia Health Sciences 

University

Augusta, Georgia 

12/27/12

Melissa A. McDiarmid, M.D., M.P.H. (chair, 2013)

Professor of Epidemiology and Preventive Medicine

Director, Occupational Health Program

University of Maryland School

of Medicine

Baltimore, Maryland

12/27/13
1. National Toxicology Program: Mission And Goals 9

Name and Title Affiliation Term Ends

Lisa Minor, Ph.D.

Consultant

In Vitro Strategies, LLC

Flemington, New Jersey 12/27/13

Richard Miller, D.V.M., Ph.D.

Vice President

Safety Assessment 

GlaxoSmithKline 

Research Triangle Park, 

North Carolina 

12/27/13

Lisa A. Peterson, Ph.D.

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

Judith Zelikoff, Ph.D.

Professor of Environmental Medicine

Director, Community Outreach

New York University School of 

Medicine

Tuxedo, New York

12/27/12

Robert E. Chapin, Ph.D.

Laboratory Director

Pfizer

Groton, Connecticut 06/30/15

iii. Scientific Advisory Committee on Alternative Toxicological Methods 

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

advisory committee established Jan. 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 page 66). 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 2013. 

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 2013 on Sept. 24, 2013, at NIEHS (http://ntp.niehs.nih.gov/go/8202). At the 

meeting, ICCVAM and NICEATM updated SACATM on the new vision, direction, and procedures for ICCVAM

and on activities by NICEATM. An overview and strategy were presented for ICCVAM to utilize adverse

outcome pathways, followed by current NICEATM projects related to the skin sensitization adverse outcome

pathway. NTP staff presented updates on Tox21 activities and on collaborations through the International

Cooperation on Alternative Test Methods (ICATM) memorandum. ICCVAM members presented reports on two

international workshops: Alternative Methods for Leptospira Vaccine Potency Testing and Alternative to the

Murine Histamine Sensitization Test (HIST) for Acellular Pertussis Vaccines.
10 National Toxicology Program Annual Report FY 2013

Table 3. NTP SACATM Membership Roster FY 2013

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, L.C.

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

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

President and Principal

Pre-Clinical Safety, Inc.

Niantic, Connecticut 11/30/14

Members of SACATM and ICCVAM, and NIEHS and NTP staff at the September 2013 

SACATM meeting 
1. National Toxicology Program: Mission And Goals 11

Name and Title Affiliation Term Ends

Michael J. Olson, Ph.D., A.T.S.

Director

Occupational Toxicology Corporate Environment, Health, Safety 

and Sustainability

GlaxoSmithKline 

Research Triangle Park, North Carolina 06/30/13

Linda A. Toth, D.V.M., Ph.D.

Associate Dean for Research and Faculty Affairs 

Professor, Department of Pharmacology 

Southern Illinois University School of 

Medicine

Springfield, Illinois

06/30/13

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

Marilyn Wind, Ph.D.

Consultant Bethesda, Maryland 06/30/15

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). Members of this committee include representatives from the

following federal agencies:

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

● U.S. Department of Agriculture (USDA)

● U.S. Department of Defense (DOD)

● U.S. Department of Energy (DOE) 

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

○ Centers for Disease Control and Prevention (CDC)

▪ Agency for Toxic Substances and Disease Registry (ATSDR)

▪ National Institute for Occupational Safety and Health (NIOSH)

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

○ National Institutes of Health (NIH)

▪ National Cancer Institute (NCI)

▪ National Institute of Environmental Health Sciences (NIEHS)

▪ National Library of Medicine (NLM)

▪ Office of the Director (OD)

● U.S. Department of the Interior (DOI)

● U.S. Department of Labor (DOL)

○ Occupational Safety and Health Administration (OSHA)

● U.S. Department of Transportation (DOT)

● U.S. Environmental Protection Agency (EPA)
12 National Toxicology Program Annual Report FY 2013

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, or other issues. These panels help ensure transparent, unbiased, and scientifically

rigorous input to the program 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 Reports Peer Review Panels

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

toxicology and carcinogenesis studies. NTP convenes an external, scientific panel to peer review draft NTP

TRs at a public meeting, with opportunity for public comment, at NIEHS. The panels are charged to peer

review the scientific and technical elements of the study and their presentation; and determine whether the

study’s experimental design and conduct support the NTP conclusions regarding the carcinogenic activity of 

the substance tested. There were no TR meetings in FY 2013. Additional information about TR review panel

meetings is available at http://ntp.niehs.nih.gov/go/36051.

NTP Monographs Peer Review Panels

Monographs are publications on a single, detailed specific topic. On Oct. 1–2, 2012, NTP convened an

external, scientific panel at NIEHS to peer review the “Draft NTP Monograph on Developmental Effects and

Pregnancy Outcomes Associated With Cancer Chemotherapy Use During Pregnancy.” The meeting was

open to the public with time scheduled for oral public comment. The panel was charged to determine whether

the scientific information cited in the draft monograph is technically correct, clearly stated, and objectively

presented; and determine whether the scientific evidence presented in the draft monograph supports the NTP

interpretation of the developmental effects and pregnancy outcomes associated with cancer chemotherapy 

use during pregnancy. The review covered the document’s main findings, cancer chemotherapy agents by

mechanism of action, long-term evaluations of growth and development, pregnancy outcomes of medical 

personnel, and background information on cancer diagnosis during pregnancy. The peer-review panel included

individuals with expertise in obstetrics and gynecology, developmental biology, gynecological cancers, breast

cancer, hematological cancers, epidemiology, and pharmacology. Mary Wolfe, Ph.D., served as the designated

federal official for the peer-review meeting. After the meeting, the panel’s input was considered in finalizing the

monograph. Additional information about this past meeting and other 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 23. On March 21-22, 2013, NTP convened an external,

scientific panel at NIEHS to peer review the draft RoC monographs for 1-bromopropane and cumene. The

meeting was open to the public with time scheduled for oral public comment. Draft RoC monographs were

prepared for each substance, which consisted of a cancer evaluation component and a substance profile. For

the draft cancer evaluation component, the panel was charged to comment on whether it is technically correct 

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

the scientific evidence is adequate for applying the listing criteria. For the draft substance profiles, the panel

was charged to comment on whether the scientific justification presented supports the NTP preliminary policy

decision on the RoC listing status of the candidate substance.
1. National Toxicology Program: Mission And Goals 13

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

scientific evidence in experimental animals and whether the information cited in the draft substance profile

supported the NTP preliminary listing recommendation for the substance in the RoC. The review individually

covered the chemical properties and human exposure, disposition and toxicokinetics, studies in experimental 

animals, mechanistic data and other relevant effects, an overall cancer evaluation, and the draft substance

profile, for both 1-bromopropane and cumene. Lori White, Ph.D., served as the designated federal officer

for the peer-review meeting. After the meeting, the input from the panel was considered in finalizing the

monographs. Additional information about this meeting and other RoC monograph peer-review meetings is

available at http://ntp.niehs.nih.gov/go/38853.
14 National Toxicology Program Annual Report FY 2013

2. Funding

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 2013 was $121.3 million.

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

through interagency agreements with other agencies (see page 15). In FY 2013, NIEHS funded 32 contracts 

(see Table 4), and held one workshop (see page 66), two special emphasis expert panel peer review meetings 

(see page 12), and three scientific advisory meetings (see pages 7–9) for NTP.

Figure 2: Past, Current, and Projected Budget
2. Funding 15

Table 4. NTP Contracts That Supported NTP Testing Activities in FY 2013

Description Contractor

ADME Chemical Disposition in Mammals

Lovelace Biomedical

Research Triangle Institute

Analytical Chemistry Support Services

Battelle Memorial

Midwest Research Institute

Research Triangle Institute

Archives and Specimen Repository Experimental Pathology Labs

Bioinformatics Methylation Project Murdock Research Institute

Chemical Sample Analysis (SEARCH Project) UNC-Chapel Hill

Evaluate Toxicity Following Early Life Exposure Southern Research Institute 

Evaluation of Alternative Toxicological Methods Integrated Laboratory Systems

Evaluation of the Toxicity of Selected Chemicals Battelle Memorial

Genetic Toxicity Integrated Laboratory Systems

Genetic Toxicity Testing Support Services Integrated Laboratory Systems

In Life Data Collection and Management System INSTEM LSS

Investigative Research Support Integrated Laboratory Systems

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

Integrated Laboratory Systems

Pathology Support and Quality Assessment Experimental Pathology Labs

Provantis Software INSTEM

Quality Assurance Oversight of NTP Toxicology Data System Validation Labscience

Quality Assessment Support/Audits and Inspections Dynamac

Reproductive Assessments by Continuous Breeding Research Triangle Institute

Research on Inhalation Toxicology of Environmental Chemicals Alion Science and Technology

Support for GeneCo Databases Thomson Reuters

Support for MATLAB LCS

Support for Preparation of the Report on Carcinogens Integrated Laboratory Systems

Toxicological and Carcinogenic Potential of Test Agents Battelle Memorial

Interagency Agreements

In FY 2013, NIEHS provided support for NTP activities through interagency agreements with NCTR and 

NIOSH. Beginning in 1992, NIEHS established an interagency agreement with FDA, to support collaborative

toxicology studies on FDA-regulated agents that were nominated to NTP. This support has been primarily to

study chemicals where FDA has no legal authority to require the regulated community to provide toxicological 

data for a product. This agreement has led to investigations of mechanisms of action, and assessments 
16 National Toxicology Program Annual Report FY 2013

of toxicity for many classes of chemicals, including cosmetics, endocrine-disrupting compounds, food 

contaminants, food cooking byproducts, dietary supplements, drugs, and anesthetics. The studies are

conducted at NCTR. In addition, the interagency agreement partially supports the NCTR Office of Regulatory

Affairs Phototoxicity Research and Testing Laboratory.

In 1997, NIEHS established an interagency agreement with NIOSH for NTP studies to characterize

and evaluate adverse effects of complex occupational exposures. For a comprehensive assessment 

of occupationally relevant exposures, NTP and NIOSH coordinate efforts to increase knowledge, 

identification, and education of occupational health research. NIOSH and NTP also work together to support

immunotoxicology projects, evaluating adverse health effect to the immune system from environmental

exposure to chemical or physical agents.

In 2007, NIEHS established an interagency agreement with the National Human Genome Research Institute

and its NIH Chemical Genomics Center (NCGC) to support high throughput screening activities for the 

collaborative Tox21 program. NIH reorganization in 2012 placed the NCGC within the newly created National

Center for Advancing Translational Sciences (NCATS), moving the NTP interagency agreement to be with

NCATS. Screening efforts are aimed at providing data for use in toxicity profiling, and for prioritization of

substances for further in-depth toxicological evaluation, identification of mechanisms of action requiring further

investigation, and development of predictive models for in vivo biological response. The use of high throughput

and high content screening assays will increase testing throughput, provide greater coverage of chemical 

and biological space, and decrease the cost of testing potentially toxic compounds. To accomplish these

goals, NTP provided sets of compounds, to be incorporated into the comprehensive Tox21 10,000-compound

library, for testing in a battery of high throughput and high content screens that were selected by the NCATS

laboratory in consultation with NTP. All test data generated from this effort will be deposited into one or more

public databases, such as PubChem.

Also in 2008, NIEHS and EPA signed the Phthalate Initiative interagency agreement, to address nominations 

of bis(2-ethylhexyl) phthalate and other phthalates to NTP for testing. Some of the study findings from this

interagency agreement, which came to an end in FY 2011, were published in FY 2013.
3. NTP Highlighted Activities 17

3. NTP Highlighted Activities

Figure 3 displays the major events for NTP from FY 2013. Below are additional activities of importance from

FY 2013.

A. Nonneoplastic Lesion Atlas

In FY 2013, NTP prepared for the launch of the NTP 

Nonneoplastic Lesion Atlas, a Web-based resource that

contains hundreds, and eventually thousands, of highquality 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, or organic materials, such as dust from cotton, 

grain, and wood. 

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

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

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

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

for nonneoplastic lesions. The NTP Nonneoplastic Lesion Atlas is planned for launch in FY 2014. For more 

information, see http://ntp.niehs.nih.gov/nnl. 

 OCT ‘12 NOV ‘12 DEC ‘12 JAN ’13 FEB ’13 MAR ’13 APR ’13 MAY ’13 JUNE ’13 JULY ’13 AUG ’13 SEPT ’13 

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

Draft NTP Monograph on Developmental

Effects and Pregnancy Outcomes Associated 

With Cancer Chemotherapy Use During

Pregnancy Peer Review Panel Meeting

See page 12

Draft RoC Monographs for 1-Bromopropane

and Cumene Peer Review Panel Meeting

See page 12

ICCVAM-NICEATM

International Workshop on Alternatives to

the Murine Histamine Sensitization Test

(HIST) for Acellular Pertussis Vaccines

See page 66

NTP at Society of Toxicology 

Meeting in San Antonio, Texas

See page 21

NTP Satellite Symposium 

on Pathology Potpourri

See page 21

Board of Scientific

Counselors Meeting

See page 7

Board of Scientific

Counselors Meeting

See page 7

Release of NTP Monograph on

Developmental Effects and Pregnancy 

Outcomes Associated With Cancer

Chemotherapy Use During Pregnancy

See page 18 SACATM

Meeting

See page 9

Figure 3: FY 2013 Highlights
18 National Toxicology Program Annual Report FY 2013

B. Realignment of NICEATM and ICCVAM

During its first 15 years, ICCVAM evaluated many alternative methods, and made formal recommendations

about their potential regulatory use to federal agencies. However, ICCVAM stakeholders raised concerns that

the methods recommended by ICCVAM were not being used for regulatory decision-making. In February 2013,

the NIEHS and NTP director announced that NIEHS would move forward with a different philosophy toward 

ICCVAM, whereby the partner regulatory agencies would drive ICCVAM activities. At the same time, NICEATM

would expand its scope to provide bioinformatic and computational toxicology support to NIEHS/NTP.

In response to this new philosophy, ICCVAM developed a draft document entitled “A New Vision and Direction

for ICCVAM,” which was made available for public comment in August 2013. The document presents

the ICCVAM areas of priority and scientific focus for immediate resource investment; plans to improve

communications with stakeholders and the public; and interest in exploring new paradigms for the validation

and utilization of alternative toxicological methods. ICCVAM is carefully considering public and SACATM

comments on the draft document, as it develops new operating procedures and plans activities for the future.

C. Chemotherapy Use During Pregnancy

In May 2013, NTP published the “NTP Monograph on Developmental

Effects and Pregnancy Outcomes Associated With Cancer Chemotherapy

Use During Pregnancy.” Many chemotherapy agents for the treatment of

cancer are known mutagens or carcinogens. However, there is a lack of 

comprehensive reviews on the pregnancy outcomes following cancer 

chemotherapy use during pregnancy in humans. The monograph provides 

a comprehensive literature review, including all available human data on

follow-up evaluations in offspring who were exposed to 

chemotherapeutics during gestation. It also includes a review of the 

developmental toxicity of these agents in laboratory animals, and reports

examining their transport via the placenta or breast milk.

Overall, the NTP evaluation found that chemotherapy for treatment of 

cancer appears to be associated with a higher rate of major malformations

following exposure during the first trimester, compared to exposure in the

second and/or third trimester only, while the rate of major malformations in the second and/or third trimester

was similar to the rate in the general population. There was an increase in the rate of stillbirth following

exposure in the second and/or third trimester, and a higher incidence of abnormally low levels of amniotic

fluid, which was primarily attributable to one chemotherapy agent (trastuzumab). Chemotherapy for treatment

of cancer did not appear to increase spontaneous preterm birth, or impair normal growth and development of

offspring during early life. While the monograph does not provide medical advice or guidance, it is intended

as a resource for clinicians and their pregnant patients. The “NTP Monograph on Developmental Effects and

Pregnancy Outcomes Associated With Cancer Chemotherapy Use During Pregnancy” is available at http://ntp.

niehs.nih.gov/go/36495.

D. CLARITY-BPA Research Program: Synergizing Academic and GuidelineCompliant Research

NIEHS and FDA have developed a consortium-based research program to improve the link between academic

research and guideline-compliant research, to evaluate the low-dose hazard of bisphenol A (BPA). Certain
3. NTP Highlighted Activities 19

plastics and epoxy resins contain BPA, and are used to make a variety of common consumer goods, such 

as water bottles, CDs, DVDs, food containers, beverage cans, and lining for pipes. The Consortium Linking

Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) utilizes a unique collaborative approach

to fill knowledge gaps, and offers a more comprehensive body of scientific information to better inform risk

assessments. Building more effective connections between risk assessors and those who conduct basic

hazard identification research could enhance the scientific basis of chemical risk assessments.

NCTR/NTP is conducting a chronic perinatal rodent toxicity study on BPA. NIEHS/NTP is managing 

consortium activities, and facilitating research by twelve grantees using samples gathered from a guidelinecompliant toxicity study. CLARITY-BPA will contribute to the understanding of potential effects of BPA. Another

outcome is that the consortium may serve as a new model for filling knowledge gaps, and improving the

scientific knowledge base on which regulatory hazard assessments are made. The CLARITY-BPA publication

in the journal Reproductive Toxicology is available at http://www.ncbi.nlm.nih.gov/pubmed/23747832.

E. Crowdsourcing Human Variability Data

NIEHS, NCATS, the University of North Carolina at Chapel Hill, 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 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, who could be anyone with an interest, 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.

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 results of the challenge will be

published in the journal Nature Biotechnology.

F. 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 consensus 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
20 National Toxicology Program Annual Report FY 2013

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.

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 2013, scientists completed

characterization of the liver transcriptome, or the collection of RNA in the liver, genomic sequencing, and 

bisulfite treatment of DNA, to map all methylated sites in the mouse genome. Ongoing bioinformatics analyses

found differentially methylated regions related to gender, strain, and heritability, and relationships with gene

expression. Analysis efforts will continue in FY 2014.

G. 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 2013.

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

Agencies in FY 2013

Notice NTP Information Cited Summary of Notice

California Office of Environmental Health

Chemical Delisted 

Effective April 19, 2013 

As Known to the State 

of California to Cause 

Reproductive Toxicity: 

Bisphenol A (BPA)

NTP-CERHR (2008). NTP-CERHR 

Monograph on the Potential Human

Reproductive and Developmental Effects 

of Bisphenol A. Research Triangle Park, 

NC, National Toxicology Program: NIH 

Publication No. 08 – 5994.

NTP (1985) Bisphenol A: reproduction 

and fertility assessment in CD-1 mice 

when administered in the feed. NTP-85-

192. Research Triangle Park, NC.

Effective April 19, 2013, the Office of Environmental

Health Hazard Assessment (OEHHA) is removing BPA 

(CAS No. 80-05-7) from the list of chemicals known to 

the State to cause cancer or reproductive toxicity for 

purposes of Proposition 65. The chemical was added 

to the list on April 11, 2013, based on reproductive

endpoints (developmental toxicity). 

April 19, 2013 — Proposition 65

http://oehha.ca.gov/prop65/law/041913BPAdelist.html

Centers for Disease Control and Prevention

Final Rule: World Trade

Center Health Program; 

Certification of Breast

Cancer in World Trade

Center Responders and 

Survivors Exposed to 

PCBs

12th edition of the Report on 

Carcinogens: polychlorinated biphenyls

are reasonably anticipated to be a

human carcinogen.

On Sept. 12, 2012, HHS published a final rule in the

Federal Register adding certain types of cancer to the 

list of World Trade Center-related health conditions

established in the World Trade Center Health Program

regulation.

April 17, 2013 — 78 FR 22794

http://www.gpo.gov/fdsys/pkg/FR-2013-04-17/

html/2013-09003.htm
3. NTP Highlighted Activities 21

Notice NTP Information Cited Summary of Notice

Final Rule: World

Trade Center Health 

Program; Addition of 

Prostate Cancer to the 

List of World Trade

Center-Related Health 

Conditions

Report on Carcinogens is a source 

used in one part of a four-part 

hierarchical methodology to apply in 

evaluating whether to propose adding 

certain types of cancer to the List of 

World Trade Center-Related Health

Conditions included in 42 CFR 88.1 (77 

FR 56138, 56142).

On May 2, 2013, the Administrator of the World Trade

Center Health Program received a petition (Petition 002) 

requesting the addition of prostate cancer to the List of 

World Trade Center-Related Health Conditions (List)

covered in the World Trade Center Health Program.

In this final rule, the Administrator adds malignant

neoplasm of the prostate (prostate cancer) to the List in 

the World Trade Center Health Program regulations. 

Sept. 19, 2013 — 78 FR 57505

http://www.gpo.gov/fdsys/pkg/FR-2013-09-19/pdf/2013-

22800.pdf

Environmental Protection Agency

Final Rule: 2017 and 

Later Model Year

Light-Duty Vehicle

Greenhouse Gas 

Emissions and 

Corporate Average Fuel 

Economy Standards

NTP 11th and 12th editions of the 

Report on Carcinogens: acetaldehyde 

and naphthalene are reasonably

anticipated to be human carcinogens;

benzene, 1,3-butadiene and

formaldehyde are known human 

carcinogens.

EPA and the National Highway Traffic Safety

Administration, on behalf of the Department of

Transportation, are issuing final rules to further reduce

greenhouse gas emissions and improve fuel economy 

for light-duty vehicles for model years 2017 and beyond.

Oct. 15, 2012 — 77 FR 62624

http://www.gpo.gov/fdsys/pkg/FR-2012-10-15/html/2012-

21972.htm 

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

niehs.nih.gov/go/regact.

H. Additional Activities

NTP participates in a number of meetings with stakeholders and the scientific community. At the 2013 annual

meeting of the Society of Toxicology (SOT) in San Antonio, Texas, staff from NIEHS/NTP and other NIEHS 

staff participated in more than 80 workshops, symposia, and platform sessions; education and information 

sessions; and poster sessions. Two NTP scientists won prestigious SOT awards: Yuanyuan (Laura) Xu, Ph.D.,

of the NTP Laboratory received the Best Postdoctoral Publication Award; and Olive Ngalame, Ph.D., also of

the NTP Laboratory, won the First Place Stem Cells Specialty Section Postdoctoral Excellence in Research

Award. A full listing of the NTP and NIEHS activities at SOT can be found at http://ntp.niehs.nih.gov/go/35370. 

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

example, the 2013 annual NTP Satellite Symposium, Pathology Potpourri, was held in Portland, Oregon, June 

15, in advance of the 32nd 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. “Proceedings of the 2013 Joint JSTP/NTP Satellite Symposium” was published in Toxicologic

Pathology, along with summaries of presentations, including diagnostic or nomenclature issues that were 

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

NTP also hosted a workshop in FY 2013 related to alternative methods development (see page 66).
22 National Toxicology Program Annual Report FY 2013

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, and 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 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 the director of OHAT.

As highlighted on page 18, in FY 2013, OHAT published the “NTP Monograph on Developmental Effects

and Pregnancy Outcomes Associated With Cancer Chemotherapy Use During Pregnancy (May 2013),” and

continued efforts to incorporate systematic review methodologies into its evaluations (http://ntp.niehs.nih.gov/

go/38673). Table 6 lists literature analysis projects that were initiated, ongoing, or completed in FY 2013.

Table 6. NTP Noncancer Health Effects Projects in FY 2013

NTP Project 

[Study Scientist] Objective and/or Summary

Completed Project

Evaluation of Developmental Effects 

and Pregnancy Outcomes Associated 

With Cancer Chemotherapy Use During

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

go/36495) 

[Howdeshell]

Many chemotherapy agents used to treat cancer are known mutagens or

carcinogens. However, there is a lack of comprehensive reviews on the pregnancy 

outcomes following cancer chemotherapy use during pregnancy in humans. This 

evaluation published as an NTP monograph provides a comprehensive literature

review of the human data, including all available data on follow-up evaluations

in offspring who were exposed to chemotherapeutics during gestation. It also 

included a review of the developmental toxicity of these agents in laboratory

animals and any reports examining their transport via the placenta or breast milk.

Ongoing Projects

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 planning a

workshop 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 impacts from high intakes of folic acid. 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.

Draft case-study protocols to assess 

the systematic review and evidence 

integration framework

[Rooney]

Case studies are being undertaken to determine if additional refinement or

revision to the Draft OHAT Approach (February 2013) might be needed. The 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.
4. Literature Analysis Activities 23

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 United States are exposed [Section 301(b)(4) of the Public Health Services

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 and consists of substances newly reviewed, in addition to those

listed in previous editions. The 12th RoC, the latest edition, was published on June 10, 2011. NTP, with

assistance from other federal health and regulatory agencies, prepares the RoC for the HHS Secretary. 

Preparation of the RoC is managed by the Office of the RoC, under the direction of Ruth Lunn, Dr.P.H.

Contract support for preparation of the RoC in FY 2013 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). The cancer evaluation for each substance is

captured in the draft RoC monographs, and summarizes and assesses the information evaluated by NTP

in making its preliminary listing decision. On an ongoing basis, NTP solicits comments on nominations and

selects nominations for RoC evaluation, referred to as candidate substances.

In FY 2013, the Office of the RoC invited public input on 20 substances nominated for future editions of the

RoC (http://ntp.niehs.nih.gov/go/rocnom); selected a new candidate topic, shift work at night, light at night, 

and circadian disruption, for review (see Table 7); and prepared and released for public comment draft RoC

monographs for four candidate substances: 1-bromopropane, cumene, ortho-toluidine, and pentachlorophenol

and byproducts of its synthesis, hereinafter referred to as pentachlorophenol (see Table 7). The scientific

reviews for the substances 1-bromopropane and cumene have been completed. The draft monographs were

peer reviewed by a panel of experts at a public meeting (http://ntp.niehs.nih.gov/go/38854), and the final RoC

monographs are available on the RoC website (1-bromopropane, http://ntp.niehs.nih.gov/go/37896; cumene, 

http://ntp.niehs.nih.gov/go/37895). 

The Office of the RoC convened a public webinar in April 2013, to obtain public and scientific input on issues

related to human exposure to pentachlorophenol, in particular, exposure to byproducts of its synthesis and

the human cancer studies of exposure to pentachlorophenol (http://ntp.niehs.nih.gov/go/pcpwebinar). The 

webinar presentations and discussions help to inform the cancer evaluation of pentachlorophenol. The draft

RoC monographs for ortho-toluidine (http://ntp.niehs.nih.gov/go/37893) and pentachlorophenol (http://ntp.

niehs.nih.gov/go/37897) were released for public comment in August 2013, and the peer review of the draft

RoC monographs is scheduled for FY 2014. Following completion of the peer review of the monographs, and 

sharing information on the reviews with NTP advisory committees, the NTP director will submit the substance

profiles for the four candidate substances to the HHS Secretary for review and approval. Preparation of the

draft monograph on trichloroethylene is ongoing.
24 National Toxicology Program Annual Report FY 2013

Table 7. Candidate Substances for the RoC

Candidate Substance

CASRN 

[Study Scientist]

Primary Uses/Exposures RoC Review Status

1-Bromopropane 

106-94-5

[Spencer]

A brominated hydrocarbon used as a solvent for cleaning

or as adhesives in a variety of industries including spray 

adhesives (used in foam cushion manufacturing), vapor 

degreaser (to clean optics, electronics, and metals), 

aerosol solvents (for aircraft maintenance), and dry 

cleaning.

Final monograph published

Peer-review meeting, March 2013

NTP BSC meeting, June 2013

Cumene 

98-82-8

[Jahnke]

An alkylated benzene found in fossil fuels and used

primarily to produce phenol and acetone.

Final monograph published

Peer-review meeting, March 2013

NTP BSC meeting, June 2013

Pentachlorophenol and 

Byproducts of Its Synthesis

87-86-5

[Jahnke]

A chlorinated aromatic compound that is primarily used 

as wood preservatives in the United States. Its use as a 

wood preservative has been limited to non-residential and

non-agricultural applications (such as the treatment of 

utility poles and cross arms) since 1984.

Draft RoC monograph released 

for public comment, August 2013

ortho-Toluidine* 

95-53-4 

[Lunn]

An arylamine used as an intermediate to manufacture 

herbicides, dyes, pigments and rubber chemicals.

Draft RoC monograph released 

for public comment, August 2013

Trichloroethylene*

79-01-6 

[Lunn]

Halogenated alkene used primarily for degreasing metals. Draft monograph in preparation 

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.

Candidate topic selected 

NTP BSC review of draft concept, 

June 2013

* Currently listed as reasonably anticipated to be a human carcinogen in the RoC.
5. Testing and Research 25

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). The 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, the BSC (see page 7), 

and the public (see http://ntp.niehs.nih.gov/go/156 for details). Research concepts are developed to outline the 

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

a chemical or substance for testing.

A concept for polycyclic aromatic compounds (PACs) was reviewed during the December 2012 BSC meeting

(see page 7). PACs are ubiquitous environmental contaminants as a result of their natural occurrence in

fossil fuel sources, and formation and release during combustion processes. Exposure to PACs is through

consumption of PAC-containing foods, such as contaminated seafood and char-grilled meat; non-dietary 

ingestion, such as house dust; inhalation of polluted air, such as cigarette smoke and diesel exhaust; or dermal 

contact in an occupational setting, such as road paving and roofing. Some PACs have been associated

with various toxicities, such as carcinogenicity, reproductive and developmental toxicity, immunotoxicity, 

and neurotoxicity. The PAC Mixtures Assessment Program was presented to the BSC to address existing

knowledge gaps. Individual PACs, defined mixtures consisting of subsets of individual PACs, and complex

PAC-containing mixtures, such as cookstove emissions and coal tar, will be assessed in a battery of in vitro

and short-term in vivo assays focused on a broad array of hazard endpoints. Available predictive models of

mixture toxicity will be evaluated for utility, by comparing modeled predictions to observed mixture responses.

The PAC Mixtures Assessment Program will provide individual chemical hazard characterization data, and

inform the refinement and development of cumulative risk assessment methods for PACs.

The full research concepts for substances can be found at http://ntp.niehs.nih.gov/go/37038. Questions about

the nomination, review, and selection process can be sent to Scott Masten, Ph.D. at masten@niehs.nih.gov.

A. Technical Reports

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

in several NTP report series: 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 substance 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 and 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.
26 National Toxicology Program Annual Report FY 2013

Table 8 lists NTP Technical Reports published in FY 2013. 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 strength of the evidence for

carcinogenic activity are included in the tables. The reports are available at http://ntp.niehs.nih.gov/. NTP 

anticipates eight draft TRs will undergo peer review in FY 2014, as shown in Table 9.

Table 8. Technical Reports Published During FY 2013

Chemical/

Exposure – 

Study Type

Technical 

Report 

Number

CASRN

Use

Levels of Evidence of Carcinogenic Activity

Male 

Rats

Female 

Rats

Male 

Mice

Female 

Mice

Aloe Vera TR-577

Recognized as a therapeutic 

dermatologic agent; extracts 

incorporated in a variety of 

topical health care products 

and cosmetics

Clear 

Evidence

Clear 

Evidence

No 

Evidence

No 

Evidence

Ginkgo biloba

Extract

TR-578

90045-36-6

Herbal supplement Some 

Evidence

Some 

Evidence

Clear 

Evidence

Clear 

Evidence

Mixtures of

3′-Azido-3′-

Deoxythymidine 

(AZT), Lamivudine 

(3TC), Nevirapine 

(NVP), and

Nelfinavir Mesylate

(NFV)

TR-569

30516-87-1 

134678-17-4 

129618-40-2 

159989-65-8

Pyrimidine nucleoside 

analog with antiviral activity 

used in the treatment of 

AIDS (Merck 1989)

N/A N/A

No 

Evidence 

(AZT)

Equivocal 

Evidence 

(AZT)

No 

Evidence 

(AZT/3TC)

Equivocal 

Evidence 

(AZT/3TC)

Some 

Evidence 

(AZT/3TC/

NVP)

Equivocal 

Evidence 

(AZT/3TC/

NVP)

No 

Evidence 

(AZT/3TC/

NFV)

No 

Evidence 

(AZT/3TC/

NFV)
5. Testing and Research 27

Chemical/

Exposure – 

Study Type

Technical 

Report 

Number/ 

CASRN

Use

Levels of Evidence of Carcinogenic Activity

Male 

Rats

Female 

Rats

Male 

Mice

Female 

Mice

Pyrogallol

TR-574

87-66-1

Naturally occurring in 

food. Used as a modifier in

oxidation dyes including hair 

dyes and colors. Also used 

as developer in photography; 

a mordant for dying wool; 

a reagent for antimony and 

bismuth; and as a reducer

for gold, silver & mercury 

salts. Used for process 

engraving and for making 

colloidal solutions of metals. 

Used in the manufacture 

of pharmaceuticals and 

pesticides. Associated with 

tobacco: reported either

as a natural component of 

tobacco, pyrolysis product

(in tobacco smoke), or

additive for one or more 

types of tobacco products.

No 

Evidence

No 

Evidence

Equivocal 

Evidence

Some 

Evidence

Trimethylolpropane 

Triacrylate

TR-576

15625-89-5

Used in the production of 

ultraviolet-curable inks,

electron beam irradiationcurable coatings, and

polymers and resins; as a 

component of photopolymer 

and flexographic printing

plates and photoresists; 

and as an ingredient 

in acrylic glues and 

anaerobic sealants. Also

used in paper and wood 

impregnates, wire and 

cable extrusion, polymerimpregnated concrete, and 

polymer concrete structural 

composites.

Equivocal 

Evidence

No 

Evidence

No 

Evidence

No 

Evidence

CLEAR Evidence SOME Evidence EQUIVOCAL Evidence NO Evidence
28 National Toxicology Program Annual Report FY 2013

Table 9. Technical Reports Expected to Undergo Peer Review in FY 2014

Chemical

Technical 

Report 

Number

CASRN

Use

Bromodichloroacetic 

acid (water disinfection 

byproduct)

TR-58

71133-14-7

Bromodichloroacetic acid is a water disinfectant byproduct. Naturally occurring

bromides in the water participate in the formation of this compound. This

chemical is formed after disinfection of water with halogenated oxidants, 

usually chlorine.

CIMSTAR 3800 (metal

working fluid) TR-586 Semisynthetic; metalworking fluid.

Cobalt

TR-581

7440-48-4

Nuclear medicine and research, industry, paints, water treatment, metallurgy. 

Associated with tobacco: reported either as a natural component of tobacco,

pyrolysis product (in tobacco smoke), or additive for one or more types of

tobacco products.

Glycidamide

TR-588

5694-00-8

Metabolite of acrylamide. Associated with tobacco: reported either as a natural

component of tobacco, pyrolysis product (in tobacco smoke), or additive for

one or more types of tobacco products.

Green tea extract TR-585 Extract of green tea used as dietary supplement.

Indole-3-carbinol

TR-584

700-06-1

Natural component of Brassica vegetables; marketed as a dietary supplement

and cancer preventative agent.

Tetrabromobisphenol A

TR-587

79-94-7 Flame retardant for plastics, paper, and textiles.

Vinylidene chloride

TR-582

75-35-4

Copolymerized with vinyl chloride or acrylonitrile for saran and saran fibers. A

widely used chemical intermediate and monomer.

B. NTP Testing

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

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

potential. For each agent studied, a project leader designs a comprehensive testing strategy to address the

identified research and testing needs. A project review committee evaluates the testing strategy and proposes

an appropriate mechanism for performing the study, such as a grant or contract. The following 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 Mike Waalkes, Ph.D.;

Program Operations Branch, led by Michelle Hooth, Ph.D.; and Toxicology Branch, led by Paul Foster, Ph.D.
5. Testing and Research 29

i. Disposition, Metabolism, and Toxicokinetic Studies

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

excretion (ADME) in the body, at differing levels of exposure, over all ages, via several routes of exposure,

and under varying genetic backgrounds, in humans and test animals. Data from NTP chemical disposition and

toxicokinetic studies are used in these studies. Substances evaluated during FY 2013 are listed in Table 10. 

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

Table 10. Ongoing and Completed Disposition, Metabolism and Toxicokinetic Studies

During FY 2013

Chemical CASRN Species/Strain Route Study 

Scientist

Bisphenol A 80-05-7 Rats:Sprague Dawley (NCTR) In-vitro Delclos

Bisphenol AF 1478-61-1 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Intravenous Sutherland

2-Butene-1,4-diol 110-64-5 N/A In-vitro Doerge

N-Butylbenzenesulfonamide 3622-84-2 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage Rider

Ephedrine and caffeine 

combination N/A Rats:Wistar Han Gavage Dunnick

1,3-Dichloro-2-propanol 96-23-1 Rats:Harlan Sprague Dawley

Mice:B6C3F1 Gavage Waidyanatha

Di(2-ethylhexyl) phthalate 117-81-7 Monkey:Rhesus Gavage Delclos

2,2’-Dimorpholinodiethyl ether 6425-39-4 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Dermal Waidyanatha

2’,2’’’-Dithiobisbenzanilide 135-57-9 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage DeVito

2-Ethylhexyl p-methoxycinnamate 5466-77-3 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage McIntyre

Furan 110-00-9 Rats:F344 (NCTR) Gavage Walker

Hydroquinone 123-31-9 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Intravenous DeVito

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats:Harlan Sprague DawleyMice:B6C3F1/N Gavage Auerbach/McIntyre

Hydroxyurea 127-07-1 Rats:Harlan Sprague Dawley Gavage McIntyre

L-beta-Methylaminoalanine 15920-93-1 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage Ryan

Methylene blue trihydrate 7220-79-3 Rats:F344/N

Mice:B6C3F1 Intravenous Chhabra

Nanoscale silver 7440-22-4 Rats:Harlan Sprague Dawley Gavage

Intravenous Walker

Octyl salicylate 118-60-5 Rats:Harlan Sprague Dawley Gavage Devito
30 National Toxicology Program Annual Report FY 2013

Chemical CASRN Species/Strain Route Study 

Scientist

Resveratrol 501-36-0

Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Intravenous Germolec

Rats:Wistar Han Gavage Germolec

Silver acetate 563-63-3 Rats:Harlan Sprague Dawley Gavage

Intravenous Boudreau

Triclosan 3380-34-5 Mice:B6C3F1/NCTR Topical 

application Fang/ Howard

Tris(2-chloroisopropyl)phosphate 13674-84-5 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Intravenous Stout

Tris(4-chlorophenyl)methane 27575-78-6 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Intravenous Surh

Tris(4-chlorophenyl)methanol 3010-80-8 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage Surh

ii. Genetic Toxicity

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

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

2013 are listed in Table 11. More information can be found at http://ntp.niehs.nih.gov/go/gt. 

Table 11. Ongoing and Completed Genetic Toxicity Studies During FY 2013

Chemical CASRN Testing Battery

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

Bisphenol AF 1478-61-1

Micronucleus

Salmonella

Black cohosh 84776-26-1 Micronucleus

Bromodichloroacetic acid 71133-14-7 Salmonella

Cell phone radiation (CDMA) N/A Micronucleus

Cell phone radiation (GSM) N/A Micronucleus

Compound Z N/A

Micronucleus

Salmonella

Dimethylamine borane 74-94-2 Salmonella

Ethanone, 1-(1,2,3,4,5,6,7,8-Octahydro-2,3,8,8-Tetramethyl-2-

Naphthalenyl)- (Iso-E Super [reg]; OTNE) 54464-57-2 Salmonella

2-Ethylhexyl p-methoxycinnamate 5466-77-3 Micronucleus

Garcinia cambogia extract 90045-23-1 Salmonella

Ionic liquid (1-Butyl-3-methylimidazolium chloride) 79917-90-1 Micronucleus

Ionic liquid (1-Ethyl-3-methylimidazolium chloride) 65039-09-0 Micronucleus

Isopropylated phenol phosphate 68937-41-7 Salmonella
5. Testing and Research 31

Chemical CASRN Testing Battery

2-Methyl-6-ethylanline 24549-06-2 Salmonella

Nelfinavir 159989-64-7 Salmonella

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

2-Nitro-1-propanol 2902-96-7 Salmonella

Perfluorobutane sulfonate 375-73-5 Micronucleus

Perfluorodecanoic acid 335-76-2 Micronucleus

Perfluorohexane sulfonate potassium salt 3871-99-6 Micronucleus

Perfluorohexanoic acid 307-24-4 Micronucleus

Perfluorononanoic acid 375-95-1 Micronucleus

Perfluorooctane sulfonate 1763-23-1 Micronucleus

Perfluorooctanoic acid 335-67-1 Micronucleus

Potassium hydroxycitrate tribasic monohydrate 232281-44-6 Salmonella

Tetrabromobisphenol A 79-94-7 Salmonella

Tris(4-chlorophenyl)methane 27575-78-6 Salmonella

Tris(4-chlorophenyl)methanol 3010-80-8 Salmonella

Wyeth 14,643 50892-23-4 Micronucleus

Zinc carbonate, basic 5263-02-5 Micronucleus

iii. Organ System Toxicity

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

immune system. Table 12 lists ongoing and completed organ systems toxicity studies during FY 2013. More

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

Table 12. Ongoing and Completed Neurotoxicity, Developmental Toxicity and Reproductive

Toxicity Studies During FY 2013

Chemical CASRN Species/Strain Route Study 

Scientist Testing Battery

Black cohosh 84776-26-1 Rats:Harlan Sprague Dawley Gavage Blystone Continuous breeding

Butyl paraben (n-Butyl-phydroxybenzoate) 94-26-8 Rats:Harlan Sprague Dawley Feed Blystone Continuous breeding

Diisobutyl phthalate 84-69-5 Rats:Harlan Sprague Dawley Feed Blystone Continuous breeding

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats:Harlan Sprague Dawley Feed Hansen, Gwinn

Teratology pilot study

Developmental 

toxicity

4-Methylimidazole 822-36-6 Rats:Harlan Sprague Dawley Feed Behl Continuous breeding
32 National Toxicology Program Annual Report FY 2013

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 or infectious 

disease, or neoplasia. Table 13 lists ongoing and completed immunotoxicity studies during FY 2013. More

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

Table 13. Ongoing and Completed Immunotoxicity Studies During FY 2013

Chemical CASRN Species/

Strain Route Study Scientist Testing Battery

Autumn sunset true color 

concentrate N/A Mice:CBA/ CaJackson

Subcutaneous

injection Howard Hypersensitivity

3’-Azido-3’-deoxythymidine 30516-87-1 Mice:B6C3F1/N Gavage Irwin

Immunosuppression–

Range finding*,

Developmental

Blasting sand (abrasive blasting

agents) N/A Rats:Harlan Sprague Dawley Inhalation Gwinn Immunosuppression

2,3-Butanedione (Diacetyl) 431-03-8 Mice:BALB/c Inhalation Morgan Immunosuppression–

Range finding*

Dermal lotion vehicle 

development N/A Mice:BALB/c Topical application Germolec Hypersensitivity

Dibenz(a,h)anthracene 53-70-3 Mice:B6C3F1/N Subcutaneous

injection Germolec

Immunosuppression–

Range finding*,

Immunosuppression–

Full protocol**,

Developmental

Double dark fudge true color

concentrate N/A Mice:CBA/ CaJackson

Subcutaneous

injection Howard Hypersensitivity

Double fudge concentrate N/A Mice:CBA/ Ca

Jackson

Subcutaneous

injection Howard Hypersensitivity

Echinacea purpurea, extract 90028-20-9 Mice:B6C3F1/N Gavage Ryan Immunosuppression–

Full protocol**

Fullerene-C60 (1 micron) 

(nanoscale material) 99685-96-8

Rats:Wistar Han

Mice:B6C3F1

Inhalation Walker Immunosuppression–

Range finding*

Fullerene-C60 (50 nanometers) 

(nanoscale material) 99685-96-8

Rats:Wistar Han

Mice:B6C3F1

Inhalation Walker Immunosuppression–

Range finding*

Genistein 446-72-0 Mice:NOD/

MrKTac Gavage Germolec Autoimmunity

Monoclonal antibody protein

therapeutics (CD-4) N/A Mice:B6C3F1 Intraperitoneal injection Germolec Immunosuppression–Full protocol**

Monoclonal antibody protein

therapeutics (CD-8) N/A Mice:B6C3F1 Intraperitoneal injection Germolec Immunosuppression–Full protocol**
5. Testing and Research 33

Chemical CASRN Species/

Strain Route Study Scientist Testing Battery

Nelfinavir mesylate 159989-

65-8 Mice:B6C3F1/N Gavage Germolec

Immunosuppression –

Range finding*;

Developmental

Nevirapine 129618-40-2 Mice:B6C3F1 Gavage Germolec Developmental

Perfluorodecanoic acid 335-76-2 Rats:Harlan 

Sprague Dawley Gavage Blystone Immunosuppression

Resveratrol 501-36-0 Mice:NOD/

MrKTac Gavage Germolec Autoimmunity

Rosewood true color 

concentrate N/A Mice:CBA/ CaJackson

Subcutaneous

injection Howard Hypersensitivity

Sodium tungstate, dihydrate 10213-10-2 Mice:B6C3F1/N Drinking water Hooth Immunosuppression–

Full protocol**

Specular hematite (abrasive

blasting agents) N/A Rats:Harlan Sprague Dawley Inhalation Gwinn Immunosuppression

3,3’,4,4’-Tetrachloroazobenzene 14047-09-7

Rats:Sprague 

Dawley Gavage Behl Immunosuppression–Range finding*

Rats:Crl:CD 

(Sprague 

Dawley)

Gavage Behl Developmental

*		 Range finding studies are performed in order to establish the potential effects of a substance on the immune system and

to determine doses that could be used in a full immunotoxicology study.

**	Full protocols are more comprehensive studies that establish potential adverse effects of a substance on the immune

system.

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. 
34 National Toxicology Program Annual Report FY 2013

Table 14. Planned or Ongoing Modified One-generation Studies

Chemical CASRN Species/

Strain

Study 

Route Planned Cohorts Study Scientist

Bisphenol AF 1478-61-1 Rats:Harlan 

Sprague Dawley Feed

Dose range finding1

Maternal transfer2

Developmental toxicity3

Fertility assessment4

Subchronic toxicity6

Sutherland

N-Butylbenzenesulfonamide 3622-84-2 Rats:Harlan 

Sprague Dawley Feed

Dose range finding1

Maternal transfer2

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

Subchronic toxicity6

Rider

Dong quai (Angelica sinensis) 

root extract 299184-76-2 Rats:Harlan Sprague Dawley Gavage

Dose range finding1

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

Subchronic toxicity6

McIntyre

2-Ethylhexyl 

p-methoxycinnamate 5466-77-3 Rats:Harlan Sprague Dawley Feed

Dose range finding1

Maternal transfer2

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

Subchronic toxicity6

McIntyre

Evening primrose oil 90028-66-3 Rats:Harlan 

Sprague Dawley Gavage

Dose range finding1

Developmental toxicity3

Fertility assessment4

Subchronic toxicity6

Germolec

Garcinia cambogia extract 90045-23-1 Rats:Harlan 

Sprague Dawley Feed

Dose range finding1

Maternal transfer2

Developmental toxicity3

Fertility assessment4

Subchronic toxicity6

Rider
5. Testing and Research 35

Chemical CASRN Species/

Strain

Study 

Route Planned Cohorts Study Scientist

Hydroquinone 123-31-9 Rats:Harlan 

Sprague Dawley Gavage

Dose range finding1

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

DeVito

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats:Harlan Sprague Dawley Feed

Dose range finding1

Developmental toxicity3

Fertility assessment4

McIntyre

Hydroxyurea 127-07-1 Rats:Harlan 

Sprague Dawley Gavage

Dose range finding1

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

Subchronic toxicity6

McIntyre

Isopropylphenyl phosphate 68937-41-7 Rats:Harlan 

Sprague Dawley Feed

Dose range finding1

Neurotoxicity assessment5

Behl

Perfluorooctane sulfonate 1763-23-1 Rats:Harlan 

Sprague Dawley Gavage Dose range finding1 Blystone

Perfluorooctanoic acid 335-67-1

Rats:Harlan 

Sprague Dawley

Mice: CD-1 Reg.

[Crl:CD1(ICR)]

Gavage Dose range finding1 Blystone

Sulfolane 126-33-0 Rats: Harlan 

Sprague Dawley

Drinking 

water

Dose range finding1

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

Subchronic toxicity6

Blystone

Triphenyl phosphate 115-86-6 Rats:Harlan 

Sprague Dawley Feed

Dose range finding1

Neurotoxicity assessment5

Behl

Valerian (Valeriana oficinalis L.) 

root extract (0.8%) 8057-49-6 Rats: Harlan Sprague Dawley Gavage Dose range finding1 DeVito

Wyeth 14,643 50892-23-4

Rats: Harlan 

Sprague Dawley

Mice: CD-1 Reg.

[Crl:CD1(ICR)]

Gavage Dose range finding1 Blystone

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 Neurotoxicity assessment: to study adverse effects in the structure or function of the central or peripheral nervous system.

6	 Subchronic toxicity: 90-day study of adverse effects in the exposed rodent.
36 National Toxicology Program Annual Report FY 2013

iv. Toxicology/Carcinogenicity Studies

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

to address specific deficiencies in the toxicology database for the chemical. Toxicology/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 of these study types 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 the toxicity studies that were ongoing, initiated, and completed during FY 

2013. Chronic toxicology/carcinogenicity studies ongoing and initiated in FY 2013 are listed in Table 18 and 

Table 19.

Table 15. Prechronic Ongoing Toxicology/Carcinogenicity Studies During FY 2013

Study CASRN Species/Strain Study Route Length Study 

Scientist

Bisphenol A 80-05-7 Rats:Harlan Sprague Dawley Gavage 4 days DeVito

Cell phone radiation (CDMA) N/A Rats:Harlan Sprague Dawley

Mice:B6C3F1

Whole body

exposure

5 days

49 days

28 days

Wyde

Cell phone radiation (GSM) N/A Rats:Harlan Sprague Dawley

Mice:B6C3F1

Whole body

exposure

5 days

49 days

28 days

Wyde

1-Butyl-3-methylimidazolium 

chloride (Ionic liquid) 79917-90-1 Rats:Harlan Sprague-DawleyMice:B6C3F1 Drinking water 90 days Ryan

1-Butyl-1-methylpyrrolidinium 

chloride (Ionic liquid) 479500-35-1 Rats:Harlan Sprague-Dawley Mice:B6C3F1 Drinking water 90 days Ryan

n-Butylpyridinium chloride 

(Ionic liquid) 1124-64-7 Rats:Harlan Sprague-Dawley Mice:B6C3F1 Drinking water 90 days Ryan

1-Ethyl-3-methylimidazolium 

chloride (Ionic liquid) 65039-09-0 Rats:Harlan Sprague-Dawley Mice:B6C3F1 Drinking water 90 days Ryan

Nanoscale silver 7440-22-4 Rats:Harlan Sprague Dawley 

(NCTR) Gavage 13 weeks Boudreau

Pentabromodiphenyl oxide

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

Tetrabromobisphenol A 79-94-7 Rats:Wistar Han Gavage 13 weeks Dunnick
5. Testing and Research 37

Table 16. Prechronic Toxicology/Carcinogenicity Studies Initiated During FY 2013

Study CASRN Species/Strain Study Route Length Study 

Scientist

1-Butyl-3-

methylimidazolium chloride 

(ionic liquid)

79917-90-1 Rats:Harlan Sprague Dawley 

Mice:B6C3F1 Drinking water 90 days Ryan

1-Butyl-1-

methylpyrrolidinium 

chloride (ionic liquid)

479500-35-1 Rats:Harlan Sprague Dawley 

Mice:B6C3F1 Drinking water 90 days Ryan

n-Butylpyridinium chloride 

(ionic liquid) 1124-64-7 Rats:Harlan Sprague Dawley Mice:B6C3F1 Drinking water 90 days Ryan

1-Ethyl-3-

methylimidazolium chloride 

(ionic liquid)

65039-09-0 Rats:Harlan Sprague Dawley 

Mice:B6C3F1 Drinking water 90 days Ryan

Pentabromodiphenyl oxide

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

Tetrabromobisphenol A 79-94-7 Rats:Wistar Han Gavage 13 weeks Dunnick

Table 17. Completed Prechronic Toxicology/Carcinogenicity Studies During FY 2013

Study CASRN Species/Strain Study Route Length Study 

Scientist

Bisphenol A 80-05-7 Rats:Sprague Dawley (NCTR) Gavage 90 days Delclos

Black cohosh 84776-26-1 Rats:Harlan Sprague Dawley Gavage 14 days Blystone
38 National Toxicology Program Annual Report FY 2013

Table 18. Chronic Toxicity/Carcinogenicity Studies Ongoing During FY 2013

Study CASRN Species/Strain Study Route Length Study 

Scientist

Aging cohort study N/A

Mice:129S1/SvImJ

Mice:B6C3F1 (Jackson)

Mice:C3H/HeJ

Mice:C57BL/6J (Jackson)

Mice:CAST/EiJ (M. m. 

castaneus)

Mice:NZO/HiLtJ

Mice:PWK/PhJ

Mice:WSB/EiJ (M. m. 

domesticus)

Mice:A/J

Mice:NOD. B10Sn-H2(b)/J

N/A 2 years Wyde

Antimony trioxide 1309-64-4 Rats:Wistar Han

Mice:B6C3F1/N Inhalation 2 years Stout

Bisphenol A 80-05-7 Rats:Sprague Dawley (NCTR) Gavage 2 years Delclos

Black cohosh 84776-26-1 Rats:Harlan Sprague Dawley 

Mice:B6C3F1/N Gavage 2 years Blystone

Bromodichloroacetic acid 

(water disinfection byproduct) 71133-14-7 Rats:F344/NTacMice:B6C3F1 Drinking water 2 years DeVito

2,3-Butanedione (Diacetyl) 431-03-8 Rats:Wistar Han

Mice:B6C3F1/N Inhalation 2 years Morgan

Cell phone radiation (CDMA) N/A Rats:Harlan Sprague Dawley

Mice:B6C3F1

Whole body

exposure 2 years Wyde

Cell phone radiation (GSM) N/A Rats:Harlan Sprague Dawley 

Mice:B6C3F1

Whole body

exposure 2 years Wyde

p-Chloro-a,a,atrifluorotoluene 98-56-6 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Inhalation 2 years Stout

CIMSTAR 3800 (metal

working fluid) N/A Rats:Wistar HanMice:B6C3F1/N Inhalation 2 years Morgan

Cobalt 7440-48-4 Rats:F344/NTac

Mice:B6C3F1 Inhalation 2 years Behl

Dibutyl phthalate 84-74-2 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Feed 2 years Blystone

Di(2-ethylhexyl) phthalate 117-81-7 Rats:Harlan Sprague Dawley Feed Perinatal 

and 2 years Foster

Furan 110-00-9 Rats:F344 (NCTR) Gavage 2 years Beland

Glycidamide 5694-00-8 Rats:F344 (NCTR)

Mice:B6C3F1/NCTR Drinking water 2 years Beland

Green tea extract N/A Rats:Wistar Han

Mice:B6C3F1 Gavage 2 years Thakur

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats:Harlan Sprague DawleyMice:B6C3F1/N Feed 2 years McIntyre
5. Testing and Research 39

Study CASRN Species/Strain Study Route Length Study 

Scientist

Indole-3-carbinol 700-06-1 Rats:Harlan Sprague Dawley

Mice:B6C3F1 Gavage 2 years Wyde

Insertional mutagenesis - 

definitive vector study N/A Mice:C57BL/6 Intravenous 14 months Germolec

Pentabromodiphenyl oxide

(technical) (DE 71) 32534-81-9 Rats:Wistar HanMice:B6C3F1 Gavage 2 years Dunnick

Perfluorooctanoic acid 335-67-1 Rats:Harlan Sprague Dawley Feed 2 years Blystone

Resveratrol 501-36-0 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage 2 years Germolec

Sodium tungstate, dihydrate 10213-10-2 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Drinking water 2 years Behl

Tetrabromobisphenol A 79-94-7 Rats:Wistar Han

Mice:B6C3F1/N Gavage 2 years Dunnick

Triclosan 3380-34-5 Mice:B6C3F1/N Dermal 2 years Fang

Trim VX (metal working fluid) N/A Rats:Wistar Han

Mice:B6C3F1/N Inhalation 2 years Morgan

Tris(2-chloroisopropyl)

phosphate 13674-84-5 Rats:Harlan Sprague DawleyMice:B6C3F1/N Feed 2 years Stout

Vinylidene chloride 75-35-4 Rats:F344/N

Mice:B6C3F1 Inhalation 2 years Wyde

Zinc carbonate, basic 5263-02-5 Rats:Harlan Sprague Dawley Feed 2 years Wyde

Table 19. Chronic Toxicity/Carcinogenicity Studies Initiated During FY 2013

Study CASRN Species/Strain Study Route Length Study 

Scientist

Triclosan 3380-34-5 Mice:B6C3F1/N Dermal 2 years Fang

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 that 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.
40 National Toxicology Program Annual Report FY 2013

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 possible with traditional toxicology parameters. Evaluating

patterns of gene expression may provide more than a link between genetics and morphology, because it is

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

toxicants. Some of the FY 2013 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. Planned or ongoing NTP toxicogenomic studies are listed

in Table 20. More information can be found at http://ntp.niehs.nih.gov/go/20358. 

Table 20. Toxicogenomic Studies Planned or Ongoing

Chemical CASRN

Species/

Strain/ 

Cell Line

Study 

Route

Study Length/Test 

Type (Platform)

Study 

Scientist

Aflatoxin B1 1162-65-8 Rats:F344/N Feed

90 days/Toxicity

NextGen sequencing

RNAseq (Illumina)

Merrick

Arsenite 7784-46-5 Human:Prostate 

(RWPE-1 cells)

Cell 

culture

30 weeks/NextGen 

sequencing

DNAseq

RNAseq (Illumina)

Merrick

Black cohosh 84776-26-1 Mice:B6C3F1/N Gavage 90 days/Toxicity 

Microarray (Affymetrix)

Cora

tert-Butylphenyl diphenyl 

phosphate 56803-37-3 Rats:Harlan Sprague Dawley Gavage 90 days/Toxicity

Microarray (Affymetrix)

Auerbach

2,3-Dibromo-7,8-

dichlorodibenzo-p-dioxin 50585-40-5 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

N,N-Dimethyl-p-toluidine 99-97-8 Rats:F344/N Gavage 5 days, 90 days/Toxicity

Microarray (Affymetrix)

Dunnick

Dong quai (Angelica sinensis) root 

extract 299184-76-2 Mice:B6C3F1/N Gavage 90 days/Toxicity

Microarray (Affymetrix) McIntyre

Estrous cycle study in corn oil 

controls 8001-30-7 Rats:Wistar Han Gavage 90 days/Microarray(Affymetrix) Merrick

2-Ethylhexyl diphenyl phosphate 1241-94-7 Rats:Harlan 

Sprague Dawley Gavage 5 days/Microarray(Affymetrix) Auerbach

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats:Harlan Sprague Dawley Feed 90 days/Toxicity 

Microarray (Affymetrix)

Auerbach
5. Testing and Research 41

Chemical CASRN

Species/

Strain/ 

Cell Line

Study 

Route

Study Length/Test 

Type (Platform)

Study 

Scientist

Isodecyl diphenyl phosphate 29761-21-5 Rats:Harlan 

Sprague Dawley Gavage 90 days/Toxicity

Microarray (Affymetrix)

Auerbach

Isopropylated phenol phosphate 68937-41-7 Rats:Harlan 

Sprague Dawley Gavage 90 days/Toxicity

Microarray (Affymetrix)

Auerbach

1,2,3,7,8-

Pentabromodibenzofuran 107555-93-1 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

2,2’,4,4’,5-Pentabromodiphenyl

ethers 5436-43-1 Rats:Wistar HanMice:B6C3F1 Gavage

GD 6 through 3 weeks/

Toxicity

Microarray (Affymetrix)

Dunnick

Pentabromodiphenyl oxide

(technical) (DE 71) 32534-81-9 Rats:Wistar Han Gavage GD 6 to PND 21/Toxicity

Microarray (Affymetrix)

Dunnick

3,3,4,4,5-Pentachlorobiphenyl 57465-28-8 Rats:Wistar Han Gavage GD 6 to PND 21/Toxicity

Microarray (Affymetrix)

Dunnick

Pentachlorodibenzofuran 57117-41-6 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

2,3,4,7,8-

Pentachlorodibenzofuran 57117-31-4 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

Phenobarbital 50-06-6 Rats:Wistar Han Gavage GD 6 to PND 21/Toxicity

Microarray (Affymetrix)

Dunnick

Tetrabromobisphenol A 79-94-7 Rats:Wistar Han Gavage

90 days/Toxicity

Microarray (uterus and

liver) (Affymetrix)

Dunnick/ 

Merrick

2,3,7,8-Tetrabromodibenzofuran 67733-57-7 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

2,2’4,4’-Tetrabromodiphenyl ether

(DE-47) 5436-43-1 Rats:Wistar Han Gavage GD 6 to PND 21/Toxicity

Microarray (Affymetrix)

Dunnick

2,3,7,8-Tetrachlorodibenzo-pdioxin 1746-01-6 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

2,3,7,8-Tetrachlorodibenzofuran 51207-31-9 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

p-Toluidine 106-49-0 Rats:F344/NTac Gavage 5 days, 90 days/Toxicity

Microarray (Affymetrix)

Dunnick

2,3,7-Tribromodibenzo-p-dioxin 51974-40-4 Mice:B6C3F1 Gavage 3 days/Immunotoxicity

PCR-Array

DeVito

Tricresyl phosphate 1330-78-5 Rats:Harlan 

Sprague Dawley Gavage 5 days /Microarray(Affymetrix)

Auerbach/

Behl

Triphenyl phosphate 115-86-6 Rats:Harlan 

Sprague Dawley Gavage 5 days/Microarray(Affymetrix)

Auerbach/

Behl
42 National Toxicology Program Annual Report FY 2013

vi. Project Review Committee Approved

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

or the internal NIEHS/NTP project review committee, but have not yet started during FY 2013.

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

FY 2013

Study CASRN Species/Strain Study 

Route Test Type/Study Length Study Scientist

N,N-Dimethyl-p-toluidine 99-97-8 Rats:F344/NTac

Mice:B6C3F1/N Gavage Microarray/5 days,13 weeks Morgan

Black cohosh 84776-26-1 Mice:B6C3F1/N Gavage

Evaluation of subchronic

macrocytic mon-regenerative 

anemia

Blystone/ 

Cora

Black cohosh 84776-26-1 Mice:B6C3F1/N Gavage

Toxicogenomic changes 

associated with subchronic

macrocytic non-regenerative 

anemia

Blystone/ 

Cora

Parabens:

Butylparaben

Ethylhexylparaben

Hexylparaben

Nonylparaben

Propylparaben

94-26-8

5153-25-3

1083-27-8

38713-56-3

94-13-3

Rats:Harlan 

Sprague Dawley Gavage

Estrogenic activity 

(uterotrophic) and liver toxicity 

signature

Blystone

p-Toluidine 106-49-0 Rats:F344/NTac Gavage Microarray/5 days Morgan

trans-Resveratrol 501-36-0 Rats:Harlan 

Sprague Dawley Gavage Modified one generation Germolec

Valerian (V. officinalis) N/A Rats:Harlan 

Sprague Dawley Gavage Prechronic toxicityPerinatal dose-range finding DeVito

C. NTP Research

In addition to testing activities, a variety of NTP research projects are underway at NIEHS, NCTR, and NIOSH.

The following NIEHS and 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 Mike Waalkes, 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 2013.
5. Testing and Research 43

Table 22. NIEHS/NTP NTP Laboratory Projects in FY 2013

NTP Labs Project 

[Study Scientist] Objective and/or Project Summary

Development of in vitro models of metal 

carcinogenesis 

[Waalkes]

To develop in vitro cell transformation models with target relevant cells using 

arsenic and cadmium. 

Epigenetics in malignant transformation

[Waalkes, 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 and hematopoietic tumor

induction. 

Formaldehyde-induced transformation of 

human myeloid progenitor cells 

[Waalkes]

To perform a proof of concept study, in vitro formaldehyde induced malignant 

transformation in hematopoietic stem cells. Companion study to the p53 study 

mentioned above.

Indium-tin-oxide and indium compounds

[Morgan] To perform various in vivo inhalation or in vitro toxicity studies.

Japan arsenic poisoning human study; 

Developmental basis of human disease

[Waalkes]

To determine the genomics of response to early life arsenic exposure in 

survivors of a baby food poisoning event in Japan in the mid-1950s from a

population now getting excess cancer typical for arsenic exposure. Human 

proof of the developmental basis of adult disease.

“Metalloestrogens” and uterine/breast

response 

[Dixon, Waalkes, Fenton]

To re-test 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, Waalkes]

(1) To develop in vitro trans-well method with metal particles and 

macrophages in one well and cells of interest (such as lung epithelium) in 

the other, and (2) to define 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 to 

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 

[Waalkes]

To study genes of interest involved with the epigenetics of malignant 

transformation using in vitro human model systems of carcinogenesis. 

MicroRNAs are thought to be a key epigenetic or post-transcriptional gene

express control mechanism.

Stem cells in toxicology and carcinogenesis 

[Waalkes]

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.

Studies on estrogen receptor in vitro 

[Waalkes] To study mammary cells and so called “metalloestrogens.” 

Toxicants and mammary gland development 

[Fenton] 

To determine the effects of different toxicants, including atrazine, on 

mammary gland development in rats and mice. 
44 National Toxicology Program Annual Report FY 2013

Genetic and epigenetic differences between individuals in the human population are proposed to be the

basis for individual susceptibility to environmental stressors. Environmental and drug safety assessments are

currently conducted with a small number of commonly used animal models that have limited genetic diversity

and are 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. NIEHS/NTP Biomolecular Screening Branch Projects in FY 2013

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, and five tissues

(brain, liver, cardiac and skeletal muscle, brown and white fat, epididymal sperm) from

the first generation offspring were collected at the average age mice would start an NTP

subchronic toxicity study and flash-frozen for DNA/RNA isolation and liver sequencing.

Progress in FY 2013 involved completion of liver transcript expression levels, genomic 

DNA sequencing, and DNA methylation mapping after bisulfite sequencing of both

parental strains and the resulting first generation offspring by high throughput sequencing

technologies and intensive computational data analyses. 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 16). 

Setting priorities in occupational toxicological research 

is based upon several sources of information that are

developed and maintained by NIOSH. 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 2013. Packing up the NIOSH mobile lab for a field study
5. Testing and Research 45

Table 24. NIOSH/NTP Research Projects FY 2013

NIOSH/NTP Projects

[Project Officer] 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 efforts will focus on completing the Longitudinal 

Investigation of Fertility and the Environment (LIFE) project, a collaborative effort

between NIOSH and the NIH Eunice Kennedy Shriver National Institute of Child Health

and Human Development. This work includes development of new biomarkers to

include in the male reproductive health profile.

Immunochemical biological

monitoring for occupational 

exposure and disease 

[Striley]

To evaluate industrial chemicals with known acute and chronic toxicities, which present 

a significant exposure risk for workers. Biological monitoring can 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. Concurrent with development of exposure assessment methods, this 

project will identify and develop new multiplex immunochemical methods to evaluate

biomarkers of occupational illness or subclinical signs of occupational illness. Biological

monitoring, through the validation and development of new methods, is a powerful tool 

for reducing risk and creating safer work practices.

Ultraviolet native fluorescencebased monitor for workplace

exposures 

[Snawder]

To develop and evaluate a readily adaptable, next generation, direct reading, personal

monitors 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 semi-volatile workplace chemicals will be helpful in rapidly assessing

chemical exposure and will result in more realistic occupational exposure assessments 

and allow for rapid interventions leading to reduced worker exposures and thus 

preventing occupational illness and disease.

Workplace exposure,

inflammation, and cardiovascular

toxicity 

[Erdely]

To investigate the role of ultrafine/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/nanosize particulates

and the development of cardiovascular diseases. Planned studies will help to 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 multi-walled

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 multi-walled carbon nanotubes have been raised

due to their high aspect ratio (length/diameter), nanoscale diameter, fiber like-shape,

durability and biopersistence. Increasing evidence has indicated potential pulmonary

health hazards associated with pulmonary exposure to multi-walled carbon nanotubes,

including inflammation, damage, fibrosis, and potential carcinogenesis. Nevertheless,

the mechanisms underlying these adverse multi-walled 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 multi-walled carbon nanotubes and carbon nanofibers. A study

of biomarkers of early pulmonary, cardiovascular, and carcinogenic effect will be carried

out among workers at these facilities.
46 National Toxicology Program Annual Report FY 2013

NIOSH/NTP Projects

[Project Officer] Objective and/or Summary

Mortality, cancer incidence and

biomarker studies

[Ruder]

To elucidate exposure-outcome associations, especially dose-response relations, 

for risk assessment and to examine relationships between biomarkers (of exposure,

susceptibility, and oncogene expression) and health effects.

Environmental Monitoring

Analytical research and 

development infrastructure

[Streicher]

To provide for the 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 being investigated as part of the

NIOSH/NTP exposure assessment interagency agreement are developed in this project.

Diacetyl exposure assessment 

[Streicher]

To develop and evaluate sampling and analytical methods for diacetyl and other 

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. Finally, 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 an indoor positioning system to provide 

remote monitoring of a worker’s exposure to volatile organic chemical (VOC)s with

position and time. The personal monitor continuously samples and analyzes the 

workers breathing zone 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 place 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) indium and indium compounds, (3) diacetyl, (4) 2-methoxy-4-

nitroaniline, and (5) 2’,2’’’-dithiobisbenzanilide. A new study will investigate worker

exposures to BPA. These chemicals have been nominated by various groups to the

NTP. We will identify possible candidate industries, labor unions, workplaces, and uses

and users; determine if there is relevance for occupational health; estimate number of

workers exposed; and perform limited workplace exposure sampling. 

Industry-wide Studies Branch 

research, development, and 

planning 

[Whelan]

To support strategic planning and feasibility studies of high priority/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 nanomaterial, it is important to gain a better understanding of actual workplace

exposures. 
5. Testing and Research 47

NIOSH/NTP Projects

[Project Officer] 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 mechanism associated with exposure. The Immunotoxicology and 

Hazard Identification Lab will achieve this goal through both individual projects and

collaborations. This research will contribute to increased identification of immunological

hazards encountered in the workplace. Further evaluation of these compounds will 

allow for better risk assessment which will ultimately establish occupational exposure

limits.

Airway fungal exposure and 

allergic sensitization in mice

[Green]

To compare the immunological health effects of lung exposure to fungal spores or to 

hyphal fragment preparations. Agriculture as well as construction and remediation 

workers are exposed to elevated levels of fungi and can experience rhinitis, respiratory 

allergic symptoms and/or asthma as a result of their exposure. This project has been

completed and had two major areas of study: (1) to determine the health effects

following aspiration of hyphal fragments from Stachybotrys chartarum and Alternaria 

alternata in the absence of intact spores in mice; and (2) to compare the ability of

aspirated spores or hyphal suspensions from Aspergillus spp, S. chartarum, and A. 

alternata to exacerbate respiratory allergy to ovalbumin.

Identification of occupational

allergens 

[Beezhold]

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. This project is intended to

address these concerns through the development of improved techniques for the 

detection of such immune reactions before adverse clinical outcomes occur, and

through the development of improved techniques for the detection and identification of

inciting occupational agents. The 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

Genetics in occupational diseases 

[Yucesoy]

To investigate susceptibility gene variants, which contribute to the development and

severity of occupational irritant contact dermatitis and asthma, using high-density and 

high throughput genotyping platforms. Previous and on-going studies in our laboratory

showed that cytokine polymorphisms have a major influence on silicosis, dementia, and

accelerated decline in lung function and vaccine efficacy. Understanding the genetic

contribution to the development, progression and outcomes of complex occupational

diseases will help improve the accuracy of risk assessment and improve safe exposure 

levels for genetically susceptible groups in the workforce.

Genetic fingerprint of mouse lung

cancer 

[Reynolds]

To determine if there are different carcinogen-specific chromosomal (genetic) markers

in spontaneously-occurring and chemically-induced mouse lung adenocarcinomas 

using in vitro and in vivo animal models. Mice were exposed by inhalation to

vanadium pentoxide, nickel oxide or cumene (a benzene derivative). Workers in the

construction and manufacturing sectors are exposed to these compounds. We are

also planning to analyze mouse lung tumors induced by single-wall carbon nanotubes.

If these experiments are successful we plan to extend these findings to tumors from

occupationally-exposed human populations. Results from these studies will be used to

establish biomarkers for early detection and therapeutic intervention of lung cancer in

worker populations.
48 National Toxicology Program Annual Report FY 2013

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-NIOSH interagency agreement supports these 

projects. Current efforts listed in Table 25 address worker exposures to welding fumes, nanosized materials, 

food flavorings, bisphenol A, indium compounds, and other industrial chemicals.

Table 25. NIEHS-NIOSH Interagency Agreement on Occupationally Relevant Exposures

in FY 2013

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, NC and Washington,

D.C.

Assess the feasibility of an

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/or unions involved in welding operations where the potential for substantial

manganese exposure exists, for exposure assessments; (2) to develop methods to identify 

specific manganese compounds, different valence states, and potential solubility contained

within various welding fumes matrices; and (3) to 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) to document high-exposure activities and

processes in the flavored food production industries, (3) to identify work practices and

procedures that affect exposure, (4) to document engineering controls, and (5) to field

test novel techniques for both gravimetric and volatile sampling.

Assessment of use of indium and 

indium compounds in the workplace

[Hines]

(1) To contact and visit companies to determine indium materials being used, jobs and

processes with potential indium exposure, exposure controls, and indium use trends 

and (2) to conduct preliminary sampling for indium, if possible.

Exposure assessment of 

engineered nanoparticles

[Geraci]

(1) To identify workplaces engaged in the synthesis, manufacture, and use of 

engineered nanomaterials and (2) to characterize workplace exposure to selected 

engineered nanoparticles.

Exposure assessment of 1-chloro4-(trifluoromethyl) benzene

(PCBTF)

[Harper]

(1) To identify worker populations at elevated risk of inhalation and surface exposure to 

PCBTF during manufacturing processes, (2) to update a previously published analytical

method for quantitatively assessing PCBTF airborne vapors and surface exposures to

allow the use of capillary column chromatography, and (3) to characterize industry-wide 

occupational exposures, including total number of workers, and evaluate patterns of

exposure to PCBTF.

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.

Exposure characterization and 

reproductive health of men 

working with bisphenol A (BPA) in

the United States 

[Hines]

(1) To determine BPA usage in industry, such as which industries and jobs use BPA and

which tasks are associated with exposure; (2) to develop air and wipe sampling methods 

for BPA using liquid chromatography mass spectrometry and liquid chromatography 

with ultraviolet detection; (3) to assess exposure to BPA among workers in these 

industries through air, wipe, and urine sample collection. If worker exposures are 

confirmed (4) to assess the reproductive health of men exposed to BPA in the

workplace, and (5) to determine if there is a relationship between occupational exposure

to BPA and reproductive health.
5. Testing and Research 49

Study 

[Study Scientist] Objective and/or Rationale

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

United States 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) evaluating higher-flow, respirable cyclones to assess health-relevant exposures.

iii. Immunotoxicology Research

The NIEHS-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.

Table 26. NIEHS-NIOSH Interagency Agreement on Immunotoxicology Studies in FY 2013

Study 

[Study Scientist] Objective and/or Rationale

NIEHS agricultural pesticide 

study 

[Green]

To support the NIEHS Agricultural Health Study, screening of 1,454 farmers serum for total 

immunoglobulin E (IgE) and mold mix specific IgE using the Phadia Immunocap assay has

been completed. Those farmers who were mold-mix-positive have recently been tested

against a 10-mold-specific IgE panel (n = 126). This component of the study is finished

and the compiled data has been forwarded to NIEHS. In further support of the NIEHS

Agricultural Health Study, the laboratory has been requested to screen an additional 640

farmers’ sera samples for total and specific IgEs. Total IgE and mold-mix-specific IgE will

be determined using Phadia ImmunoCap assays as previously described. The data derived

from this analysis will be sent to NIEHS and prepared for publication in an allergy journal.

A marker for Aspergillus terreus

exposure 

[Green]

To develop new and improved methods for detecting fungal exposure. In this project

the emerging opportunistic fungal pathogen, Aspergillus terreus, has been used as a

model fungal species. Terrelysin, a cytolysin and potential biomarker of fungal infection,

was characterized and a recombinant protein produced. The recombinant terrelysin was

then used to immunize mice to produce terrelysin specific monoclonal antibodies. Seven

specific monoclonal antibodies were produced and used to characterize the production of

terrelysin. Monoclonal antibodies binding sites are currently being determined in epitope

mapping experiments. Several manuscripts have been recently published that report these

findings. The development of a sensitive immunoassay that can be used in the serological

detection of this biomarker is anticipated.

Animal model for airway 

exposure to dry fungal aerosols

[Green]

To develop a murine model of dry fungal exposure to better mimic natural human

exposures to fungi. In collaboration with aerosol scientists at NIOSH, an acoustical

generation system has been developed. In preliminary experiments, histopathology and

bronchoalveolar lavage fluid analysis demonstrated pulmonary deposition of conidia

following dry fungal bioaerosol exposure. The system has been optimized for the

generation of fungal spores. Once the system has been fully characterized, experiments

are planned to further characterize the immune responses associated with various fungal 

species that frequently colonize water-damaged buildings or are occupationally relevant.
50 National Toxicology Program Annual Report FY 2013

Study 

[Study Scientist] Objective and/or Rationale

Characterization of fungal 

diversity in the indoor built

environment 

[Green]

To investigate and characterize the diversity of fungal bioaerosols in the indoor built

environment using large-scale ribosomal RNA sequencing in collaboration with the Kansas

City Safe and Healthy Homes Partnership Project. 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 this analysis have been compiled and provide

detailed insight into the diversity of previously overlooked fungal bioaerosol sources in

the Kansas City Environment. Future studies in collaboration with Assured Bio, Inc., are

planned for 2013. These studies aim to further evaluate indoor environments using large 

scale ribosomal RNA approaches from samples collected in contaminated and noncontaminated Atlanta, Georgia, homes. 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.

The role of genetic variation in 

environmental and occupational 

diseases: irritant contact 

dermatitis

[Yucesoy]

(1) To investigate whether the 24-hour irritant patch test is predictive of occupational 

hand dermatitis caused by high exposure to hand washing in health care workers, and

(2) to investigate association between genetic variations in specific candidate genes (with

emphasis on variants of cytokines, major histocompatibility complex region, antioxidant

enzyme genes and genes related to skin barrier integrity) and irritation threshold levels of

the subjects with development of irritant contact dermatitis. This study is in collaboration

with Case Western Reserve and West Virginia Universities.

The role of genetic variation in 

environmental and occupational 

diseases: allergic contact 

dermatitis 

[Yucesoy]

(1) To investigate genetic factors in individuals predisposed to develop allergic contact 

dermatitis, specifically induced by nickel and (2) to investigate genetic factors involved in 

the development of allergic contact dermatitis in individuals sensitized to weak allergens, 

individuals sensitized to allergens that require metabolism in the skin, and individuals who react

to more than three allergens of the standard screening series. This study is in collaboration

with Case Western Reserve University and Dartmouth-Hitchcock Medical Center.

The role of genetic variation in 

environmental and occupational 

diseases: occupational asthma

[Yucesoy]

(1) To investigate whether genetic variations in specific candidate genes (such as cytokine,

major histocompatibility complex region, antioxidant enzyme genes) are associated with

asthma induced by diisocyanates, and (2) to investigate potential associations between

genetic variations in candidate genes and occupational asthma caused by low molecular

weight agents. This project is in collaboration with the Universities of Montreal and Cincinnati.

The role of genetic variation in 

environmental and occupational 

diseases: chronic beryllium

disease

[Yucesoy]

To investigate the contribution of genetic variations in the major histocompatibility complex

region to the development of beryllium sensitization and chronic beryllium disease. This

study is in collaboration with National Jewish Medical and Research Center.

Investigations into health 

effects caused by exposure

to indoor air reaction products 

(supportive animal studies) 

[Wells, Anderson]

(1) To identify and measure the reaction products of gas-phase compounds present in the 

indoor environment, especially oxygenated organics; (2) to further develop and validate 

a novel in vitro exposure methods utilizing realistic indoor chemistry scenarios to expose 

cells and tissues to these indoor air reaction products; (3) to complete both in vitro and

in vivo assays to assess adverse health effects caused by indoor air reaction products;

and (4) to further investigate the role of structurally similar indoor air chemicals present in 

mixtures in the indoor environment.

Alternative methods for 

chemical allergen identification

and assessment: use of 

immunochemical techniques 

for investigation of diisocyanate 

allergic diseases

[Siegel]

(1) To develop an amine based probe for kinetic assessment of chemical binding

(haptenation) that will complement the thiol based probe previously reported by our

laboratory, (2) to assess potential for inclusion of a metabolic activation step in our kinetic

based assays for identification of prohaptens, (3) to expand the compilation of chemical

allergens assessed by kinetic electrophilic reactivity analyses for comparison to reported

allergenic potencies (murine local lymph node assay exposure concentration values), and 

(4) to use in-house developed monoclonal antibodies against diisocyanate bound proteins

to identify potential haptenation targets.
5. Testing and Research 51

Study 

[Study Scientist] Objective and/or Rationale

Analysis of mycotoxins in dust 

samples from a water-damaged 

building

[Park]

(1) To develop a cost-effective method using a state-of-the art technology, ultraperformance liquid chromatography-tandem mass spectrometry, for simultaneously 

analyzing multiple fungal toxins in environmental samples, and (2) to perform experiments 

to examine stability of the mycotoxins in floor dust stored in different temperature conditions

at different points of time using the developed method. The developed method will be

applied to quantify fungal toxins in floor dust samples that will be collected from a funded

school study in 2014, and samples previously collected from water damaged buildings for

which we have existing data on fungal and bacterial components as well as occupants’

health. The ultra-performance liquid chromatography-tandem mass spectrometry 

technology does not require significant sample preparation, and thus the technology

enables us to develop rapid and robust methods to screen multiple mycotoxins in samples.

The project provides us with an opportunity to examine potential roles of exposure to fungal

toxins on occupants’ health in water-damaged buildings.

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. These NCTR-NTP studies, funded by NCTR voluntary allocations, are listed in Table 27. Table 28 lists 

projects that are funded through FDA and NTP interagency agreements.

Table 27. NCTR/NTP Projects in FY 2013

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Biochemical and Molecular Basis of Toxicology

Biological based dose-response

modeling for the thyroid axis in the 

fetus and neonate

[Fisher]

(1) To create biological based dose-response models for the hypothalamicpituitary-thyroid axis in the developing rat and human as a function of iodide status, 

(2) to interface the these models with physiologically-based pharmacokinetic 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) to 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.

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) to determine whether or not interstrain-specific susceptibility of mice

to nonalcoholic steatohepatitis-induced hepatocarcinogenesis is associated with 

differences in individual hepatic epigenetic phenotypes, (3) to determine the role 

of epigenetic dysregulation in the etiology and pathogenesis of nonalcoholic 

steatohepatitis-induced hepatocarcinogenesis in mice induced by tamoxifen

administration, and (4) to determine whether or not aberrant epigenetic markers

can be used as targets for prevention of nonalcoholic steatohepatitis-induced

hepatocarcinogenesis in mice.
52 National Toxicology Program Annual Report FY 2013

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

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 to determine whether the expression of 

ATP binding cassette transporters is correlated with clinical aggressiveness of the

tumor, (2) to 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) to assess the epigenetic regulation of ATP

binding cassette transporters in pancreatic cancer.

Mechanism of tumorigenic pyrrolizidine

alkaloids and development of liquid 

chromatography-electrospray 

ionization/multi-stage mass 

spectrometry (LC/ESI/MS/MS)

methodology for detection and 

quantification of pyrrolizidine alkaloids

[Fu]

(1) To validate the proposed mechanism by which pyrrolizidine alkaloids induce

tumors in rodents, (2) to develop an LC/ESI/MS/MS method for detection and

quantification of dehaloperoxidase-derived DNA adducts in rodents, (3) to develop

an LC/ESI/MS/MS method for detection and quantification of Genotoxic pyrrolizidine

alkaloids in herbal plants and herbal dietary supplements, and (4) to develop an LC/

ESI/MS/MS method for detection and quantification of dehaloperoxidase-derived

hemoglobin adducts in rodents.

Sequencing Quality Control Project

[Su]

To assess the technical performance of different next-generation sequencing 

technologies and various bioinformatics strategies for handling and analyzing

the massive sequence data sets by using the reference RNA samples previously

established by the MicroArray Quality Control project and (2) to profile RNA samples

isolated from cells with or without treatment by nanoparticles and toxicants of known

mechanisms of toxicity to further evaluate their performance in assessing the 

safety and toxicity of FDA-regulated products by using next-generation sequencing

technologies. Next-generation sequencing is expected to revolutionize transcriptome 

studies because of its claimed higher sensitivity and specificity, the capability of

simultaneously detecting individual exons and alternative splicing, and the possibilities

of genome-wide quantification (through single-molecule sequencing).This collaborative

project is a natural extension of the FDA-led MicroArray Quality Control project.

Study of drug-induced liver toxicity 

using state-of-the-art in vitro liver 

models including primary rat and 

mouse hepatocytes and stem cells

[Guo]

To obtain signature gene and protein expression patterns of each cell type for

comparison to toxin-induced changes. In addition, the contribution of each cell type

to overall liver toxicity from agent exposure can be determined once these isolated

cell types are available reliably.

Dose-response genotoxicity of 

ethylmethane sulfonate in mice using 

the 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) to measure 

genotoxicity using a design to detect the maximum responses, (3) to measure the 

effects of ethylmethane sulfonate exposure in neonatal as well as adult animals, and 

(4) to measure genotoxicity in the major target tissues for ethylmethane sulfonate

carcinogenicity.

Neurotoxicology

Evaluation and characterization 

of blood-brain barrier pathology

in 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine-probenecid-induced

Parkinson’s disease-like conditions 

in a mouse model and its potential 

amelioration by endoplasmic reticulum

stress reducers (molecular chaperones) 

and other putative anti-Parkinson 

therapeutics

[Sarkar]

(1) To characterize and evaluate the role of key neurovascular elements such as 

pericytes, astrocytes, and endothelial cells and basement membrane proteins in the

expression of Parkinson’s disease-like pathology; and (2) to determine the ability

of endoplasmic reticulum stress-reducers and related heat shock protein inhibitors

to alter the expression of Parkinson’s disease-like pathology and flavonoids

(Mangiferin and Morin), antioxidants (N-acetyl cysteine and acetyl-l-carnitine),

superoxide scavengers (such as tetramethylpyrazine), MAP kinase inhibitors (such

as SB239063), synthetic dyes (Azure A and Thionin), and metal chelators (clioquinol 

and VK-28) to provide neuroprotection against Parkinson’s disease-like pathology.
5. Testing and Research 53

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Developing more complete genomic 

and histological evaluations of 

vascular damage in the brain

meninges and choroid plexus after 

neurotoxic insult

[Bowyer]

(1) To determine whether these two regions of meninges associated vasculatures 

differ with respect to gene expression, (2) to determine gene expression patterns 

present in the larger arteries compared to the arterioles and the meningeal layers 

(pia and some arachnoid membrane), (3) to evaluate the changes in meninges

associated vasculatures during neonatal and juvenile periods, (4) to determine

whether the gene expression profiles in the various components of meninges

associated vasculatures respond differently to neurotoxic insults, and (5) to 

determine the possibilities of developing bioinformatic methods for predicting the

relative genomic contributions of the various meninges associated vasculatures

components (large arteries, arterioles, and meninges) to the overall genomic 

response of the entire meninges associated vasculatures under environmentally 

induced hyperthermia, amphetamine exposure, or control conditions.

Methods development for highresolution dedicated positron emission 

tomography to rodent neuroplasticity 

and toxicity during development 

[Wang]

To use high-resolution dedicated positron emission tomograph to screen and 

evaluate in vitro and in vivo measurements from a broad range of pathophysiological

or pharmacological parameters using specific tracers in the developing rat and three

different age groups of developing rats (gestational day 18 female rats, post-natal 

day 7 rat pups, and post-natal day 35 rats).

Assessment of gaseous anesthetics in 

the developing nonhuman primate 

[Wang]

(1) To evaluate whether prolonged exposure to gaseous anesthetics nitrous oxide or 

isoflurane increases neuronal cell death, and to determine if combinations of nitrous

oxide and isoflurane have additive effects in the developing nonhuman primate;

(2) to determine if a relatively high dose or prolonged exposure of the developing 

nonhuman primates to nitrous oxide or isoflurane alone, or their combination will

induce long-term behavioral deficits or pathological changes; (3) to determine

the effectiveness of using noninvasive imaging techniques (high resolution 

dedicated positron emission tomography) and magnetic resonance imaging to 

monitor pathological changes in this model; and (4) to identify potential, underlying 

mechanisms that could link alteration of mitochondrial function and elevation of 

reactive oxygen species to gaseous anesthetic-induced neuronal cell death. 

In vitro assay to predict developmental 

neurotoxicity of pediatric anesthetics 

[Wang]

To use rodent in vitro organotypic and primary culture models: (1) to examine the 

toxicity of anesthetics including propofol (gamma-amino butyric acid (GABA)-A

agonist), baclofen (GABA-B agonist), diazepam (GABA-A agonist), pentobarbital

(GABA-A agonist and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

receptor antagonist), etomidate (GABA-A agonist), sevoflurane (N-methyl-Daspartate antagonist and GABA agonist), fentanyl (opiate agonist) and anesthetic 

combinations commonly used in pediatric surgical procedures; (2) to determine

the utility of in vitro culture systems to predict in vivo outcomes in subsequent

studies; (3) to determine the dose and time-course over which the potential 

neurotoxic effects of anesthetics are expressed in the developing brain; (4) to

determine effective ways to protect against anesthetic-induced developmental 

neurotoxicity that have potential clinical utility; (5) to identify mechanisms that link 

altered N-methyl-D-aspartate receptor function and/or elevation of reactive oxygen 

species to anesthetic-induced neuro-apoptosis; and (6) to identify biomarkers such

as genomic pathway signatures and determine their validity for predicting in vitro 

outcomes of pediatric anesthetic exposure.

Methylphenidate (ritalin) exposure

during pregnancy: assessment of 

neurotoxicity in offspring 

[Ferguson]

To determine the neurobehavioral toxicity associated with pre- and early postnatal

treatment with methylphenidate in rats using a range of behaviors at preweaning,

adolescent, and adult ages.
54 National Toxicology Program Annual Report FY 2013

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Effect of pediatric anesthetics on 

zebrafish embryos: neurotoxicity

verses gene expression changes and 

neuronal kinase cyclin-dependant 

kinase 5 as a mediator of toxicity 

[Kanungo]

(1) To determine whether ketamine has neurotoxic effects (on neurogenesis and 

axonogenesis) in zebrafish and (2) to determine if the window of such effects

varies between early and late differentiating neurons (sensory and motor neurons,

respectively).

Methods development for toxicity

assays using the zebrafish embryo as

a model system: whole animal high 

throughput assays for chemical testing 

[Kanungo]

(1) To use the established high throughput assay system using zebrafish embryos

for morphological and behavioral endpoints of toxicity and subtle organ-specific

toxicities to study the effect of methamphetamine on zebrafish embryos, especially

relating to sensory and motor neuron development; (2) to determine if carbon

nanotubes pass through the blood brain barrier in zebrafish embryos, and if so, do

these nanomaterials generate reactive oxygen species, deplete dopamine and its 

metabolites (dihydroxyphenylacetic acid and homovanillic acid), and alter markers

of oxidative stress; and (3) to determine the effect of nicotine on zebrafish embryos,

especially relating to sensory and motor neuron development and the mechanism of 

action.

Long-term consequences of 

neonatal ketamine anesthesia in 

rhesus monkeys: extended cognitive 

assessments 

[Paule]

(1) To continue monitoring the cognitive capabilities of rhesus monkey subjects that

were exposed to a single, 24-hour duration of ketamine-induced anesthesia during 

the first week postpartum; and (2) to extend the functional domains that are being

assessed beyond learning, the ability to perform simple visual discriminations,

motivation, and speed of psychomotor processing, to include performance of a 

temporal discrimination task (timing task), a counting task, and reversal learning 

tasks (cognitive flexibility).

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) to characterize

acute effects of psychomodulators (amphetamine, ketamine) in normal rats using 

1H-magnetic resonance spectroscopy S (preclinical biomarker development).

Nanotoxicology

Antimicrobial properties of zinc oxide

and titanium dioxide nanoparticles 

against multidrug-resistant 

Staphylococcus and Enterococcus

spp. and their cytotoxic and genotoxic 

potential in bacteria and normal

human epidermal keratinocytes and 

primary intestinal cells

[Khan]

(1) To study the mechanism of antimicrobial properties of titanium dioxide and

zinc oxide nanoparticles in multidrug-resistant Staphylococcus and Enterococcus 

spp., (2) to study synergy between nanoparticles and antibiotics, (3) to evaluate

the cytotoxic and genotoxic potential of nanoparticles in bacterial and normal

human epidermal keratinocytes and primary intestinal cell lines, and (4) to study 

the transcriptomic gene expression in normal human epidermal keratinocytes and 

intestinal cell lines. 

Immunological effects of nanoparticles 

on induction of pro-inflammatory

responses to Candida albicans by

vaginal epithelial cells

[Wagner]

(1) To measure the effects of graphene and poly(lactic-co-glycolic acid) 

nanoparticles on messenger RNA and protein expression of inflammatory cytokines

and signal transduction proteins by vaginal epithelial cells stimulated with C. 

albicans and (2) to measure oxidative effects and DNA damage in vaginal epithelial 

cells by nanoparticles.

Determination of cytotoxicity and 

genotoxicity of nanomaterials of 

interest to the FDA and mechanism of 

action

[Fu]

To develop a set of cell-free and cell-based in vitro tests that can be used to rapidly

identify nanomaterials of interest to the FDA that elicit oxidative damage and (2) 

to determine if in the presence of nano-metal materials, endogenous and dietary 

antioxidants can display pro-oxidative activity.
5. Testing and Research 55

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Development of methods for 

determining nanoparticle penetration/

permeation into vaginal mucosal 

tissue

[Zhang] 

(1) To develop/adapt methods to deliver nanoscale material into rat vaginal tract 

and (2) to develop the analytical methods to quantify vaginal mucosal penetration of 

select nanomaterials following intravaginal lavage. 

Neurotoxicity assessment of silver 

nanoparticles in PC-12 cells and in 

rats 

[Ali]

(1) To evaluate the neurotoxicity of different sizes of silver nanoparticles using 

cultured PC-12 cells; (2) to determine if in vitro exposure to silver nanoparticles 

selectively induces specific genomic changes in cultured PC-12 cells using

microarrays; (3) to determine if single or multiple doses of silver nanoparticles 

produce reactive oxygen species, alterations in lipid peroxidation, or changes in 

antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) or 

glutathione levels in the rat brain; (4) to determine if single or multiple doses of silver

nanoparticles induce specific genomic changes (microarrays), neurotransmitter

concentrations, or 3-nitrotyrosine formation in the rat brain; and (5) to determine if

multiple doses of silver nanoparticles produce morphological alterations in bloodbrain barrier, brain, or other visceral organs of the rat.

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 the FDA (Salmonella

Ames test, mouse lymphoma assay, and in vivo mouse micronucleus assay); and (2) 

to 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

and/or changes in antioxidant enzyme activity (catalase, superoxide dismutase, 

glutathione peroxidase) and glutathione levels in mice and rat brains; and (5)

selective pattern of deposition and damage produces in different regions of rat and 

mice brain using in vivo magnetic resonance imaging.

Study of nanoparticles migration 

from food-contact nanomaterials: 

characterization and quantification of

silver nanoparticles in stimulants 

[Trbojevich]

(1) To quantify the migration of nanoparticles from nanocomposites used in foodcontact material and (2) to characterize and quantify silver nanoparticles in food 

stimulants.

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) to explore the possible mechanisms

underlying genotoxicity of engineered nanomaterials by conducting genomic

analysis, (3) to identify potential improvements to the assay and general strategies 

for evaluating nanomaterials, and (4) to 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.
56 National Toxicology Program Annual Report FY 2013

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Assessment of size and shape 

dependent-toxicity of silver 

nanoparticles as measured by

changes in the permeability at the

gastrointestinal surface 

[Khare]

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: (1) determining the effect of nanomaterials on

the permeability of intestinal epithelial cells in vitro and ileal mucosa ex vivo and

(2) measuring 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.

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) to qualify the discovered biomarkers.

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 that, 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) to

compare the Pig-A assay results with the in vivo Comet and micronucleus formation 

assays, and hypoxanthine-guanine phosphoribosyltransferase lymphocyte gene

mutation assays.

Development of predictive 

mitochondrial biomarkers for druginduced cardiotoxicity using a system 

biology approach

[Desai]

(1) To determine doxirubicin-induced changes in plasma troponin T, creatinine

kinase-MB, and cardiolipin levels in rats, and to correlate with non-invasive

measurements of heart rate, heart rate variability, and electrocardiogram; (2)

to determine morphological changes in cardiac mitochondria in left ventricular 

region by electron microscopy; (3) to quantify analyte profiling in the heart using

transcriptional profiling of approximately 906 mitochondria-related genes using

MitoChip (genomics); (4) to conduct protein profiling by 2D-high performance liquid

chromatography/tandem mass spectrometry (proteomics); and (5) to measure 

endogenous metabolites (creatinine, creatine, lactate, Krebs cycle intermediates,

small ketone bodies in plasma) by nuclear magnetic resonance imaging and mass

spectrometry (metabolomics).

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 enhances the sensitivity

and reproducibility of the in vivo alkaline-comet assay for use in preclinicalhazard 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

pre-clinical and clinical predictive 

biomarkers of toxicity

[Desai]

(1) To develop MitoChip for various mammalian species, including rat, non-human

primate, and human; (2) to 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) to characterize species-specific transcriptional profiles to predict risk

of drug toxicity or disease onset in different mammalian species.

Further evaluation of the types of 

genetic events detected by the mouse

lymphoma assay and the role of the 

assay in mechanistically based risk

assessment 

[Moore]

(1) To determine if the L5178Y TK+/- mouse lymphoma assay adequately detects 

both aneuploidy and mitotic recombination, (2) to determine if L5178Y mouse

lymphoma cells have active recombinase functions that lead to a large proportion

of mutants that result from recombinase-mediated rearrangements, and (3) to

determine the fundamental genetic mechanism(s) causing the small and large 

colony thymidine kinase mutant phenotypes.
5. Testing and Research 57

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Validation of recently established gptdelta mice at NCTR 

[Manjanatha]

(1) To dose gpt-delta transgenic mice with low, intermediate, or high doses of 

cyclophosphamide and bleomycin and 100 mg/kg ethylnitrosourea (cumulative

dose) as a positive controls for induction of mutations in the transgenes gpt, red 

and gam genes (spi- selection); (2) to characterize recovered mutants from target 

tissues for generation of gpt and spi mutational spectra; and (3) to analyze red 

blood cells for phosphatidylinositol glycan complementation group A (Pig-A) mutant

frequencies and erythrocytes for micronucleus frequencies. 

Cancer mutations as biomarkers

of cancer risk: human studies with 

implications for personalized medicine 

[Parsons]

(1) To develop the information necessary for the rational use of oncogene 

mutations as quantitative biomarkers of cancer risk; specifically allele-specific

competitive blocker polymerase chain reaction will be used to determine normal

and pathological levels of relevant oncogene mutations in multiple human tissues 

and tumors, (2) to compare the information derived from human tissues with 

data generated in a parallel rodent protocol as an approach for incorporating 

carcinogenesis-relevant data into the rodent for human extrapolation necessary 

in cancer risk assessment, (3) to validate a streamlined allele-specific competitive

blocker polymerase chain reaction methodology and develop the methodology

necessary to measure oncogene mutation frequency in cell-free DNA isolated 

from plasma, and (4) to communicate to the regulatory risk-assessment 

community the regulatory significance of data on tumor-associated mutations in

rodents and humans.

Development of a new safety 

evaluation method using microRNA 

expression analysis as a biomarker for

detecting carcinogens 

[Chen]

(1) To determine microRNA expression profiles of the tumor target tissues of rats

and mice treated with the genotoxic carcinogens: aristolochic acid, riddelliine and 

comfrey, and the non-genotoxic carcinogens: propiconazole and triadimeforn, and 

the non-carcinogen myclobutanil using microarray technologies; (2) to develop a

polymerase chain reaction array containing the primers, which are specifically used

to amplify carcinogensis-related microRNAs, and to use the polymerase chain 

reaction array to conduct time-course and dose-response studies for microRNA 

expression alterations in tissues of rats treated with carcinogens; and (3) to define

the microRNA biomarker genes that are associated with carcinogen exposure by

predicting their target genes and determining their biological functions.

QT interval correction via mixedeffects modeling 

[George]

To develop an appropriate non-linear mixed effects pharmacokinetic model in order 

to examine the effects of bitter orange/synephrine extract on electrocardiogram data

from a rodent study. QT interval is a measure of time in the heart’s electrical cycle.

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) to examine the mechanisms(s) 

responsible for embryotoxicity associated with selected known or suspect

embryotoxins that affect differentiation into osteoblasts.

Assessing acetaminophen-induced 

liver injury and the influence of dietary

supplements: potential synergistic 

interactions 

[Shi]

Using a battery of dietary supplements: (1) to determine the dose-response

toxicological effect in vivo in mice following repeated dosing with each dietary 

supplement in the absence and presence of acetaminophen, and (2) where dietary

supplements and acetaminophen administration resulted in greater hepatotoxicity, to 

examine the mechanism of action using genomics, proteomics, and metabolomics

approaches for in vivo samples from mice and in vitro samples from primary 

hepatocyte cultures.

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.
58 National Toxicology Program Annual Report FY 2013

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Effect of fetal or postnatal exposure to 

oxybenzone on reproduction in male

rats 

[Nakamura]

(1) To evaluate the expression of genes involved in testosterone synthesis in testes 

and prostate of adult males treated with oxybenzone perinatally and as young

adults, (2) to evaluate the expression of androgen-receptor genes in prostate and 

testes of rats treated with oxybenzone both perinatally and as young adults, and (3)

to evaluate expression of DNA methyltransferase genes in the testes and prostate of 

rats treated with oxybenzone both perinatally and as young adults.

Evaluate the impact of Deepwater 

Horizon oil-contaminated Gulf seafood 

residues in edible tissues on the

human intestinal microbiota of the

consumer 

[Cerniglia]

(1) To determine whether polycyclic aromatic hydrocarbon residues in edible tissues

of Deepwater Horizon oil-contaminated seafood adversely affect the human intestinal 

microbiota, (2) to determine if the human intestinal microbiota metabolize polycyclic

aromatic hydrocarbons that are toxic components of Deepwater Horizon oil, and (3)

to identify, characterize, and determine the toxicity of polycyclic aromatic hydrocarbon

metabolites generated from degradation by human intestinal microbiota.

3D- and 4D- quantitative spectrometric 

data-activity relationship modeling 

applied to various toxicological 

endpoints 

[Beger]

(1) To develop 3D- and 4D-quantitative spectrometric data-activity relationship models 

for endocrine disruptors, lowest-observed-adverse-effects level and no observedadverse-effects level, and other relevant toxicological endpoints; and (2) to determine 

how the technique used to predict 13C or 15N nuclear magnetic resonance spectra 

affects 3D-quantitative spectrometric data-activity relationship modeling. 

Table 28. NIEHS-NCTR Interagency Agreement Studies in FY 2013

Study 

CASRN 

[Principal Investigator]

Objective and/or Rationale

AIDS Program

Perinatal carcinogenicity of drug 

combinations used to prevent mother-to-child

transmission of HIV

30516-87-1, 134678-17-4, 129618-40-2, 

159989-65-8

[Beland]

To determine the carcinogenicity, genotoxicity, and metabolism of

antiretroviral drug combinations administered to mice transplacentally and

perinatally. Various combinations of the drugs AZT, lamivudine, mevirapine

and Nelfinavir mesylate are being studied. The studies include 14-day rangefinding and two-year chronic in pregnant C57BL6N females and in B6C3F1

hybrid offspring.

Dietary Supplements

Thirteen-week studies to determine the 

pathogenesis of the whole leaf extract of 

the Aloe vera plant in the cecum and large 

intestine of the F344 rat 

85507-69-3 

[Boudreau]

To investigate the pathogenesis of Aloe vera extracts and gel, with and 

without added Aloin A, in the cecum and large intestine of the F344 rat. 

Senna, having similar components to those in Aloe vera, will also be studied

to determine if it exerts comparable effects when administered in the drinking

water of F344 rats.

Food Contaminants

Assessment of molecular changes in 

male and female Sprague Dawley rats 

orally exposed to bisphenol A (BPA) from

gestational day 6 through postnatal day 90 

80-05-7

[Camacho]

To determine BPA-induced molecular changes in gene expression, protein 

levels, and epigenetic modifications in tissues collected from Sprague Dawley

rats orally exposed to BPA from gestational day 6 through postnatal day 90.
5. Testing and Research 59

Study 

CASRN 

[Principal Investigator]

Objective and/or Rationale

Evaluation of toxicity of BPA in male and 

female Sprague Dawley (NCTR) rats 

exposed orally from gestational day 6 through 

postnatal day 90 

80-05-7 

[Delclos]

(1) To assess the toxicity of BPA in rats dosed perinatally via gavage, and (2) 

to evaluate estrogenic endpoints from the first filial offspring generation.

Evaluation of various diets on endpoints 

critical to the evaluation of BPA and other 

endocrine-active agents 

[Delclos]

To evaluate reproductive and developmental endpoints in F0-F2 generation 

in CD-1 mice with various chows (such as Purina Mills 5K96, NIH-41, Purina

Mills 5001), some of which have low isoflavone levels, in preparation of

studies on BPA in CD-1 mice.

Two-year chronic toxicology study of BPA 

administered by gavage to Sprague Dawley

(NCTR) 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 Sprague Dawley 

(NCTR) 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. These evaluations will be conducted by investigators funded by

NIEHS. 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 and 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 reveal potential effects undetected by

standard toxicological evaluations.

Neurobehavioral effects of BPA across age

and sex 

80-05-7 

[Ferguson]

(1) To characterize how 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 across three

human-relevant dose and one dose of the reference estrogen; and (2) to 

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.

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 exposed in NTP 

study number C10119 to melamine, cyanuric acid and melamine cyanurate.

The initial toxicity was reported in the kidneys of the pets and children 

exposed to adulterated foods.

Assessment of nephrotoxicity from an 

exposure to melamine, cyanuric acid, and 

its combination in newborn F344 rats from

postnatal day 1 to weaning 

108-78-1, 108-90-5

[Gamboa]

(1) To determine if a combined exposure to melamine and cyanuric acid

in newborn F344 rats is more nephrotoxic than exposure to the individual

compounds, (2) to establish the dose-response curve for the combined

exposure, and (3) to investigate the longer-term effects of these lesions 

during a recovery period.
60 National Toxicology Program Annual Report FY 2013

Study 

CASRN 

[Principal Investigator]

Objective and/or Rationale

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

Thirteen-week dermal toxicity of triclosan in 

B6C3F1 mice 

3380-34-5

[Fang]

(1) To determine the toxicity of dermally applied triclosan with ethanol as the 

vehicle and (2) to determine the possible toxicity and phototoxicity of triclosan

under normal use conditions.

Nanoscale Material Program

Thirteen-week study to evaluate the 

toxicology of silver nanoparticles in Sprague 

Dawley rats 

744-22-4

[Boudreau]

To determine if exposure over a 13-week period to nanoscale (10, 70, and 

107 nm) silver particles induces toxicity.

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 and focus specifically on fertility and early embryonic development to

implantation, and (2) to compare the results of a typical Segment I, II, and III 

study design with results from a modified one-generation study proposed by

the 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 to

compare the results of a typical Segment I, II, and III study design with results 

from a modified one-generation study proposed by the NTP.

Effect of oxybenzone on pre and postnatal

development in Sprague Dawley rats 

(Segment III) 

131-57-7

[Inselman]

(1) To determine the potential toxicity of oxybenzone on pre and early

postnatal development in male and female rats, and (2) to compare the 

results of a typical Segment I, II, and III study design with results from a 

modified one-generation study proposed by the NTP.
6. Alternative Methods Development 61

6. Alternative Methods Development

A. Tox21

In FY 2008, NTP established a high throughput screening initiative, representing

a new paradigm in toxicological testing. On Feb. 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, the NIEHS/NTP

formally entered into a partnership with NCGC, now at NCATS; and the EPA National 

Center for Computational Toxicology within the Office of Research and Development.

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 (qHTS) assays, using phylogenetically lower animal species, such as fish and

worms, as well as high throughput, whole-genome, 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, and upregulation of various stress response pathways, such as 

antioxidant, hypoxia, and heat shock, in a variety of cell types; or to 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 that

would be used 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 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
62 National Toxicology Program Annual Report FY 2013

available at http://www.epa.gov/ncct/dsstox. Chemical analyses to determine the identity, purity, and stability of

all compounds in the library were initiated in FY 2012 and will continue through FY 2014.

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’s PubChem (http://pubchem.ncbi.

nlm.nih.gov), EPA’s ACToR (http://www.epa.gov/ACToR), and NTP’s CEBS (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, as a monitor of the technical quality of a screen.

The emphasis during Phase II is on assays measuring nuclear receptor signaling in agonist and antagonist 

mode, such as 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; induction of stress-response pathways, such as 

antioxidant, DNA damage, heat shock, and hypoxia; and assays measuring specific toxicity endpoints, such as

mitochondrial membrane changes, caspase activation, aromatase inhibition, and upregulation of nuclear factor

kappa beta, a transcription factor that has an important role in the immune system.

In FY 2013, 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 via 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. 

The Tox21 Phase II screening is being conducted in a new, high-speed, robotics screening facility dedicated at

the NCGC in FY 2011. It was built with funds provided by NIEHS/NTP. This robotics facility was used to screen

the 10K compound library in triplicate (30,000 compounds 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 NIEHSNCATS-UNC DREAM Toxicogenetics Challenge (see page 19 for more details about the challenge).

Table 29 describes the NTP Tox21 projects in FY 2013 that are being carried out by NIEHS/NTP staff. This

includes projects in the NTP WormTox Facility, which develops toxicological assays using the nematode

Caenorhabditis elegans (C. elegans), and evaluates their utility as medium throughput screening tools. The 

use of C. elegans is consistent with the NTP strategy to reduce the number of mammals used in testing. In

FY 2012, screening using the C. elegans growth assay was completed on the EPA ToxCast Phase II chemical 

library of 700 compounds. The data from this screen have been analyzed, and a manuscript describing the

results is in the process of completion.
6. Alternative Methods Development 63

Table 29. NTP Tox21 Projects in FY 2013

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 proliferatoractivated receptor coactivator 

pathway 

[Merrick]

To develop an assay that would detect compounds that interfere with the estrogen-related 

receptor/peroxisome proliferator-activated receptor gamma coactivator pathway, a critical 

pathway for metabolic homeostasis. Two steps are required to develop the stable cell line

and the first step to produce stable cell lines expressing peroxisome proliferator-activated

receptor gamma coactivator-1-alpha has been accomplished, with an expectation that the

assay will be functional in late FY 2014.

High content screening with 

HepaRG cells 

[Ferguson]

To establish metabolically functional human HepaRG liver cells in 96- or 384-well format to

multiplex high content screening assays in collaboration with the NCGC.

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.

Stem cells projects

[Ferguson]

To screen for chemical toxicity in human or mouse stem cell lines (undifferentiated or 

differentiated) by qHTS at the 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 would be employed with larger sets of chemicals for hazard

identification and chemical prioritization for toxicity testing.

Assay Interpretation

Validation of high throughput

screening estrogen receptor 

(ER) qHTS transactivation 

assay 

[Casey]

(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 (EPA

and Organisation for Economic Co-operation and Develop). 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 2014. (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, but there are differences in the

percentages of compounds classified as active and negative. Understanding the factors

contributing to differences in performance of these assays is critical to their regulatory

acceptance and utilization. Once the quality of the data is deemed to be sufficient

(anticipated to occur in FY 2014), results can be used to assess and further develop

quantitative structure-activity relationship models for ER binding.

Data Analysis

Modeling mixtures of androgen

receptor- and estrogen 

receptor-active 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. (3) Collaborative work with the University of

North Carolina at Chapel Hill involves providing data for their structure-activity relationship 

mixture model. 
64 National Toxicology Program Annual Report FY 2013

NTP Project Title 

[Study Scientist] Objective and/or Summary

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 publication describing this pipeline

is expected to be published in FY 2014.

Prioritization of Tox21 

compounds for genotoxicity

[Hsieh]

To develop a prioritization approach that not only includes compounds that show clear 

evidence of activity in the qHTS genotoxic assays, but also weakly active compounds

based on chemical structure-activity relationship analysis.

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, 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 with general usage scheduled for 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.

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.

Genomic signatures forecasting 

chemical carcinogenicity project

[Auerbach]

To develop transcriptomic signatures related to carcinogen treatment using NTP’s 

DrugMatrix data in combination with a machine-learning approach. Signatures are being

validated using an independent set of data from the Japanese Toxicogenomics Project.

Unsupervised, data-driven 

analysis of Tox21 assay data 

project

[Auerbach]

To employ unsupervised data analysis (data organization based on patterns and performed

by software) methods to identify chemicals that exhibit similar biological properties to well

characterized toxicants among the qHTS assays used to screen the 10K library.

Next generation sequencing in 

toxicology project

[Merrick]

To develop 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]

To develop the world’s largest public database for skin sensitization. This 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 structureactivity 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 with the

EPA to evaluate submitted studies and expand the database even further are underway.

In silico prediction of 

metabolism project

[Ferguson]

To evaluate various in silico methods of predicting the extent of xenobiotic metabolism,

identity metabolites and prioritize chemicals in the Tox21 10K library. Computational

methods will be used to ‘bin’ the 10K library and develop sub-sets of chemicals likely to be

appreciably metabolized in humans.

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) to facilitate extrapolation of findings from model

species to humans. To accomplish this, an effort has been initiated to select a set of genes

(~1,000 to 1,500) 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 2014 for comment by the scientific community.
6. Alternative Methods Development 65

NTP Project Title 

[Study Scientist] Objective and/or Summary

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. 

Screening for aromatase 

inhibitors in the Tox21 10K

library

[Merrick]

To screen aromatase inhibitors in the Tox21 10K library, collaboration was established with

Dr. Shiuan Chen at Beckman Institute, City of Hope, CA to develop an “AroER Tri-screen.” 

The 10K library was screened in this assay and the resulting data are being analyzed.

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. 

Analysis of NTP’s 52 

compounds in EPA’s ToxCast 

Phase II program 

[Casey]

To screen 52 compounds in the EPA’s 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. These analyses will be used to enable chemical prioritization and hazard

predictions. Preliminary analyses have been conducted, and compounds and assay

systems for potential targeted testing have been identified.

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: (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 EPA’s ToxCast Phase II 700-compound library on

C. elegans development and (2) to compare the relative toxicities to those in higher 

organisms. The in vivo studies were completed in FY 2013 and data analysis is scheduled 

for completion in FY 2014.

Ionic liquids project

[Boyd]

(1) To describe the effects of ~200 ionic liquids on C. elegans feeding, growth, and 

reproduction and (2) to compare the results in C. elegans to results in rodents and other 

toxicological models. 

Flame retardants project

[Boyd]

(1) To describe the effects of eight flame retardants on C. elegans feeding, growth, and 

reproduction and (2) to compare the results in C. elegans to results in rodents.

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.

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.
66 National Toxicology Program Annual Report FY 2013

B. ICCVAM

The Interagency Coordinating Committee on the Validation of

Alternative Methods (ICCVAM) is composed of representatives from

15 U.S. federal regulatory and research agencies that generate or 

use toxicological and safety testing information. The purpose of 

ICCVAM is to promote 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. In addition to its support

of ICCVAM, NICEATM:

● Supports NIEHS/NTP activities, especially those related to Tox21.

● Conducts analyses and evaluations, and coordinates independent validation studies on promising test 

methods.

● Provides information to test method developers, regulators, and regulated industry, through its website

and workshops on topics of interest.

In FY 2013, the NICEATM website was integrated into the NTP website. Information on NICEATM activities is

located at http://ntp.niehs.nih.gov/go/niceatm. The new NICEATM website includes resources for test method

developers at http://ntp.niehs.nih.gov/go/resources. Information on ICCVAM activities is available on the NTP

website at http://ntp.niehs.nih.gov/go/iccvam.

Upon the retirement of Rear Adm. William Stokes in December 2012, Warren Casey, Ph.D., became acting

director of NICEATM for the remainder of FY 2013. Casey also served as administrative director of ICCVAM.

NICEATM received contract support during FY 2013 from Integrated Laboratory Systems Inc.

i. Workshops

NICEATM worked with ICCVAM member agencies and

international partners to organize the International Workshop

on Alternatives to the Murine Histamine Sensitization Test

(HIST) for Acellular Pertussis Vaccines. The workshop was

held Nov. 28–29, 2012, at the William H. Natcher Conference

Center at NIH in Bethesda, Maryland. Workshop participants

reviewed and discussed data generated by an international

study that compared the performance of 12 in vitro assays. 

Two cell-based assays that appeared most promising for

future acceptance or adoption were recommended for 

further development and optimization. Workshop participants

supported development of a standardized Chinese hamster 

ovary cell aggregation assay as an alternative to the murine 

histamine sensitization test for calibration of pertussis toxin

reference standards. The workshop report is in press in 

Biologicals and will be published in early 2014. Links to

the workshop report, summary, presentations, and other 

information are at http://ntp.niehs.nih.gov/go/HISTwksp. 

Follow-up workshops are planned for August 2014 and 

mid-2015.

ICCVAM Member Agencies

CPSC

USDA

DOD

DOE

DOI

DOL

DOT

OSHA

EPA

HHS

CDC

FDA

NIH

ATSDR

NIOSH

NIEHS

NCI

NLM

OD
6. Alternative Methods Development 67

ii. Publications

The following FY 2013 publications describe NICEATM and ICCVAM activities.

Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). 2012. ICCVAM

Test Method Evaluation Report: Identifying Chemical Eye Hazards with Fewer Animals. NIH Publication No.

12-7930. Research Triangle Park, N.C.: National Institute of Environmental Health Sciences. 

Johnson N, Doelling V, Jones B, eds. 2013. International Workshop on Alternative Methods for Vaccine

Potency Testing: State of the Science and the Way Forward. Biologicals 41(5):279–338.

Kolle SN, Basketter DA, Casati S, Stokes WS, Strickland J, van Ravenzwaay B, Vohr HW, Landsiedel

R. 2013. Performance standards and alternative assays: practical insights from skin sensitization. Regul 

Toxicol Pharmacol 65(2):278–285.

A complete list of articles on NICEATM and ICCVAM activities published in scientific journals is available at

http://ntp.niehs.nih.gov/go/niceatm-pubs. Reports published by NICEATM are available at http://ntp.niehs.nih.

gov/?objectid=E2E87900-F051-C3CA-C6C4A81E33A38907.

iii. NICEATM Activities

NICEATM Support of Tox21

Following a NICEATM nomination, the estrogen receptor (ER) transactivation (TA) test method was adapted to

a high throughput format by NCATS and used to screen the Tox21 10K chemical library. NICEATM analyzed

the results and determined that the high throughput BG1Luc ER TA test method performed well when 

compared against the validated manual method.

NICEATM also compared data from the BG1Luc ER TA test method to an ER beta-lactamase method, which

was already being used in the Tox21 program. The ER beta-lactamase method differs from the BG1Luc ER TA

method in that the cell line used includes a partial ER receptor that contains only the ligand binding site of the ER.

Results from the BG1Luc ER TA and ER beta-lactamase were compared both to each other and to reference

data. The study found that the two methods produced data of acceptable quality and results that agreed mostly,

but not completely, with each other and with the reference data. Understanding the factors contributing to

differences in performance of these assays is critical to their regulatory acceptance and utilization.

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 pesticide formulations. NICEATM is reviewing the data for quality control purposes and using modified

Klimisch criteria to evaluate the reliability of the data.

Scientists within NICEATM and NIEHS are collaborating with scientists at Procter and Gamble to develop

an approach that can 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

an integrated testing strategy developed by Procter and Gamble. The open-source integrated testing strategy

will be described in a short communication in the journal ALTEX in 2014. Downloadable files to run the

analysis, along with documentation and sample data, are available at http://ntp.niehs.nih.gov/go/its.
68 National Toxicology Program Annual Report FY 2013

NICEATM scientists worked with immunotoxicologists from NIEHS/NTP, EPA, and other academic institutions

and international organizations, to create a standardized ontology for in vivo immunotoxicity data. Using the 

standardized ontology, NICEATM is working with EPA to create a comprehensive database consisting of highquality 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 from 52 chemicals selected

by EPA and NIEHS/NTP, to support future validation of high throughput, in vitro test methods and in silico

models of estrogenic activity. Studies selected included data for a number of different endpoints that indicate

estrogenic activity. Information from references is being extracted and compiled using a standardized ontology.

A semiautomated, graphical user interface is being developed to evaluate the quality of the data in an efficient

and standardized manner, according to modified Klimisch criteria (Schneider et al. 2009. “ToxRTool,” a new

tool to assess the reliability of toxicological data. Toxicol Lett 189(2),138–144). The NICEATM database will be

made available on the NTP website at http://ntp.niehs.nih.gov/go/40658.

NICEATM activities initiated in FY 2013 to support risk-based, chemical testing prioritization include

construction of reverse toxicokinetic models to assist in vitro to in vivo extrapolation, and development of 

approaches to estimate bioconcentration potential and exposure.

iv. Test Method Evaluation Activities

ICCVAM received no formal test method nominations or submissions in FY 2013. 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 that 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 2013 are

summarized in Table 30.

Table 30. Test Method Evaluation Activities in FY 2013

Test Method ICCVAM Recommendations/Agency Status

OptiSafe test for identifying 

potential eye irritants

NICEATM evaluated the validation study data for OptiSafe, an in vitro test method for

identification of potential eye irritants. Initial review of the data indicates that the OptiSafe

method may compare favorably to other in vitro eye-safety testing methods. ICCVAM will

reactivate its Ocular Toxicity Working Group to more thoroughly evaluate this test method to

determine its potential regulatory utility and whether additional activities are necessary.

ICCVAM plan for the

evaluation of alternative 

skin-sensitization test 

methods and testing 

strategies

ICCVAM is developing a plan for the evaluation of alternative, skin-sensitization test methods

and testing strategies. Proposed activities include collaboration with international partners

to support ongoing development and validation of in vitro skin-sensitization test methods, 

evaluation of alternative test method and testing strategy submissions, and promotion of

validated methods through workshops, webinars, and guidance documents. The newly

established ICCVAM Skin Sensitization Working Group is considering comments received on

this proposal and will advance recommendations for appropriate ICCVAM activities for the next

several years. More information is available at http://ntp.niehs.nih.gov/go/40498.
6. Alternative Methods Development 69

v. 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). In FY 2013, NICEATM and ICCVAM were

involved in the following OECD activities:

● Incorporation of ICCVAM performance standards into 2013 updates of OECD test guidelines

describing in vitro methods to identify potential dermal corrosives.

● A presentation by Casey, representing NICEATM and ICCVAM, at the November 2012 Validation

Management Group for Non-Animal Testing meeting, part of the OECD Task Force on Endocrine

Disrupters Testing and Assessment.

● Participation of Casey at the November 2012 meeting of the OECD Thyroid Scoping Effort Expert

Group. This meeting initiated an effort to identify available assays for the detection of potential thyroid

disruptors, and assess their suitability for regulatory use or potential future test guideline development.

As a follow-up activity, NICEATM staff collected information on available thyroid assays and submitted

it to the group for inclusion in a larger report that will be reviewed by OECD in 2014.

Ongoing international validation studies that include participation of NICEATM or ICCVAM are listed in Table 31. 

Casey represented NICEATM and ICCVAM at an ICATM coordination meeting in February 2013.

Table 31. Participation in International Validation Studies

Test Method Type of 

Test

Lead 

Organization NICEATM-ICCVAM Involvement

EpiOcular (MatTek) and

SkinEthic (L’Oreal)

Ocular 

irritation EURL ECVAM

NICEATM and ICCVAM members served on the validation

management team and provided comments on study design, 

chemical selection, and test method performance criteria.

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 design, 

chemical selection, test method protocols, and study reports.

In vitro tests for assessing 

skin sensitization potential 

of chemicals

Allergic 

contact 

dermatitis

EURL ECVAM

NICEATM and ICCVAM members served on the validation

management team and provided comments on study design, 

chemical selection, and test method protocols.

In vitro tests for assessing 

skin sensitization potential 

of chemicals

Allergic 

contact 

dermatitis

JaCVAM

NICEATM and ICCVAM members served on the validation

management team and provided comments on study design, 

chemical selection, and test method protocols.

Short-time-exposure test 

for identifying potential eye 

irritants

Ocular 

irritation JaCVAM

NICEATM prepared a summary review document that

presents an evaluation of short-time-exposure test-method 

performance based on test substances with corresponding in

vivo data (http://ntp.niehs.nih.gov/iccvam/docs/ocutox_docs/

STE-SRD-NICEATM-508.pdf). The evaluation underwent 

an independent peer review coordinated by NTP. The

reviewers made a number of comments with respect to the

usefulness and limitations of the short-time-exposure method 

for consideration by regulatory agencies when such data are

submitted.
70 National Toxicology Program Annual Report FY 2013

7. Public Tools

NTP provides a variety of resources for public use. Those available in FY 2013 are described below.

A. Nonneoplastic Lesion Atlas

NTP Nonneoplastic Lesion Atlas is a web-based resource containing hundreds, and eventually 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, and record of nonneoplastic rodent lesions, to 

improve the consistency of the NTP nonneoplastic lesion database. The atlas is planned for launch in FY 2014.

See page 17 for more information about the atlas.

B. Chemical Effects in Biological Systems (CEBS) Database

CEBS 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 peer-reviewed 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 continues to add legacy NTP data to CEBS. Phase II Tox21 data will also be publicly available through

CEBS, as the 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 CEBS or download for import into other tools. CEBS 

can be accessed at http://cebs.niehs.nih.gov.
7. Public Tools 71

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 NTP Archives maintains 7.7 million 

histopathology slides, 4.8 million paraffin-embedded tissue blocks, 213,000 bags of formalin-fixed tissues and

organs, 117,000 selected frozen tissue specimens; and study data including 4.4 million pages of paper records, 

10.8 million pages of data on microfiche, and 2.4 million pages of digital or electronic records on CDs or DVDs.

It also houses histological images, including more than 38,000 kodachrome slides and 27,000 digital images, 

in a publically accessible, searchable, Web-based database, as well as 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.

A recent gene expression study, using archival material, showed that a gene expression signature, comprised 

of 14 different transcripts, could be reliably measured from RNA extracted from four-year-old, formalin-fixed,

paraffin-embedded (FFPE) liver tissues in an NTP 90-day rat study. FFPE RNA from liver, kidney, and lung,

to a lesser extent, were of sufficient quality for molecular analysis to measure gene expression by quantitative

polymerase chain reaction that favorably compared to results of fresh frozen tissues from identical animals.

These studies demonstrated the usefulness of archival tissue blocks for gene profiling. A more systematic

query of archival materials, from different organs and storage times, is planned for the future, to more precisely 

determine the value in transcript profiling of the NTP Archives.

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

ntparchives2014_508.pdf.

D. The NCGC BioPlanet

No single, 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 through NTP (see page 16), built an

integrated pathway resource that hosts information on approximately 1,600 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 2014.

E. DrugMatrix and ToxFX

Related to the goal of developing analysis tools and approaches, to allow an integrated assessment of high 

throughput screening endpoints and associations with findings from traditional toxicology and cancer models,

NTP acquired DrugMatrix, a toxicogenomics reference database, as well as the accompanying extensive
72 National Toxicology Program Annual Report FY 2013

frozen tissue archives and informatics system. 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). To date, more than 475 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,

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) in 

collaboration with researchers at Harvard University. Finally, all the RNA expression, such as transcriptomics,

data from DrugMatrix has been integrated in NextBio, a database and analysis tool, allowing analysis of the

data within the context of all existing genomic and transcriptomic data. All DrugMatrix data have been made

available through an easily accessible FTP portal (ftp://anonftp.niehs.nih.gov/drugmatrix). 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 NTP data files for

their own purposes, including use in publications. Meta Data Viewer is available at http://ntp.niehs.nih.gov/go/

tools_metadataviewer. 
8. Appendices 73

8. Appendices

A. Program Contact Information

National Toxicology Program: 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 main website: http://www.niehs.nih.gov

NCTR main website: http://www.fda.gov/AboutFDA/CentersOffices/OC/OfficeofScientificandMedicalPrograms/

NCTR/default.htm

NIOSH main website: http://www.cdc.gov/niosh

Staff directory: http://directory.psc.gov/employee.htm

B. Frequently Used Abbreviations

3TC lamivudine

ADME	 absorption, distribution, metabolism, and excretion

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

CDMA 	 code division multiple access

CEBS Chemical Effects in Biological Systems 

CLARITY-BPA Consortium Linking Academic and Regulatory Insights on BPA Toxicity

CPSC U.S. Consumer Product Safety Commission

DOD Department of Defense

DOE Department of Energy

DOI Department of the Interior

DOL	 Department of Labor

DOT Department of Transportation

ECVAM	 European Centre for the Validation of Alternative Methods

EPA Environmental Protection Agency

ER estrogen receptor 

FDA U.S. Food and Drug Administration

FFPE 	 formalin fixed, paraffin embedded
74 National Toxicology Program Annual Report FY 2013

FY	 fiscal year

GABA	 gamma-amino butyric acid

GD gestational day

GMM	 genetically modified model

GSM	 global system for mobile communication

HHS U.S. Department of Health and Human Services

HIST murine histamine sensitization test

IARC International Agency for Research on Cancer

ICATM	 International Cooperation on Alternative Test Methods

ICCVAM	 Interagency Coordinating Committee on the Validation of Alternative Methods

IgE	 immunoglobulin E

JaCVAM	 Japanese Center for the Validation of Alternative Methods

KoCVAM	 Korean Center for the Validation of Alternative Methods

LC/ESI/MS/MS	 liquid chromatography-electrospray ionization/multi-stage mass spectrometry

LIFE Longitudinal Investigation of Fertility and the Environment

MOU	 Memorandum of Understanding

N/A	 not applicable

NCATS National Center for Advancing Translational Sciences

NCEH National Center for Environmental Health

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

NFV	 nelfinavir mesylate

NHGRI National Human Genome Research Institute

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

OECD Organisation for Economic Co-operation and Development

OHAT	 Office of Health Assessment and Translation

OSHA Occupational Safety and Health Administration

PAC polycyclic aromatic compounds

PCB	 polychlorinated biphenyl

PCBTF	 chloro-4-(trifluoromethyl) benzene
8. Appendices 75

Pig-A phosphatidylinositol glycan complementation group A 

PND postnatal day

qHTS quantitative high throughput screening

RoC Report on Carcinogens

SACATM	 Scientific Advisory Committee on Alternative Toxicological Methods

SOT Society of Toxicology

TA transactivation

TOX	 NTP Toxicity Report

TR NTP Technical Report

UNC University of North Carolina

U.S. United States

USDA U.S. Department of Agriculture

VOC	 volatile organic compound
76 National Toxicology Program Annual Report FY 2013

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

C. FY 2013 Bibliography

i. Reports

Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). 2012. ICCVAM Test Method

Evaluation Report: Identifying Chemical Eye Hazards With Fewer Animals. NIH Publication No. 12-7930. Research

Triangle Park, NC: National Institute of Environmental Health Sciences.

National Institute of Occupational Safety and Health. 2013. Current Intelligence Bulletin 65: Occupational Exposure to 

Carbon Nanotubes and Nanofibers. DHHS (NIOSH) Publication No. 2013-145. Cincinnati, OH: National Institute for

Occupational Safety and Health. 

National Toxicology Program. 2013. National Toxicology Program Monograph on Developmental Effects and Pregnancy

Outcomes Associated With Cancer Chemotherapy Use During Pregnancy. Research Triangle Park, NC: National Institute

of Environmental Health Sciences.

National Toxicology Program. 2013. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis 

Studies of Ginkgo biloba Extract (CAS No. 90045-36-6) in F344/N Rats and B6C3F1/N Mice (gavage studies). NTP TR

578, NIH Publication No. 13-5920. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2013. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis 

Studies of Mixtures of 3′-Azido-3′-Deoxythymidine (AZT), Lamivudine (3TC), Nevirapine (NVP), and Nelfinavir Mesylate

(NFV) (CAS Nos. 30516-87-1, 134678-17-4, 129618-40-2, 159989-65-8) in B6C3F1 Mice (transplacental exposure

studies). NTP TR 569, NIH Publication No. 13-5911. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2013. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis 

Studies of a Noncolorized Whole Leaf Extract of Aloe barbadensis Miller (Aloe Vera) in F344/N Rats and B6C3F1 Mice

(drinking water studies). NTP TR 577, NIH Publication No. 13-5910. Research Triangle Park, NC: National Toxicology

Program.

National Toxicology Program. 2013. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis 

Studies of Pyrogallol (CAS No. 87-66-1) in F344/N Rats and B6C3F1/N Mice (dermal studies). NTP TR 574, NIH

Publication No. 13-5916. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2013. National Toxicology Program Technical Report on the Toxicology and Carcinogenesis 

Studies of Trimethylolpropane Triacrylate (technical grade) (CAS No. 15625-89-5) in F344/N Rats and B6C3F1/N Mice

(dermal studies). NTP TR 576, NIH Publication No. 13-5918. Research Triangle Park, NC: National Toxicology Program.

National Toxicology Program. 2013. Report on Carcinogens Monograph on 1-Bromopropane. NIH Publication No.

13-5982. Research Triangle Park, NC: National Institute of Environmental Health Sciences.

National Toxicology Program. 2013. Report on Carcinogens Monograph on Cumene. NIH Publication No. 13-5983, ISSN

2331-267X. Research Triangle Park, NC: National Institute of Environmental Health Sciences.

ii. Journal Publications

1 Anderson SE, Franko J, Wells JR, Lukomska E, Meade BJ. 2013. Evaluation of the hypersensitivity potential of

alternative butter flavorings. Food Chem Toxicol 62:373–381.

1 Anderson SE, Khurshid SS, Meade BJ, Lukomska E, Wells JR. 2013. Toxicological analysis of limonene reaction

products using an in vitro exposure system. Toxicol Vitr27(2):721–730.
8. Appendices 77

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

7 Annab LA, Bortner CD, Sifre MI, Collins JM, Shah RR, Dixon D, Karimi Kinyamu H, Archer TK. 2012. Differential

responses to retinoic acid and endocrine disruptor compounds of subpopulations within human embryonic stem cell

lines. Differentiation 84(4):330–343 [Online 18 August 2012].

4 Antunes AMM, Wolf B, Oliveira MC, Beland FA, Marques MM. 2013. 2′-Deoxythymidine adducts from the anti-HIV

drug nevirapine. Molecules 18(5):4955–4971.

2 Ashley K, Harper M. 2013. Analytical performance issues Closed-Face Filter Cassette (CFC) sampling-guidance on

procedures for inclusion of material adhering to internal sampler surfaces. J Occup Environ Hyg 10(3):D29-D33.

5,7 Attene-Ramos MS, Huang R, Sakamuru S, Witt KL, Beeson GC, Shou L, Schnellmann RG, Beeson CC, Tice RR,

Austin CP, Xia M. 2013. Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high

throughput screening. Chem Res Toxicol 26(9):1323–1332.

5,7 Attene-Ramos MS, Miller N, Huang R, Michael S, Itkin M, Kavlock RJ, Austin CP, Shinn P, Simeonov A, Tice RR,

Xia M. 2013. The Tox21 robotic platform for the assessment of environmental chemicals—from vision to reality. Drug

Discov Today 18(15–16):716–723.

7 Balbus JM, Barouki R, Birnbaum LS, Etzel RA, Gluckman SPD, Grandjean P, Hancock C, Hanson MA, Heindel JJ,

Hoffman K, Jensen GK, Keeling A, Neira M, Rabadan-Diehl C, Ralston J, Tang KC. 2013. Early-life prevention of

non-communicable diseases. Lancet 381(9860):3–4.

3 Bandele O, Camacho L, Ferguson M, Reimschuessel R, Stine C, Black T, Olejnik N, Keltner Z, Scott M, Gamboa

da Costa G, Sprando R. 2013. Performance of urinary and gene expression biomarkers in detecting the nephrotoxic

effects of melamine and cyanuric acid following diverse scenarios of co-exposure. Food Chem Toxicol 51:106–113

[Online 2 October 2012].

7 Behl M, Rao D, Aagaard K, Davidson TL, Levin ED, Slotkin TA, Srinivasan S, Wallinga D, White MF, Walker VR,

Thayer KA, Holloway AC. 2013. Evaluation of the association between maternal smoking, childhood obesity, and

metabolic disorders: a National Toxicology Program workshop review. Environ Health Perspect 121(2):170–180.

3 Beland FA, Mellick PW, Olson GR, Mendoza MC, Marques MM, Doerge DR. 2013. Carcinogenicity of acrylamide in

B6C3F(1) mice and F344/N rats from a 2-year drinking water exposure. Food Chem Toxicol 51:149–159 [Online 27

September 2012].

7 Bergman T, Ryden A, Law RJ, de Boer J, Covaci A, Alaee M, Birnbaum L, Petreas M, Rose M, Sakai S, Van den Eede

N, van der Veen I. 2012. A novel abbreviation standard for organobromine, organochlorine and organophosphorus

flame retardants and some characteristics of the chemicals. Environ Int 49:57–82.

1 Bernstein DI, Kashon M, Lummus ZL, Johnson VJ, Fluharty K, Gautrin D, Malo JL, Cartier A, Boulet LP, Sastre J,

Quirce S, Germolec D, Tarlo SM, Cruz MJ, Munoz X, Luster MI, Yucesoy B. 2013. CTNNA3 (alpha-catenin) gene

variants are associated with diisocyanate asthma: a replication study in a Caucasian worker population. Toxicol Sci 

131(1):242–246.

7 Bhusari S, Malarkey DE, Hong HH, Wang Y, Masinde T, Nolan M, Hooth MJ, Lea IA, Vasconcelos D, Sills RC,

Hoenerhoff MJ. 2013. Mutation spectra of Kras and Tp53 in urethral and lung neoplasms in B6C3F1 mice treated with

3,3’,4,4’-tetrachloroazobenzene. Toxicol Pathol; doi:10.1177/0192623313491169 [Online 23 May 2013].

7 Binder AK, Rodriguez KF, Hamilton KJ, Stockton PS, Reed CE, Korach KS. 2013. The absence of ER-beta results

in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles. Endocrinology 

154(6):2174–2187.
78 National Toxicology Program Annual Report FY 2013

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

4 Binienda ZK, Sarkar S, Mohammed-Saeed L, Gough B, Beaudoin MA, Ali SF, Paule MG, Imam SZ. 2013. Chronic

exposure to rotenone, a dopaminergic toxin, results in peripheral neuropathy associated with dopaminergic damage. 

Neurosci Lett 541:233–237 [Online 19 March 2013].

7 Birnbaum LS. State of the science of endocrine disruptors. 2013. Environ Health Perspect 121(4):A107.

7 Birnbaum LS. 2013. When environmental chemicals act like uncontrolled medicine. Trends Endocrinol Metab

24(7):321–323.

7 Birnbaum LS. 2013. 15 years out: reinventing ICCVAM. Environ Health Perspect 121(2):A40.

7 Birnbaum LS. 2013. Designing safer chemicals. Environ Health Perspect 121(1):A9.

7 Birnbaum LS, Aungst J, Schug TT, Goodman JL. 2013. Working together: research- and science-based regulation of

BPA. Environ Health Perspect 121(7):A206-A207.

7 Birnbaum LS, Thayer KA, Bucher JR, Wolfe MS. 2013. Implementing systematic review at the National Toxicology

Program: status and next steps. Environ Health Perspect 121(4):A108-A109.

7 Blackshear PE, Pandiri AR, Ton TV, Clayton NP, Shockley KR, Peddada SD, Gerrish KE, Sills RC, Hoenerhoff MJ.

2013. Spontaneous mesotheliomas in F344/N rats are characterized by dysregulation of cellular growth and immune

function pathways. Toxicol Pathol; doi: 10.1177/0192623313501894 [Online 26 August 2013].

7 Boekelheide K, Blumberg B, Chapin RE, Cote I, Graziano JH, Janesick A, Lane R, Lillycrop K, Myatt L, States JC,

Thayer KA, Waalkes MP, Rogers JM. 2012. Predicting later-life outcomes of early-life exposures. Environ Health

Perspect 120(10):1353–1361.

3 Boudreau MD. 2013. “Nondecolorized” qualifier is a misnomer for the Aloe vera whole leaf extract test material.

Toxicol Sci 133(2):343.

3 Boudreau MD, Mellick PW, Olson GR, Felton RP, Thorn BT, Beland FA. 2013. Clear evidence of carcinogenic activity

by a whole-leaf extract of Aloe barbadensis miller (Aloe vera) in F344/N rats. Toxicol Sci 131(1):26–39 [Online 13

September 2012].

4 Bowyer JF, Patterson TA, Saini UT, Hanig JP, Thomas M, Camacho L, George NI, Chen JJ. 2013. Comparison of the

global gene expression of choroid plexus and meninges and associated vasculature under control conditions and after

pronounced hyperthermia or amphetamine toxicity. BMC Genomics 14:147 [Online 19 March 2013].

7 Boyle MC, Boyle MH. 2013. Meeting report: Urinary pathology; sixth Research Triangle Park rodent pathology course.

Vet Pathol 50(3):563–568.

4 Bull RJ, Kolisetty N, Zhang X, Muralidhara S, Quinones O, Lim KY, Guo Z, Cotruvo JA, Fisher JW, Yang X, Delker D,

Snyder SA, Cummings BS. 2012. Absorption and disposition of bromate in F344 rats. Toxicology 300(1–2):83–91.

7 Buse E, Haeger JD, Svensson-Arvelund J, Markert UR, Faas MM, Ernerudh J, Dixon D, Cline JM, Pfarrer C. 2013.

The placenta in toxicology. Part I: Animal models in toxicology: placental morphology and tolerance molecules in the 

cynomolgus monkey (Macaca fascicularis). Toxicol Pathol; doi:10.1177/0192623313482208 [Online 2 April 2013].

1 Buskirk AD, Templeton SP, Nayak AP, Hettick J, Law BF, Green B, Beezhold D. 2013. Melanin modulates the

pulmonary immune response to Aspergillus fumigatus conidia. J Immunotoxicol; doi:10.3109/1547691X.2013.819054

[Online 6 August 2013].
8. Appendices 79

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

1 Callahan A, Baron E, Fekedulegn D, Kashon M, Yucesoy B, Johnson VJ, Domingo DS, Kirkland B, Luster MI, and

Nedorost S. 2013. Winter season, frequent hand washing, and irritant patch test reactions to detergents are associated

with hand dermatitis in healthcare workers. Dermatitis 24(4):170–175.

4 Cao J, Rebuli ME, Rogers J, Todd KL, Leyrer SM, Ferguson SA, Patisaul HB. 2013. Prenatal bisphenol A exposure

alters sex-specific estrogen receptor expression in the neonatal rat hypothalamus and amygdala. Toxicol Sci

133(1):157–173.

7 Carlin DJ, Rider CV, Woychik R, Birnbaum LS. 2013. Unraveling the health effects of environmental mixtures: an

NIEHS priority. Environ Health Perspect 121(1):A6-A8.

7 Cesta MF, Hard GC, Boyce JT, Ryan MJ, Chan PC, Sills RC. 2013. Complex histopathologic response in rat kidney

to oral beta-myrcene: An unusual dose-related nephrosis and low-dose alpha2u-globulin nephropathy. Toxicol Pathol

41(8):1068–1077 [Online 28 March 2013].

7 Chatterjee S, Ganini D, Tokar EJ, Kumar A, Das S, Corbett J, Kadiiska MB, Waalkes MP, Diehl AM, Mason RP.

2013. Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic 

steatohepatitis. J Hepatol 58(4):778–784.

4 Chen S, Wan L, Couch L, Lin H, Li Y, Dobrovolsky VN, Mei N, Guo L. 2013. Mechanism study of goldensealassociated DNA damage. Toxicol Lett 221(1):64–72 [Online 13 June 2013].

7 Cline JM, Dixon D, Ernerudh J, Faas MM, Gohner C, Hager JD, Markert UR, Pfarrer C, Svensson-Arvelund

J, Buse E. 2013. The placenta in toxicology. Part III: Pathologic assessment of the placenta. Toxicol Pathol; 

doi:10.1177/0192623313482207 [Online 26 March 2013].

2 Connor TH, MacKenzie BA, DeBord DG. 2012. Clarification about hazardous drugs. Am J Health Syst Pharm

69(22):1949–1950.

7 Cora MC, Neel JA, Grindem CB, Kissling GE, Hess PR. 2013. Comparison of automated versus manual neutrophil

counts for the detection of cellular abnormalities in dogs receiving chemotherapy: 50 cases (May to June 2008). J Am

Vet Med Assoc 242(11):1539–1543.

7 Cote I, Anastas PT, Birnbaum LS, Clark RM, Dix DJ, Edwards SW, Preuss PW. 2012. Advancing the next generation

of health risk assessment. Environ Health Perspect 120(11):1499–1502.

4 Cuevas E, Trickler WJ, Guo X, Ali SF, Paule MG, Kanungo J. 2013. Acetyl l-carnitine protects motor neurons and

Rohon-Beard sensory neurons against ketamine-induced neurotoxicity in zebrafish embryos. Neurotoxicol Teratol

39:69–76 [Online 31 July 2013].

1 Dahm MM, Evans DE, Schubauer-Berigan MK, Birch ME, Deddens JA. 2013. Occupational exposure assessment

in carbon nanotube and nanofiber primary and secondary manufacturers: mobile direct-reading sampling. The Ann

Occup Hyg 57(3):328–344.

7 Das S, Kumar A, Seth RK, Tokar EJ, Kadiiska MB, Waalkes MP, Mason RP, Chatterjee S. 2013. Proinflammatory

adipokine leptin mediates disinfection byproduct bromodichloromethane-induced early steatohepatitic injury in obesity.

Toxicol Appl Pharmacol 269(3):297–306.

7 De Arras L, Seng A, Lackford B, Keikhaee MR, Bowerman B, Freedman JH, Schwartz DA, Alper S. 2013. An

evolutionarily conserved innate immunity protein interaction network. J Biol Chem 288(3):1967–1978.
80 National Toxicology Program Annual Report FY 2013

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

2 Decker JA, DeBord DG, Bernard B, Dotson GS, Halpin J, Hines CJ, Kiefer M, Myers K, Page E, Schulte P, Snawder J.

2013. Recommendations for biomonitoring of emergency responders: focus on occupational health investigations and

occupational health research. Mil Med 178(1):68–75 [Online 30 January 2013].

4 De Conti A, Tryndyak V, Koturbash I, Heidor R, Kuroiwa-Trzmielina J, Ong TP, Beland FA, Moreno FS, Pogribny IP.

2013. The chemopreventive activity of the butyric acid prodrug tributyrin in experimental rat hepatocarcinogenesis

is associated with p53 acetylation and activation of the p53 apoptotic signaling pathway. Carcinogenesis 

34(8):1900–1906.

7 Dere E, Anderson LM, Coulson M, McIntyre BS, Boekelheide K, Chapin RE. 2013. SOT symposium highlight:

translatable indicators of testicular toxicity: inhibin B, micrornas, and sperm signatures. Toxicol Sci 136(2):265–273

[Online 19 September 2013].

4 Desai VG, Herman EH, Moland CL, Branham WS, Lewis SM, Davis KJ, George NI, Lee T, Kerr S, Fuscoe JC. 2013.

Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F(1) mouse model. Toxicol Appl Pharmacol

266(1):109–121 [Online 13 November 2012].

4 Dorne JL, Doerge DR, Vandenbroeck M, Fink-Gremmels J, Mennes W, Knutsen HK, Vernazza F, Castle L, Edler L,

Benford D. 2013. Recent advances in the risk assessment of melamine and cyanuric acid in animal feed. Toxicol Appl 

Pharmacol 270(3):218–229.

7 Eastmond DA, Vulimiri SV, French JE, Sonawane BE. 2013. The use of genetically modified mice in cancer risk

assessment: challenges and limitations. Crit Rev Toxicol 43(8):611–631.

7 Elmore SA, Berridge BR, Boyle MC, Cora MC, Hoenerhoff MJ, Kooistra L, Laast V, Morrison J, Rao D, Rinke M,

Yoshizawa K. 2013 Proceedings of the 2012 National Toxicology Program Satellite Symposium. Toxicol Pathol 

41(2):151–180.

7 Elmore SA, Hoenerhoff M, Katsuta O, Kokoshima H, Maronpot R, Nagai H, Satoh H, Tanaka Y, Tochitani T, Tsuchiya

S, Yoshizawa K. 2013. Proceedings of the 2013 Joint JSTP/NTP satellite symposium. J Toxicol Pathol 26(2):231–257.

1 Erdely A, Dahm M, Chen BT, Zeidler-Erdely, PC, Fernback JE, Birch ME, Evans DE, Kashon ML, Deddens JA,

Hulderman T, Bilgesu SA, Battelli L, Schwegler-Berry D, Leonard HD, McKinney W, Frazer DG, Antonini JM,

Porter DW, Castranova V, Schubauer-Berigan MK. 2013. Carbon nanotube dosimetry: From workplace exposure

assessment to inhalation toxicology. Part Fibre Toxicol 10(1):53.

2 Esswein EJ, Breitenstein M, Snawder J, Kiefer M, Sieber WK. 2013. Occupational exposures to respirable crystalline

silica during hydraulic fracturing. J Occup Environ Hyg 10(7):347–356.

7 Euling SY, White LD, Kim AS, Sen B, Wilson VS, Keshava C, Keshava N, Hester S, Ovacik MA, Ierapetritou MG,

Androulakis IP, Gaido KW. 2013. Use of genomic data in risk assessment case study: II. Evaluation of the dibutyl

phthalate toxicogenomic data set. Toxicol Appl Pharmacol 271(3):349–362.

4 Ferguson SA, Law CD, Abshire JS. 2012. Developmental treatment with bisphenol A causes few alterations on

measures of postweaning activity and learning. Neurotoxicol Teratol 34(6):598–606 [Online 9 October 2012].

4 Ferguson SA, Maier KL. 2013. A review of seasonal/circannual effects of laboratory rodent behavior. Physiol Behav

119:130–136 [Online 19 June 2013].

4 Ferguson SA, Sarkar S, Schmued LC. 2013. Longitudinal behavioral changes in the APP/PS1 transgenic Alzheimer’s

disease model. Behav Brain Res 242:125–134 [Online 9 January 2013].
8. Appendices 81

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

4 Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME. 2013. Evaluation of iodide deficiency in

the lactating rat and pup using a biologically based dose-response model. Toxicol Sci 132(1):75–86 [Online 5 January

2013].

7 Flake GP, Moore AB, Flagler N, Wicker B, Clayton N, Kissling GE, Robboy SJ, Dixon D. 2013. The natural history of

uterine leiomyomas: morphometric concordance with concepts of interstitial ischemia and inanosis. Obstet Gynecol Int

2013:285103 [Online 30 September 2013].

7 Gao XH, Yu LD, Castro L, Tucker CJ, Moore AB, Xiao H, Dixon D. 2012. An essential role of p27 downregulation

in fenvalerate-induced cell growth in human uterine leiomyoma and smooth muscle cells. Am J Physiol Endocrinol 

Metabol 303(8):E1025-E1035.

4 Genter MB, Newman NC, Shertzer HG, Ali SF, Bolon B. 2012. Distribution and systemic effects of intranasally

administered 25 nm silver nanoparticles in adult mice. Toxicol Pathol 40(7):1004–1013.

4 Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett

JM, Fisher JW. 2013. An animal model of marginal iodine deficiency during development: the thyroid axis and

neurodevelopmental outcome. Toxicol Sci 132(1):177–195.

7 Gohner C, Svensson-Arvelund J, Pfarrer C, Hager JD, Faas M, Ernerudh J, Cline JM, Dixon D, Buse E, Markert UR.

2013. The placenta in toxicology. Part IV: Battery of toxicological test systems based on human placenta. Toxicol

Pathol; doi:10.1177/0192623313482206 [Online 2 April 2013].

7 Graves JP, Edin ML, Bradbury JA, Gruzdev A, Cheng J, Lih FB, Masinde TA, Qu W, Clayton NP, Morrison JP, Tomer

KB, Zeldin DC. 2013. Characterization of four new mouse cytochrome P450 enzymes of the CYP2J subfamily. Drug

Metab Dispos 41(4):763–773 [Online 15 January 2013].

2 Guo NL, Wan YY, Denvir J, Porter DW, Pacurari M, Wolfarth MG, Castranova V, and Qian Y. 2012. Multi-walled

carbon nanotube-induced gene signatures in the mouse lung: potential predictive value for human lung cancer risk

and prognosis. J Toxicol Environ Health A 75(18):1129–1153.

7 Gwinn WM, Wei Q, Shines CJ, Bousquet RW, Taylor GJ, Waalkes MP, Morgan DL. 2013. Macrophage solubilization

and cytotoxicity of indium-containing particles in vitro. Toxicol Sci 135(2):414–424.

7 Hakk H, Szabo DT, Huwe J, Diliberto J, Birnbaum LS. 2012. Novel and distinct metabolites identified following a single

oral dose of alpha- or gamma-hexabromocyclododecane in mice. Environ Sci Technol 46(24):13494–13503.

7 Hall AP, Elcombe CR, Foster JR, Harada T, Kaufmann W, Knippel A, Kuttler K, Malarkey DE, Maronpot RR, Nishikawa

A, Nolte T, Schulte A, Strauss V, York MJ. 2012. Liver hypertrophy: a review of adaptive (adverse and non-adverse)

changes-conclusions from the 3rd international ESTP expert workshop. Toxicol Pathol 40(7):971–994.

6 Hannas BR, Howdeshell KL, Furr J, Gray LE Jr. 2013. In utero phthalate effects in the female rat: a model for MRKH

syndrome. Toxicol Lett 223(3):315–321 [Online 28 March 2013].

3 Hansen DK, George NI, White GE, Abdel-Rahman A, Pellicore LS, Fabricant D. 2013. Questionable conclusions in

the article “cardiovascular toxicity of Citrus aurantium in exercised rats’’ response. Cardiovasc Toxicol 13(2):182–183.

3 Hansen DK, George NI, White GE, Abdel-Rahman A, Pellicore LS, Fabricant D. 2013. Cardiovascular toxicity of citrus

aurantium in exercised rats. Cardiovasc Toxicol 13(3):208–19 [Online 12 February 2013].

7 Harry GJ. 2013. Microglia during development and aging. Pharmacol Ther 139(3):313–326.
82 National Toxicology Program Annual Report FY 2013

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 MOU

6 Funded by the NIEHS/EPA Interagency Agreement 

7 Funded by NIEHS voluntary allocations to the NTP

4 He W, Zhou YT, Wamer WG, Hu X, Wu X, Zheng Z, Boudreau MD, Yin JJ. 2013. Intrinsic catalytic activity of Au

nanoparticles with respect to hydrogen peroxide decomposition and superoxide scavenging. Biomaterials 34(3):765–

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4 He Z, Ferguson SA, Cui L, Greenfield LJ Jr, Paule MG. 2013. Role of neural stem cell activity in postweaning

development of the sexually dimorphic nucleus of the preoptic area in rats. PLoS One 8(1):e54927 [Online 6 February

2013].

7 Hobbs CA, Mercado-Feliciano M, Cora MC, McIntyre BS, Stout MD, Foster PM, Shepard KG, Swartz CD, Garantziotis

S, Witt KL, Smith-Roe SL. 2013. Black cohosh extract is genotoxic in rodents and induces hematological changes

associated with disruption of folate metabolism. Environ Mol Mutagen 54:S27.

7 Hoenerhoff MJ, Pandiri AR, Snyder SA, Hong HHL, Ton TV, Peddada S, Shockley K, Witt K, Chan P, Rider C,

Kooistra L, Nyska A, Sills RC. 2013. Hepatocellular carcinomas in B6C3F1 mice treated with Ginkgo biloba extract

for two years differ from spontaneous liver tumors in cancer gene mutations and genomic pathways. Toxicol Pathol 

41(6):826–841.

7 Hong SP, Fuciarelli AF, Johnson JD, Graves SW, Bates DJ, Waidyanatha S, Smith CS. 2013. Toxicokinetics of

methyleugenol in F344 rats and B6C3F1 mice. Xenobiotica 43(3):293–302.

7 Humble MM, Young MJ, Foley JF, Pandiri AR, Travlos GS, Copeland WC. 2013. Polg2 is essential for mammalian

embryogenesis and is required for mtDNA maintenance. Hum Mol Genet 22(5):1017–1025 [Online 1 December 2012].

7 Humblet O, Korrick SA, Williams PL, Sergeyev O, Emond C, Birnbaum LS, Burns JS, Altshul LM, Patterson Jr DG,

Turner WE, Lee MM, Revich B, Hauser R. 2013. Genetic modification of the association between peripubertal dioxin

exposure and pubertal onset in a cohort of Russian boys. Environ Health Perspect 121(1):111–117.

4 Imam SZ, Trickler W, Kimura S, Binienda ZK, Paule MG, Slikker W Jr, Li S, Clark RA, Ali SF. 2013. Neuroprotective

efficacy of a new brain-penetrating C-abl inhibitor in a murine Parkinson’s disease model. PLoS One 8(5):e65129

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4 James SJ, Shpyleva S, Melnyk S, Pavliv O, Pogribny IP. 2013. Complex epigenetic regulation of Engrailed-2 (EN-2)

homeobox gene in the autism cerebellum. Transl Psychiatry 3:e232.

4 Jevtovic-Todorovic V, Absalom AR, Blomgren K, Brambrink A, Crosby G, Culley DJ, Fiskum G, Giffard RG, Herold

KF, Loepke AW, Ma D, Orser BA, Planel E, Slikker W, Soriano SG, Stratmann G, Vutskits L, Xie Z, Hemmings HC.

2013. Anaesthetic neurotoxicity and neuroplasticity: An expert group report and statement based on the BJA Salzburg

Seminar. Br J Anaesth 111(2):143–151.

4 John K, Pratt M, Beland FA, Churchwell MI, McMullen G, Olivero OA, Pogribny IP, Poirier MC. 2012. Benzo[a]pyrene

(BP) DNA adduct formation in DNA repair-deficient p53 haploinsufficient [Xpa(-/-)p53(/-)] and wild-type mice fed BP

and BP plus chlorophyllin for 28 days. Carcinogenesis 33(11):2236–2241.

4 Johnson N, Doelling V, Jones B, eds. 2013. International Workshop on Alternative Methods for Vaccine Potency

Testing: State of the Science and the Way Forward. Biologicals 41(5):279–338.

4 Jones YL, Peters SM, Weland C, Ivanova NV, Yancy HF. 2013. Potential use of DNA barcodes in regulatory science:

identification of the U.S. Food and Drug Administration’s “Dirty 22,” contributors to the spread of foodborne pathogens.

J Food Prot 76(1):144–149 [Online 16 January 2013].

5,7 Judson R, Kavlock R, Martin M, Reif D, Houck K, Knudsen T, Richard A, Tice RR, Whelan M, Xia MH, Huang RL,

Austin C, Daston G, Hartung T, Fowle JR, Wooge W, Tong WD, Dix D. 2013. Perspectives on validation of high

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5 Funded by the NTP/NHGRI/EPA/FDA Interagency MOU

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7 Davis BJ, Fenton SE. 2013. The mammary gland. In: Haschek and Rousseaux’s Handbook of Toxicologic Pathology,

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7 Rao DB, Herbert RA, Brix AE. 2013. Practice of toxicologic pathology: nomenclature. In: Haschek and Rousseaux’s

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B, eds). Elsevier, Inc., Academic Press, Chapter 16, 539–550.

7 Schug TT, Howard SG, Taylor KW, Heindel JJ. 2012. Obesity and diabetes: role of environmental chemical exposures.

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Exposures. (Weiss B, ed). RSC Publishing, 201–240.

7 Stokes WS, Marsman D. 2013. Animal welfare considerations for biomedical research and testing. In: Laboratory

Animal Welfare. American College of Laboratory Animal Medicine Series. (Bayne T, ed). New York:Elsevier.

7 Tokar EJ, Boyd WA, Freedman JH, Waalkes MP. 2013. Toxic effects of metals. In: Casarett and Doull’s Toxicology:

The Basic Science of Poisons, 8th Edition. (Klaassen CD, ed). New York:McGraw-Hill, 981–1030.

7 White SS, Fenton SE. 2013. Breast cancer—importance of life stage with respect to environmental influences. In:

Issues in Toxicology Series: Aging and Vulnerability to Environmental Chemicals: Age-related Disorders and Their

Origins in Environmental Exposures. (Weiss B, ed). Cambridge, United Kingdom:The Royal Society of Chemistry,

Chapter 11, 285–320.
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