National Toxicology Program

U.S. Department of Health and Human Services

Annual Report 2012
National Toxicology Program

ANNUAL REPORT

for

Fiscal Year 2012

National Institute of Environmental Health Sciences 

National Institutes of Health

National Center for Toxicological Research

Food and Drug Administration

National Institute for Occupational Safety and Health

Centers for Disease Control and Prevention

September 2013

Department of Health and Human Services

National Toxicology Program

NIH Publication No. 13-5970

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. NIEHS Strategic Plan ................................................................................................................ 17

B. Systematic Review Process ...................................................................................................... 17

C. Health Effects of Low-Level Lead.............................................................................................. 18

D. Studies of Botanical Dietary Supplements ................................................................................ 18

E.	 Diversity-Outbred Mice and Population Based Models for Toxicology and Disease ................. 19

F.	 Mouse Methylome Project......................................................................................................... 20

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

H. Additional Activities.................................................................................................................... 22

4. Literature Analysis Activities ........................................................................................................ 23

A. Non-Cancer Health Effects ....................................................................................................... 23

B. Report on Carcinogens.............................................................................................................. 24

5. Testing and Research .................................................................................................................... 26

A. Technical Reports ...................................................................................................................... 27

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

i.	 Disposition, Metabolism, and Toxicokinetic Studies .........................................................33

ii. Genetic Toxicity ................................................................................................................34

iii. Organ System Toxicity......................................................................................................35

iv. Toxicology/Carcinogenicity Studies ..................................................................................41

v. Toxicogenomic Studies.....................................................................................................46

C. NTP Research ........................................................................................................................... 48

i.	 Assessing Exposure to Substances in the Workplace .....................................................51

ii. Comprehensive Assessment of Occupationally-Relevant Exposures..............................55

iii. Immunotoxicology Research ............................................................................................56

iv. NTP Research in Partnership with NCTR ........................................................................59
6. Alternative Methods Development................................................................................................ 70

A. Tox 21........................................................................................................................................ 70

B.	 ICCVAM..................................................................................................................................... 74

i.	 Workshops........................................................................................................................74

ii. Publications ......................................................................................................................75

iii. International Validation Activities ......................................................................................76

7. Public Tools .................................................................................................................................... 78

A. Chemical Effects in Biological Systems (CEBS) Database ....................................................... 78

B. NTP Archives............................................................................................................................. 78

C. The NCGC BioPlanet ................................................................................................................ 79

D.	 DrugMatrix® and ToxFX®.......................................................................................................... 79

E.	 MetaDataViewer........................................................................................................................ 80

8. Appendices ..................................................................................................................................... 81

A. Frequently Used Abbreviations.................................................................................................. 81

B. Agency Staff and Contact Information....................................................................................... 83

C. FY 2012 Bibliography ................................................................................................................ 87
Letter from the NIEHS/NTP Director 1

Letter from the NIEHS/NTP Director

The NTP, an interagency program, provides scientific knowledge and functions as a truly

translational program to safeguard public health. The NTP is involved in activities to 

evaluate substances of potential concern for our health and to develop new methods to 

improve such evaluation procedures. For example, the NTP is taking on the challenge of

studying mixtures instead of testing one chemical at a time. In addition, a current project

is examining the effects of exposures throughout a life span, to expand our knowledge

about effects of prenatal exposures and how they may link to adult disease. The NTP is

also leading the Tox21 initiative with the National Institutes of Health’s National Center for 

Advancing Translational Science, the United States Environmental Protection Agency, 

and the Food and Drug Administration. Tox21 is a high throughput-testing program that shows great promise 

for moving toxicology into a predictive science. 

As it has done in the past, in fiscal year 2012 (FY 2012), the NTP through scientific collaborations with

federal agencies continued to enhance our knowledge about substances that affect health in our homes,

workplaces, and other environments. The NTP translated and communicated these findings to the public,

industry, federal regulatory agencies, and others so the information could be used to protect public health. The 

NTP disseminated reports on studies of dietary supplements, skin care products, and widely used industrial

chemicals through publication of nine NTP Technical Reports. The final NTP Monograph on Heath Effects of 

Low-level Lead was published, providing an overview of the science to date and conclusions on the potential 

health effects from low-level exposures to lead. The NTP also carried out two widely attended workshops: (1)

the International Workshop on Alternative Methods for Human and Veterinary Rabies Vaccine Testing: State 

of the Science and Planning the Way Forward and (2) the International Workshop on Alternative Methods for 

Leptospira Vaccine Potency Testing: State of the Science and the Way Forward.

Of broad interest both nationally and internationally, the NIEHS Office of Health Assessment and Translation

initiated modifications in how it would conduct NTP literature-based analysis of existing studies to assess

if a substance may be of concern for human health. This effort focuses on incorporating systematic review 

methodologies to increase transparency in conducting and communicating these assessments. In addition, 

revisions to the multi-step process used to prepare the Biennial Report on Carcinogens were put in place and 

the evaluation of substances for future reports has begun.

The ability of the NTP to stay on the cutting edge of scientific research and to pioneer the development and

application of new technologies is the reflection of a staff truly dedicated to the NTP and its mission. I am

pleased to share through this report some of our achievements in FY 2012.

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/

newchems/pubs/invntory.htm) lists more than 83,000 chemicals as 

being available for sale and use in the United States. New chemicals 

are continuously introduced into the Unites States 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, or soil. 

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

Department of Health and Human Services, HHS) established the 

National Toxicology Program (NTP) in 1978 as a focal point to coordinate toxicology testing in the Federal 

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

1. Coordinate toxicology testing programs within the federal government.

2. Strengthen the science base in toxicology.

3. Develop and validate improved testing methods. 

4. 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. The 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 (see Table X). The American people and

government agencies at state and Federal levels rely on the 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, the

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 NIEHS/NTP 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, the 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, within the Division of NTP, at the NIEHS (919-541-7539 or wolfe@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 2012

A. Organizational Structure and Oversight

Three agencies form the core for the NTP (Figure 1): the National Institute of Environmental Health Sciences

(NIEHS) of the National Institutes of Health, the National Institute for Occupational Safety and Health (NIOSH) 

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

through its National Center for Toxicological Research (NCTR). 

The NTP is located administratively at the NIEHS, and Linda Birnbaum, PhD, DABT, ATS, serves as the 

Director of the NIEHS and the NTP Director. John Bucher, PhD, serves as associate director of the NTP 

and director of the Division of the NTP at NIEHS, which is dedicated toward carrying out NTP activities. The 

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 in innovation and the application

of research to solve public health problems.

John Howard, MD, MPH, JD, LLM, is the director of NIOSH, and Gayle Debord, PhD, Chief, Biomonitoring

and Health Assessment Branch, manages the NTP program at NIOSH. Staff within the following divisions 

participate in NTP activities: Division of Surveillance, Hazard Evaluations, and Field Studies; Division of

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

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

transfer that knowledge into practice for the betterment of workers. NIOSH’s participation in the 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., PhD, is the director of NCTR, and Paul Howard, PhD, Associate Director, Office of

Scientific Coordination, manages the NTP program at NCTR. 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. The NCTR conducts an

array of studies that reflect the NTP mission statement and are critical in supporting FDA product centers and

their regulatory roles.

Figure 1. Organizational Structure of NTP

Assistant Secretary for Health

NTP Core Agencies Policy Oversight External Science Oversight and Review

NTP Director

• CDC/NIOSH • NTP Executive Committee

• FDA/NCTR

• NIH/NIEHS

• Board of Scientific Councilors

• Scientific Advisory Committee on 

Alternative Toxicological Methods

• 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 2012

B. Training Programs

The 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 2012, NTP staff mentored 19 

postdoctoral fellows at the NIEHS.

Table 1. NTP Training Program Postdoctoral Fellows in FY 2012

Training Program Fellow

Applied toxicology and carcinogenesis 

Minerva Mercado-Feliciano

Kristen Ryan

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

Erin Quist

C. Advisory Boards and Committees

Three formal groups (NTP Executive Committee, NTP Board of Scientific Counselors, and Scientific Advisory

Committee on Alternative Toxicological Methods) provide policy, or science oversight and peer review, to 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. Agency for Toxic Substances and Disease Registry/National Center for Environmental Health 

(ATSDR/NCEH)

● 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 Institute of Environmental Health Sciences (NIEHS)

● National Institute for Occupational Safety and Health (NIOSH)

● Occupational Safety and Health Administration (OSHA)

To enhance agency interactions, the NTP uses agency Points of Contact in lieu of formal committees 

to streamline communication and better utilize agency staff. Agency Points of Contact have a dedicated 

responsibility and time commitment, are knowledgeable about the NTP mission and 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 the NTP on the merit of its programs and activities. The Secretary of HHS 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 the NTP’s activities. The BSC charter and current roster are available at http://

ntp.niehs.nih.gov/go/164. Dr. Lori White, Designated Federal Officer, manages the BSC. Table 2 provides the

BSC membership roster for FY 2012.

BSC members and NTP staff at the June 2012 BSC meeting. 

The BSC met twice in FY 2012 

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

At the meeting held on December 

15, 2011, the BSC reviewed draft 

testing concepts for the study of 

sulfolane, phenolic 

bensotriazoles, and 

trimethylsilyldiazomethane. The 

BSC heard reports on an NTP 

Workshop: Role of Environmental 

Chemicals in the Development of 

Diabetes and Obesity and on a 

revised process for preparing the 

Report on Carcinogens (RoC). 

They reviewed a concept for a 

RoC workshop on permanent hair

dyes and heard updates on 

NTP’s progress since the April 

2011 meeting from the NIEHS/

NTP Director and NTP Associate 

Director.
8 National Toxicology Program Annual Report 2012

The second BSC meeting was held on June 21–22, 2012. The BSC voted unanimously to approve the 

concept for a contract for genetic toxicity testing. NTP staff presented an initiative to incorporate environmental 

enrichment into NTP studies and the recommendations for seven draft NTP Technical Reports from a 

peer-review panel convened on February 8-9, 2012. The BSC reviewed draft concepts on five substances

nominated for review for the RoC: pentachlorophenol, cumene, trichloroethylene, ortho-toluidine, and

1-bromophenol. The NIEHS/NTP Director and NTP Associate Director provided reports, updating the BSC on 

NTP’s progress since the December 2011 meeting. 

The BSC formed a working group, which met on August 28–29, 2012, in Raleigh, NC, to evaluate the Draft

NTP Approach for Reaching Conclusions for Literature-Based Evidence Assessments. The NIEHS Office

of Health Assessment and Translation (OHAT), in concert with the Office of Liaison, Policy and Review,

developed this approach during 2012 to incorporate elements of systematic review methodology into literaturebased evaluations. The working group, chaired by Dr. Lynn Goldman, dean of the George Washington School

of Public Health, prepared a report with recommendations for revisions to the approach. The BSC reviewed 

and approved the report, enabling formal transmittal to the NTP. NTP staff discussed changes to the draft 

approach based upon the working group’s input and presented next steps toward finalization of the approach.

Table 2. NTP Board of Scientific Counselors Membership Roster FY 2012

Name and Title Affiliation Term Ends

Robert E. Chapin, PhD

Laboratory Director

Pfizer

Groton, CT 06/30/15

David C. Dorman, DVM, PhD

Professor 

College of Veterinary Medicine

North Carolina State University

Raleigh, NC 06/30/15

David A. Eastmond, PhD (chair)

Professor and Chair

Department of Cell Biology and Neuroscience

University of California

Riverside, CA 12/27/12

Elaine M. Faustman, PhD

Professor and Director

Institute for Risk Analysis and Risk Communication

Department of Environmental and Occupational Health Sciences

University of Washington

Seattle, WA 12/27/12

Miguel C. Fernández, MD, FACEP, FAAEM, FACMT, FAACT

Professor of Surgery

Division of Emergency Medicine

Director, South Texas Poison Center

University of Texas Health Science 

Center

San Antonio, TX

06/30/13

Jack R. Harkema, DVM, PhD, DACVP

Distinguished Professor

Department of Pathobiology and Diagnostic Investigation

Michigan State University

East Lansing, MI 06/30/15

Dale Hattis, PhD

Research Professor

George Perkins Marsh Institute

Clark University

Worcester, MA 06/30/15

Dana Loomis, PhD

Professor and Chair

Department of Epidemiology

University of Nebraska

Medical Center

Omaha, NE

12/27/12

Stephen W. Looney, PhD

Professor

Department of Biostatistics

Department of Oral Health and Diagnostic Science

Georgia Health Sciences 

University

Augusta, GA 

06/30/12
1. National Toxicology Program: Mission And Goals 9

Name and Title Affiliation Term Ends

Melissa A. McDiarmid, MD, MPH

Professor of Epidemiology and Preventive Medicine

Director, Occupational Health Program

University of Maryland

School of Medicine

Baltimore, MD

06/30/13

Lisa Minor, PhD

Consultant

In Vitro Strategies, LLC 

Flemington, NJ 6/30/13

Richard Miller, DVM, PhD

Vice President

Safety Assessment 

GlaxoSmithKline 

Research Triangle Park, NC 06/30/13

Mitzi Nagarkatti, PhD

Professor and Chair

Department of Pathology, Microbiology and Immunology

University of South Carolina 

School of Medicine

Columbia, SC

12/27/11

Ruthann A. Rudel, MS

Senior Scientist

Toxicology and Environmental Health Risk Assessment

Silent Spring Institute

Newton, MA 12/27/11

Sonya Sobrian, Ph.D.

Associate Professor

Department of Pharmacology

Howard University

Washington, DC 06/30/15

Gina M. Solomon, MD, MPH

Senior Scientist

Natural Resources Defense 

Council

San Francisco, CA 

12/27/11

Justin G. Teeguarden, PhD

Senior Scientist

Fundamental and Computational Sciences Directorate

Pacific Northwest National

Laboratory

Richland, WA

12/27/11

Judith Zelikoff, PhD

Professor of Environmental Medicine

Director, Community Outreach

New York University

School of Medicine

Tuxedo, NY

12/27/12

iii. Scientific Advisory Committee on Alternative Toxicological Methods

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

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

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

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

(NICEATM), and the Director of NIEHS regarding statutorily mandated duties of ICCVAM and activities of

NICEATM (see page 74). 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 SACATM membership roster for

FY 2012. SACATM typically meets once a year and members serve rotating terms of up to four years. Dr. Lori

White, Designated Federal Officer, manages SACATM.

SACATM met once during FY 2012 at the NIEHS, on September 5–6, 2012 (http://ntp.niehs.nih.gov/

go/8202). At that meeting, SACATM voted unanimously to support ICCVAM’s high priority rating for

the nomination of the Electrophilic Contact Allergen Identification Screening test and to use a contract

mechanism for continuation of administrative and scientific support for NICEATM. SACATM voted to accept

the report prepared by the Implementation Working Group with advice on implementation of ICCVAMrecommended methods. They were provided information on high-throughput adaptation and performance of 
10 National Toxicology Program Annual Report 2012

the BG1Luc ER TA agonist and antagonist assays and federal agency research, development, translation, 

and validation activities relevant to the NICEATM-ICCVAM Five-Year Plan. Additionally, they commented on

a draft NICEATM-ICCVAM Five-Year Plan. They heard a report on the International Workshop on Alternative 

Methods for Human and Veterinary Rabies Vaccine Testing: State of the Science and Planning the Way 

Forward and about plans for an upcoming vaccine workshop. Liaisons from the European Union Reference

Laboratory for Alternatives to Animal Testing /European Center for the Validation of Alternative Methods,

the Korean Center for the Validation of Alternative Methods, the Japanese Center for the Validation of

Alternative Methods, and Health Canada presented updates on the activities of their groups.

SACATM formed the Implementation Working Group to assess implementation of ICCVAMrecommended alternative methods. Dr. Joy Canvagnaro chaired the working group, which met eight

times by teleconference during FY 2012. The working group prepared a report with recommendations

for ICCVAM and NICEATM. Most of the recommendations concerned improvements in ICCVAM data

collection regarding implementation of ICCVAM-recommended alternative methods by both industry and

regulatory agencies. The group also recommended specific actions to enhance communication regarding

implementation with the regulatory agencies, specifically the EPA and the FDA. SACATM reviewed and

approved the report at the September 2012 meeting.

Members of SACATM, ICCVAM, and NIEHS/NTP staff at the September 2012 SACATM meeting.
1. National Toxicology Program: Mission And Goals 11

Table 3. NTP SACATM Membership Roster FY 2012

Name and Title Affiliation Term Ends

Laura Andrews, PhD, DABT 

Vice President

Pharmacology and Toxicology 

Genzyme Corporation 

Framington, MA 06/30/12

Tracie E. Bunton, DVM, PhD

Consultant

Eicarte LLC

Gettysburg, PA 06/30/15

Joy Cavagnaro, PhD, DABT, RAC, ATS, RAPS

President and Founder

Access BIO, L.C.

Boyce, VA 11/30/14

Joan M. Chapdelaine, Ph.D.

Consultant Scott Township, PA 06/30/15

Eugene L. Elmore, PhD 

Senior Project Scientist

Department of Radiation Oncology 

University of California

Irvine, CA 06/30/12

Mark G. Evans, DVM, PhD, ACVP

Research Fellow

La Jolla Laboratories 

Pfizer

San Diego, CA 06/30/15

Steven R. Hansen, DVM, MS, MBA, DABT, ABVT

Senior Vice President

Animal Health Services

ASPCA Animal Poison Control 

Center

Urbana, IL

06/30/12

Gwendolyn Y. McCormick, DVM, MS, DACLAM

Attending Veterinarian and Distinguished Research Fellow

Animal Resources Department

Boehringer Ingelheim 

Pharmaceuticals, Inc

Ridgefield, CT

06/30/12

Steven M. Niemi, DVM

Director

Center for Comparative Medicine

Massachusetts General Hospital

Charlestown, MA 06/30/13

Ricardo Ochoa, DVM, PhD, ACVP

President and Principal

Pre-Clinical Safety, Inc.

Niantic, CT 11/30/14

Michael J. Olson, PhD, ATS

Director

Occupational Toxicology Corporate Environment, Health, Safety and 

Sustainability 

GlaxoSmithKline 

Research Triangle Park, NC 06/30/13

Linda A. Toth, DVM, PhD

Associate Dean for Research and Faculty Affairs 

Professor, Department of Pharmacology 

Southern Illinois University 

School of Medicine

Springfield, IL

06/30/13

Daniel M. Wilson, PhD, DABT

Mammalian Toxicology Consultant

Toxicology and Environmental Research and Consulting

The Dow Chemical Company

Midland, MI 11/30/14

Marilyn Wind, PhD

Consultant Bethesda, MD 06/30/15

Interagency Coordinating Committee on the Validation of Alternative Methods

ICCVAM is a permanent interagency committee of the NIEHS under NICEATM. The committee was formally

established by the ICCVAM Authorization Act of 2000 (42 U.S.C. 285l-3). The purpose of ICCVAM 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
12 National Toxicology Program Annual Report 2012

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

○ Centers for Disease Control and Prevention

▪ Agency for Toxic Substances and Disease Registry (ATSDR)

▪ National Institute for Occupational Safety and Health (NIOSH)

○ 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

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

● U.S. Department of Labor

○ Occupational Safety and Health Administration (OSHA)

● U.S. Department of Transportation

● U.S. Environmental Protection Agency (EPA)

iv. Special Emphasis Panels

The NTP uses ad hoc scientific experts, referred to as special emphasis panels (SEPs), as needed, to provide

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

new/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. On February 8–9, 2012, the NTP convened an external, scientific panel

to peer review seven draft NTP TRs at a public meeting at the NIEHS with opportunity for public comment. The 

panel was charged to (1) peer review the scientific and technical elements of the study and their presentation

and (2) determine whether the study’s experimental design and conduct supported the NTP’s conclusions 

regarding the carcinogenic activity of the substance tested. The panel reviewed the draft TRs for NTP studies 

on N,N-dimethyl-p-toluidine, Ginkgo biloba extract, β-picoline, pyrogallol, trimethylolpropane triacrylate,

3′-azido-3′-deoxythymidine (AZT), and mixtures of 3′-azido-3′-deoxythymidine (AZT), lamivudine (3TC), and 

nevirapine (NVP). Ms. Danica Andrews served as the Designated Federal Official for the peer-review meeting.
1. National Toxicology Program: Mission And Goals 13

Additional information about this past meeting and other TR review panel meetings is available at http://ntp.

niehs.nih.gov/go/36051.

NTP Monographs Peer Review Panels

Monographs are publications of a single, detailed specific topic. On November 17-18, 2011, the NTP convened

an external, scientific panel to peer review the Draft NTP Monograph on Health Effects of Low-level Lead

at the NIEHS. The panel was charged (1) to determine whether the scientific information cited in the draft

monograph is technically correct, clearly stated, and objectively presented and (2) to determine whether the

scientific evidence presented in the draft monograph supports the NTP’s conclusions regarding health effects

of low-level lead. The meeting was open to the public with time scheduled for oral public comment. The panel 

consisted of experts to review the topics of exposure and the major health effect areas of immune, kidney,

cardiovascular, neurological, and reproductive and developmental, for both of the life stages children and 

adults. Ms. Danica Andrews served as the Designated Federal Official for the peer-review meeting. After the

meeting, the input from the panel was considered in finalizing the monograph. Additional information about

this past meeting and other NTP Monograph review panel meetings is available at http://ntp.niehs.nih.gov/

go/36639. 

The final monograph on low-level lead was released in June 2012 and can be found at http://ntp.niehs.nih.gov/

go/36443.
14 National Toxicology Program Annual Report 2012

2. Funding

Current and Projected Research Capacity

The 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 2012 was $126.8 million.

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

through Interagency Agreements with other agencies (see page 16). In FY 2012, the NIEHS funded 40 

contracts (see Table 4), held two workshops (see page 74), two special emphasis expert panel peer-review

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

Figure 2: Past, Current and Projected Budget

FY 2013 ($120.8 million projected)

NIEHS

NCTR

NIOSH

10.7% ($13.5) 9.9% ($12)

6.4% ($7.7) 5.4% ($6.9)

83.7% ($101.1)

FY 2010 ($122.6 million)

4.2% ($5.2)

13.1% ($16)

82.7% ($101.4)

FY 2012 ($126.8 million)

83.9% ($106.4)

FY 2011 ($126 million)

($12.7)

4.8% ($6.1)

85.1% ($107.2)

10.1%
2. Funding 15

Table 4. NTP Contracts that Supported NTP Testing Activities in FY 2012

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

Bioinformatics support Kelly Scientific

Chemistry support Research Triangle Institute

Evaluate the toxicological potential for selected agents Battelle Memorial

Evaluation of alternative toxicological methods Integrated Laboratory Systems

Genetic toxicity in bacteria and rodents Integrated Laboratory Systems

High throughput sequencing Lieber Institute

Inhalation toxicology of environmental chemicals Alion Science and Technology

Investigative research support Integrated Laboratory Systems

NTP computer and user support Vistronix, Inc.

NTP information systems support Scimetrika

Pathology support

Charles River

Experimental Pathology Labs

Integrated Laboratory Systems

Ramazzini

Pathology support for quality assessment Experimental Pathology Labs

Potential for environmental & therapeutic agents Virginia Commonwealth

Production of B6C3F1 mice Taconic Farms

Provantis software INSTEM

Provision for animals and specialized services

Charles River

Harlan Laboratories

Jackson Laboratories

Taconic Farms

QA and auditing Labscience

Quality assessment support for conducts of audits Dynamac

Reproductive assessments by continuous breeding Research Triangle Institute

Scientific software Leadscope, Inc.

Scientific software support DrugMatrix

Thomson Reuters Scientific

Statistical support SRA International

Studies to evaluate effects of chemicals Research Triangle Institute

Technical reports preparation support services Biotechnical Sciences

Toxicological & carcinogenic potential of chemicals Battelle Memorial

Toxicological & carcinogenic potential of lab animals Battelle Memorial
16 National Toxicology Program Annual Report 2012

Interagency Agreements

In FY 2012, the NIEHS provided support for NTP activities through interagency agreements with NIOSH 

and NCTR. Beginning in 1992, the NIEHS established an interagency agreement with FDA to support 

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

been primarily to study chemicals where the FDA has no legal authority to require the regulated community to 

provide toxicological data for a product. This agreement has led to assessments of toxicity for many classes 

of chemicals, including cosmetics, endocrine-disrupting compounds, food contaminants, food cooking

by-products, dietary supplements, drugs, and anesthetics and investigations of mechanisms of action. The 

studies are conducted at the NCTR. In addition, the interagency agreement partially supports the NCTR/Office

of Regulatory Affairs Phototoxicity Research and Testing Laboratory.

In 1997, the NIEHS began 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, the NTP and NIOSH are coordinating efforts to increase knowledge,

identification, and education of occupational health research. NIOSH and NTP are also working together

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

environmental exposure to chemical or physical agents.

In 2008, the NTP entered into a partnership with the National Human Genome Research Institute’s NIH 

Chemical Genomics Center (NCGC, now the NIH National Center for Advancing Translational Sciences) 

and the EPA’s National Center for Computational Toxicology (NCCT), through a five-year memorandum of

understanding (see http://ntp.niehs.nih.gov/go/28213 for information about the memorandum). This interagency 

partnership is the basis for the United States Tox21 program (see page 70) and supports the NCGC BioPlanet 

(see page 79).

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

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

findings from this interagency agreement, which came to an end in FY 2011, were published in FY 2012.
3. NTP Highlighted Activities 17

3. NTP Highlighted Activities

Figure 3 displays the major events for the NTP from FY 2012. Below are additional activities of importance

from FY 2012.

Figure 3: FY 2012 Highlights

A. NIEHS Strategic Plan

In FY 2012, the NIEHS released its Strategic Plan that set the Institute’s scientific and

governance direction for the next five years (2012–2017). The NIEHS initiated the

strategic planning process in 2011 and solicited stakeholder input before completion.

The strategic plan includes the institute’s mission, vision, major themes, crosscutting

themes, and strategic goals. As a division of NIEHS, the NTP’s toxicology research 

will continue to be integrated into the basic and translation programs at NIEHS to 

protect public health and solve health problems. The NIEHS strategic plan can be 

found at http://www.niehs.nih.gov/about/strategicplan/. 

B. Systematic Review Process

In FY 2012, the NIEHS’ Office of Health Assessment and Translation (OHAT) and Office of Liaison, Policy and

Review initiated an effort to incorporate systematic review methodology into NTP literature-based evaluations. 

Systematic reviews use a standardized methodology to identify relevant research and to report and critically 

appraise data from the studies that are included in a review. The systematic review format helps provide a 

structure to guide identification and determination of literature for inclusion, as well as extraction of data from

studies, assessment of study quality and reporting, and synthesis of data toward reaching a conclusion. 

Currently, systematic reviews are most utilized in clinical epidemiology for health care interventions; however,

there is increasing interest in adopting this format in the environmental health sciences and risk assessment.

 OCT ‘11 NOV ‘11 DEC ‘11 JAN ’12 FEB ’12 MAR ’12 APR ’12 MAY ’12 JUNE ’12 JULY ’12 AUG ’12 SEPT ’12 

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

Health Effects of Lowlevel Lead Peer Review 

Panel Meeting

See page 13

Technical Reports Peer 

Review Panel Meeting

See page 12

ICCVAM-NICEATM

International Workshop on 

Alternative Methods for Leptospira 

Vaccine Potency Testing

See page 75

ICCVAM-NICEATM

International Workshop on 

Alterative Methods for Human and 

Veterinary Rabies Vaccine Testing

See page 74

NTP at Society of Toxicology 

Meeting in San Antonio, TX

See page 22

NTP Satellite Symposium on 

Pathology Potpourri

See page 22

Board of Scientific

Counselors Meeting

See page 7

Board of Scientific

Counselors Meeting

See page 7

Release of NTP Monograph on

Health Effects of Low-Level Lead

See page 18

Release of 

NIEHS Strategic Plan

See page 17

SACATM

Meeting

See page 9
18 National Toxicology Program Annual Report 2012

OHAT’s goal is to provide a framework for carrying out literaturebased assessments and enhance the transparency in reaching and 

communicating NTP conclusions. The systematic review format includes 

seven steps: (1) prepare the topic, (2) search for and select studies for

inclusion, (3) extract data from studies, (4) assess the quality of individual 

studies, (5) rate the confidence in the body of evidence, (6) translate

confidence ratings into level of evidence for health effect, and (7) integrate

evidence to reach hazard identification conclusions. OHAT held two public

webinars in FY 2012 on March 20 and April 23 to discuss issues relating to

systematic review with a focus on approaches for assessing the quality of 

individual studies and risk of bias for individual studies and across studies.

With an initial introduction to the BSC at its meeting in June 2012, the NTP convened a BSC working group

(see page 8) to provide input regarding a draft approach in August 2012, with plans to present their report to 

the BSC and request public input in December 2012. In FY 2013, the goal is to obtain additional public input 

on a revised draft approach prior to OHAT moving forward with its implementation and evaluation. (http://ntp.

niehs.nih.gov/go/38673).

C. Health Effects of Low-Level Lead

In FY 2012, the NTP released the NTP Monograph on Health Effects of 

Low-Level Lead. Prior to publication, an ad hoc, scientific panel peer reviewed

the draft NTP monograph at a public meeting held November 17–18, 2011, at 

NIEHS (http://ntp.niehs.nih.gov/go/37090, see page 13). The NTP selected 

low-level lead for evaluation because of (1) the availability of a large number of 

epidemiological studies of lead, (2) a nomination by NIOSH for an assessment 

of lead at lower levels of exposure, and (3) public concern for effects of 

lead in children and adults. OHAT carried out the evaluation and produced 

the monograph. The monograph summarizes the evidence in humans and 

presents conclusions on health effects in children and adults associated 

with low-level lead exposure (less than 10 micrograms of lead per deciliter 

of blood). The assessment focused on epidemiological evidence at low 

blood lead levels because health effects at higher blood lead levels are well 

established. Overall, the NTP concluded that there is sufficient evidence that

low blood lead levels are associated with adverse health effects in children and adults. The monograph can be 

found at http://ntp.niehs.nih.gov/go/36443. 

D. Studies of Botanical Dietary Supplements

Ginkgo biloba extract 

Ginkgo biloba extract has been used for centuries in traditional Chinese medicine and 

today as a botanical supplement with the potential to improve brain function and for its 

antioxidant and anticancer effects. However, this botanical supplement has the potential for 

consumption over extended periods of time, with a general lack of sufficient data on longterm carcinogenicity risk. The NIEHS supported NTP studies that looked at the long-term

effects of Ginkgo extract in male and female F344/N rats and B6C3F1/N mice. Ginkgo biloba

extract was given orally to the animals for up to two years. At the end of the two-year studies, 
3. NTP Highlighted Activities 19

the NTP found an increase in liver cancer in male and female mice, and in cancer of the thyroid gland in male 

and female rats and male mice. The NTP Technical Report on the Toxicology and Carcinogenesis Studies of 

Ginkgo Biloba Extract was peer reviewed on February 8–9, 2012 (http://ntp.niehs.nih.gov/go/36144) and will be 

published in 2013 (http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/TR578_508.pdf). 

In a companion study, the NTP examined the genetic profile of liver tumors in mice treated with Ginkgo biloba

extract for two years. Exposure of B6C3F1/N mice to the extract in the two-year NTP carcinogenicity bioassay 

resulted in a dose-dependent increase in liver tumors, including development of cancerous tumors. This NTP 

study provides a molecular context for the genetic changes associated with the development of liver tumors 

in mice exposed to the Ginkgo biloba extract, and it illustrates the marked differences between those tumors

and tumors that developed spontaneously in the mice. Reports in the scientific literature tout health benefits of

Ginkgo biloba extract, although the NTP’s findings in this companion study suggest that long-term use of high

doses of the extract may pose potential health risks. The publication in Toxicologic Pathology is available at 

http://dx.doi.org/10.1177/0192623312467520.

Black cohosh extract

Black cohosh extract is used as a remedy for pain and gynecological ailments; modern

preparations are commonly sold as ethanolic extracts available as dietary supplements. 

The NIEHS supported NTP studies in rodents (female B6C3F1/N mice and Wistar Han 

rats) to characterize the general toxicity of black cohosh extract and to address suspected

estrogenic/anti-estrogenic activity. 

Increasing doses of black cohosh extract caused increasing severity of a specific type

of anemia along with increasing biomarkers of chromosomal damage in red blood

cells. Effects were more severe in mice (seen at all exposure dose levels) than rats. 

Dose-dependent thymus and liver toxicity was also observed in rats. Apparent effects 

on puberty were observed, but were not associated with alteration in estrogenic/antiestrogenic activity. These are the first studies to observe adverse effects of black cohosh extract in rodents.

Further studies are underway to determine the reproductive toxicity, immune toxicity, and carcinogenicity of 

black cohosh extract. The publication in Toxicology and Applied Pharmacology is available at http://dx.doi.

org/10.1016/j.taap.2012.05.022.

E. Diversity-Outbred Mice and Population Based Models for Toxicology and 

Disease

While there is great genetic diversity in human populations, many toxicology studies utilize mouse models that 

are genetically homogenous. Using genetically diverse mouse populations for studies can better indicate how 

human populations will respond to a substance. Such population-based models can be useful for quantifying 

how both genetic and epigenetic diversity impact the way animals react to potential toxins. Epigenetic diversity 

refers to differences in the expression of the genome that do not involve alterations in the actual DNA 

sequence. For example, previous NTP studies suggest that there are significant, population-based differences

for benzene-induced genotoxicity (direct or indirect damage to genes) and hematotoxicity (damage to blood) 

among mice. Benzene is a known toxicant that harms humans and mice.

To obtain a genetically diverse mouse population, NIEHS/NTP researchers crossed different mouse breeds 

to create the diversity-outbred mice. The resulting mouse population has greater genetic diversity than what 

exists in human populations. The diversity-outbred mice should allow the NTP to perform rodent epidemiology 
20 National Toxicology Program Annual Report 2012

studies in a controlled environment. Scientists will be able to link adverse effects (e.g., cancer or toxicity to

different organ systems) of exposure to a substance with particular genetic or epigenetic differences in the 

mouse population. 

Research to determine the utility of diversity-outbred mice for the NTP research and testing program continued 

in FY 2012. This work involved 28-day-old diversity-outbred male mice exposed to inhaled benzene (0, 1,

10, or 100 ppm; 75 mice/group) for five days per week for four weeks, in two independent studies. NTP staff

scientists evaluated the resulting data throughout FY 2012 and expect to complete the evaluation in FY 2013. 

Preliminary results indicate one specific gene is responsible for both increased resistance and susceptibility to

genotoxicity and hematotoxicity from benzene exposure.

F. Mouse Methylome Project

Susceptibility to toxicity or disease and how that is inherited depends on more than DNA sequence variations. 

The methylome, a component of the epigenome, is one of the other 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. DNA methylation is an important epigenetic modification of the genome that plays a major role

in development, toxicity, and disease. Presently, there is no mouse reference database for the methylome, 

which significantly handicaps an understanding of the mouse model in toxicology and environmentally related

diseases and in designing and conducting research to understand the associated mechanisms. 

The Mouse Methylome project, which is being carried out by NIEHS/NTP, will use Next Generation sequencing

technologies to create a high-resolution map of the mouse liver methylome from three different mouse strains 

(two parental strains C57BL/6N and C3H/HeN and their first generation hybrid offspring B6C3F1/N). These

strains show dramatically different incidences of spontaneous liver tumors, which often confound two-year 

toxicology and carcinogenesis studies. This variable incidence of liver cancer may be due in part to differences 

in the epigenetic machinery and also to 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, along with samples of four other tissues (brain, cardiac and skeletal muscle,

brown and white fat, epididymal sperm), from all mice in this project 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 2012, scientists completed

characterization of the liver transcriptome (the collection of RNA in the liver) and genomic sequence. Efforts 

will continue in FY 2013 to measure site-specific methylation of DNA.

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 2012.
3. NTP Highlighted Activities 21

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

Agencies in 2012

Notice NTP Information Cited Summary of Notice

California Office of Environmental Health

Changes to the 

Proposition 65:

added to list of 

chemicals known to

the State to cause 

cancer: isopyrazam

and 3,3’,4,4’- 

tetrachloroazobenzene

NTP Toxicology and 

Carcinogenesis Studies of 

3,3’,4,4’-Tetrachloroazobenzene 

(TCAB) (CAS No. 14047-09-7) 

Technical Report Series No. 558. 

NIH Publication No. 11-5899. 

http://ntp.niehs.nih.gov/ntp/htdocs/

LT_rpts/TR558.pdf

Effective July 24, 2012, the Office of Environmental Health

Hazard Assessment is adding two chemicals, isopyrazam 

(CAS No. 881685-58-1) and 3,3’,4,4’-tetrachloroazobenzene 

(CAS No. 14047-09-7), to the list of chemicals known to the

State to cause cancer for purposes of the Safe Drinking

Water and Toxic Enforcement Act of 1986 (commonly known

as Proposition 65). http://oehha.ca.gov/prop65/prop65_

list/072012listR.html

Centers for Disease Control and Prevention

Final Rule: World Trade

Center Health (WTC) 

Program; addition

of certain types of 

cancer to the list of 

WTC-related health 

conditions

Five main sources, including the 

NTP 12th Edition of the Report 

on Carcinogens, were used to 

evaluate whether to add cancers 

to the WTC-Related Health 

Conditions list. http://ntp.niehs.nih.

gov/?objectid=035E57E7-BDD9-

2D9B-AFB9D1CADC8D09C1

In accordance with WTC Health Program regulations, which 

establish procedures for adding a new condition to the list 

of covered health conditions, this final rule adds to the List

of WTC-Related Health Conditions the types of cancer 

proposed for inclusion by the notice of proposed rulemaking.

September 12, 2012 – 77 FR 567138 http://www.gpo.gov/

fdsys/pkg/FR-2012-09-12/pdf/2012-22304.pdf

U.S. Environmental Protection Agency

Final Rule: national

emission standards 

for hazardous air 

pollutants from coaland oil-fired electric

utility steam generating 

units and standards of 

performance for fossilfuel-fired electric utility,

industrial-commercialinstitutional, and small 

industrial-commercialinstitutional steam 

generating units

NTP Toxicology and 

Carcinogenesis Studies of Nickel 

Sulfate Hexahydrate (Cas No. 

10101-97-0) in F344/N Rats and 

B6C3F1 Mice (Inhalation Studies)

http://ntp.niehs.nih.gov/ntp/htdocs/

LT_rpts/tr454.pdf

NTP Toxicology and 

Carcinogenesis Studies of Nickel 

Subsulfide (CAS NO. 12035-72-2) 

in F344/N RATS and B6C3F1 Mice 

(Inhalation Studies) http://ntp.niehs.

nih.gov/ntp/htdocs/LT_rpts/tr453.pdf

On May 3, 2011, under authority of Clean Air Act (CAA)

sections 111 and 112, the EPA proposed both national 

emission standards for hazardous air pollutants (NESHAP) 

from coal- and oil-fired electric utility steam generating units

(EGUs) and standards of performance for fossil-fuel-fired

electric utility, industrial-commercial-institutional, and small 

industrial-commercial-institutional steam generating units (76 

FR 24976). After consideration of public comments, the EPA 

is finalizing these rules in this action.

February 16, 2012 – 77 FR 9304 http://www.gpo.gov/fdsys/

pkg/FR-2012-02-16/pdf/2012-806.pdf

Final Rule: 2017

and later model year 

light-duty vehicle 

greenhouse gas 

emissions and 

corporate average fuel 

economy standards

11th Edition of the Report on 

Carcinogens: acetaldehyde

and naphthalene are listed as 

reasonably anticipated to be 

human carcinogens; benzene,

1,3-butadiene, and formaldehyde 

are listed as known human

carcinogens. http://ntp.niehs.nih.

gov/ntp/roc/toc11.htm

EPA and NHTSA, on behalf of the U.S. 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. 

On May 21, 2010, President Obama issued a Presidential

Memorandum requesting that NHTSA and EPA develop

through notice and comment rulemaking a coordinated

National Program to improve fuel economy and reduce 

greenhouse gas emissions of light-duty vehicles for model 

years 2017-2025, building on the success of the first phase of

the National Program for these vehicles for model years 2012-

2016. This final rule, consistent with the President’s request,

responds to the country’s critical need to address global 

climate change and to reduce oil consumption.

October 15, 2012 – 77 FR 62624 http://www.gpo.gov/fdsys/

pkg/FR-2012-10-15/pdf/2012-21972.pdf
22 National Toxicology Program Annual Report 2012

Notice NTP Information Cited Summary of Notice

Occupational Safety and Health Administration

Final Rule: Hazard

Communication

Utilized the classification listing

that NTP uses on Safety Data 

Sheets; based classification to

determine carcinogenicity on 

the 12th Edition of the Report on 

Carcinogens. http://ntp.niehs.nih.

gov/?objectid=03C9CE38-E5CDEE56-D21B94351DBC8FC3

In this final rule, OSHA is modifying its Hazard

Communication Standard (HCS) to conform to the United 

Nations’ Globally Harmonized System of Classification and

Labelling of Chemicals (GHS). OSHA has determined that the 

modifications will significantly reduce costs and burdens while

also improving the quality and consistency of information 

provided to employers and employees regarding chemical 

hazards and associated protective measures.

March 26, 2012 – 77 FR 17574 http://www.gpo.gov/fdsys/pkg/

FR-2012-03-26/pdf/2012-4826.pdf

A complete listing of NTP studies used by Federal and state regulatory agencies is at: http://ntp.niehs.nih.

gov/go/regact.

H. Additional Activities

The NTP participates in a number of meetings with stakeholders and the scientific community. At the 2012

Society of Toxicology (SOT) meeting in San Antonio, TX, NIEHS’ Division of the NTP and other NIEHS staff

participated in approximately 100 workshop/symposium/platform sessions, education/information sessions,

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

go/35370. Several posters were presented by ICCVAM on alternative methods development. Information

about NICEATM and ICCVAM activities at the 2012 SOT Meeting can be found at http://iccvam.niehs.nih.gov/

meetings/SOT12/sotablst.htm.

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

For example, the NTP hosted the 2012 annual NTP Satellite Symposium, entitled “Pathology Potpourri,” at the 

Society of Toxicologic Pathology on June 23. The goal of the NTP Symposium is to present current diagnostic 

pathology or nomenclature issues to the toxicological pathology community in a fun and interactive manner. 

The NTP also hosted two workshops related to alternative methods development (see page 74).
4. Literature Analysis Activities 23

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 or is specifically carcinogenic.

A. Non-Cancer Health Effects 

The NTP has made a commitment to studying non-cancer health effects. To accomplish this, the NIEHS 

established the Office of Health Assessment and Translation (OHAT) within the Division of NTP to serve

as an environmental health resource to the public, and to regulatory and health agencies. This office

conducts evaluations to assess the evidence that environmental chemicals, physical substances, or mixtures 

(collectively referred to as “substances”) cause adverse health effects and 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 1998–2010 are now carried out by OHAT.

OHAT also organizes workshops or state-of-the-science evaluations to address issues of importance in

environmental health sciences. OHAT assessments are published as NTP Monographs. The OHAT evaluation

process can be found at http://ntp.niehs.nih.gov/go/38138. Kristina Thayer, PhD, is the director of OHAT.

As highlighted on page 18, in FY 2012, OHAT published the final NTP Monograph on Heath Effects of Lowlevel Lead and focused on incorporating systematic review methodologies into its evaluation. OHAT will ask

for public comment on the framework document called the “Draft OHAT Approach for Systematic Review and

Evidence Integration for Literature-based Health Assessments” (http://ntp.niehs.nih.gov/go/38673) in FY 2013. 

Table 6 lists literature analysis projects that were completed, ongoing, or initiated in FY 2012.

Table 6. NTP Non-Cancer Health Effects Projects in FY 2012

NTP Project 

[Study Scientist] Objective and/or Summary

Completed Project

NTP Monograph on Health Effects of

Low-level Lead (http://ntp.niehs.nih.gov/

go/36443) 

[Rooney]

This evaluation summarizes the evidence in humans and presents conclusions 

on health effects in children and adults associated with low-level lead exposure 

as indicated by less than 10 micrograms of lead per deciliter of blood (<10 μg/dL).

The assessment focuses on epidemiological evidence at blood lead levels <10 

μg/dL and <5 μg/dL because health effects at higher blood lead levels are well

established.

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]

The 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.
24 National Toxicology Program Annual Report 2012

NTP Project 

[Study Scientist] Objective and/or Summary

Draft NTP Monograph on Developmental

Effects and Pregnancy Outcomes 

Associated with Cancer Chemotherapy 

Use During Pregnancy (http://ntp.niehs.

nih.gov/go/36495)

[Howdeshell]

The overall goal of this monograph is to summarize the reports of effects of 

gestational exposure to cancer chemotherapy on pregnancy outcomes to serve 

as a resource for the clinical and patient communities.

Initiated Project

Air Pollution and Children’s Health (http://

ntp.niehs.nih.gov/go/37853)

[Howdeshell]

This project focuses on exposures related to outdoor air pollution and their

association with emerging children’s health outcomes.

B. Report on Carcinogens

The Report on Carcinogens (RoC) is a congressionally mandated listing of substances (1) that either are 

known to be human carcinogens or may reasonably be anticipated to be human carcinogens and (2) 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 Secretary of Health and Human Services has delegated preparation of the 

RoC to the NTP, with assistance from other Federal health and regulatory agencies. Preparation of the RoC 

is managed by the Office of the RoC (ORoC) under the direction of Ruth Lunn, DrPH, within the NIEHS

Division of the NTP. Integrated Laboratory Systems, Inc. (ILS), provided contract support for preparation of 

the RoC in FY 2012. The 12th RoC, the latest edition, was published on June 10, 2011.

Preparation of the RoC

During the early part of FY 2012, the NTP made revisions to the multi-step process used to prepare the RoC. 

The development of the process included opportunity for public input on the proposed process and NTP BSC 

review of the revised process (for more information see http://ntp.niehs.nih.gov/go/37153). The NTP published 

a Federal Register notice in October 2011 inviting written comments on the proposed process for preparing 

the RoC and convened a listening session in November 2011 to receive oral public comments. The revised 

process for the preparation of the RoC was presented to the NTP BSC on December 11, 2011 (http://ntp.niehs.

nih.gov/go/9741), and the final process was released in January 2012 (available at http://ntp.niehs.nih.gov/go/

rocprocess). 

With the revised process, the NTP maintains critical elements of the previous process including external 

scientific and public involvement, scientific rigor, and external peer review. New elements of the revised

process include the concept document and the monograph. Each substance proposed for review for the RoC 

will have a concept document prepared for public comment and comment from the NTP BSC. The concept 

document will outline the reason for a substance’s review for the RoC and lay out the proposed approach for 

obtaining external scientific and public inputs in development of the cancer evaluation component of the draft

RoC monograph. The monograph is an integrated document that will contain a cancer evaluation component 
4. Literature Analysis Activities 25

and substance profile. The monograph will undergo public peer review. This revised process should enhance

transparency and enable the NTP to publish the RoC in a timelier manner.

In January 2012, the NTP invited public input on 15 nominations for the 13th and future editions of the RoC 

(http://ntp.niehs.nih.gov/go/rocnom). Based upon input by the public and NTP’s interagency partners, ORoC 

identified five substances for evaluation, hereafter referred to as “candidate substances”. A draft concept

document was developed for each candidate substance to outline the rationale and proposed approach for its 

review. The NTP BSC reviewed the draft concepts at the June 21–22, 2012 public meeting (http://ntp.niehs.

nih.gov/go/9741). Following the meeting, the NTP Director approved the five candidate substances for formal

review for the RoC, these are listed in Table 7.

During the last part of FY 2012, ORoC initiated preparation of draft RoC monographs for the five candidate

substances and established a website to share information regarding their reviews and to receive public input 

(accessed from http://ntp.niehs.nih.gov/go/37893). ORoC will complete the preparation of draft monographs for 

several candidate substances during FY 2013, after which they will be released for public comment and peer 

review by an external panel at a public meeting. 

Table 7. Candidate Substances for the RoC 

Candidate Substance

CASRN

[Study Scientist]

Primary Uses/Exposures

1-Bromopropane 

106-94-5

[Spencer]

A brominated hydrocarbon used as a solvent cleaner to degrease electronics, precision optics, 

and metals, as a solvent vehicle in industries that use aerosolized adhesives (e.g., foam 

cushion manufacturing), as a spot remover in the textile industry, and as a solvent in the dry 

cleaning industry. 

Cumene 

98-82-8

[Jahnke]

An alkylated benzene found in fossil fuels and used primarily to produce phenol and acetone.

Pentachlorophenol 

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.

ortho-Toluidine* 

95-53-4 

[Lunn]

An arylamine used as an intermediate to manufacture herbicides, dyes, pigments and rubber 

chemicals.

Trichloroethylene*

79-01-6 

[Lunn]

Halogenated alkene used primarily for degreasing metals; it is also used as a solvent for

applications related to adhesives, painting, lacquering, and varnishes. It was commonly used 

in the dry cleaning industry from the 1930s to the 1950s, but is rarely used today. 

* Currently listed as reasonably anticipated to be a human carcinogen in the RoC.
26 National Toxicology Program Annual Report 2012

5. Testing and Research

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

of issues important to public health. The 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 (nomination website: 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 the NTP. The review 

and selection of nominations for study is a multi-step process with input from NTP participating Federal 

agencies, the NTP BSC (see page 7), and the public (see http://ntp.niehs.nih.gov/go/156 for details). Table 8 

lists the substances whose draft research concepts were reviewed in FY 2012 by the NTP BSC. 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. The full research concepts for 

the substances in Table 8 can be found at http://ntp.niehs.nih.gov/go/37038. Questions about the nomination, 

review, and selection process can be sent to Dr. Scott Masten (masten@niehs.nih.gov).

Table 8. Research Concepts Reviewed by the BSC in FY 2012

Substance 

CASRN

[Nominator]

Nomination Rationale Proposed Studies

[Study Scientist]

Octrizole and related phenolic 

benzotriazoles

3147-75-9

[NIEHS]

High production volume and use as UV 

stabilizers in a wide variety of industrial 

and consumer products; possess

physico-chemical properties that 

suggest persistence in the environment 

and potential to bioaccumulate; limited

toxicological data, and reproductive and 

developmental effects data for several 

class members suggest potential hazard

•	 Short-term in vivo and in vitro toxicity studies 

on multiple (10 or more) class members to 

prioritize individual compounds for further 

study

•	 For one or more selected class members:

◦ ADME and toxicokinetics studies by oral

and dermal routes of administration

◦ Reproductive and developmental toxicity 

studies

◦ Subchronic toxicity studies

◦ Chronic toxicity and carcinogenicity 

studies, if warranted based on above

[Blystone]

Sulfolane

126-33-0

[Alaska Department of

Environmental Conservation, 

Fairbanks North Star Borough

Mayor, Alaska Department of

Health and Social Services, 

Alaska State Senator John

Coghill, National Center for 

Environmental Health / Agency 

for Toxic Substances and 

Disease Registry]

High production volume and use in oil 

and gas refining; public concern related

to groundwater contamination in Alaska;

inadequate data to evaluate potential 

adverse health effects from long-term 

exposures

•	 Short-term oral toxicity studies to evaluate 

species sensitivity

•	 ADME and toxicokinetics studies by multiple

routes of administration

•	 Perinatal exposure studies to evaluate 

reproductive and developmental toxicity, 

immune toxicity, and developmental 

neurotoxicity

•	 Chronic toxicity and carcinogenicity studies

[Blystone]
5. Testing and Research 27

Substance 

CASRN

[Nominator]

Nomination Rationale Proposed Studies

[Study Scientist]

Trimethylsilyldiazomethane

18107-18-1

[Occupational Safety and 

Health Administration]

Low volume production and use as 

a reagent in synthetic and analytical 

chemistry; developed and promoted

as a safer substitute for diazomethane;

inadequate toxicological data; case

reports of severe lung injury and death in

exposed laboratory personnel

•	 Chemistry studies to evaluate stability and 

reactivity

•	 Acute inhalation toxicity studies

[Gwinn]

A. Technical Reports

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

several NTP report series: Technical Reports (TR), NTP Toxicity Reports (TOX), and Genetically Modified

Models Reports (GMM). TRs present the results of NTP’s long-term, generally 2-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 the NTP in

transgenic mouse strains (e.g., 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 reports of the TR, TOX, and GMM series 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 studies, are also available on the “NTP Study 

Reports” page on the website.

The following tables (Tables 9–10) list Technical Reports completed or published in FY 2012. The NTP held a 

peer-review meeting on February 8-9, 2012, to review the seven reports listed in Table 9, see page 12 for more 

details. The 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 the 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/. The NTP anticipates seven draft NTP Technical Reports will undergo 

peer review in FY 2013, as shown in Table 11.
28 National Toxicology Program Annual Report 2012

Table 9. Technical Reports Peer Reviewed during FY 2012

Chemical/ 

Exposure – 

Study Type

Technical 

Report 

Number/ 

CASRN

Use

Levels of Evidence of 

Carcinogenic Activity

Male 

Rats

Female 

Rats

Male 

Mice

Female 

Mice

3’-Azido-3’-

deoxythymidine 

(AIDS)*

GMM-14

30516-87-1

Pyrimidine nucleoside analog 

with antiviral activity used in the 

treatment of AIDS (Merck 1989)

N/A N/A N/A N/A

3’-Azido-3’-

deoxythymidine 

(AIDS)* in 

combination with 

Lamivudine and 

Nevirapine

GMM-16

30516-87-1

Pyrimidine nucleoside analog 

with antiviral activity used in 

the treatment of AIDS (Merck

1989), special combination 

to study AIDS therapeutics 

including Lamivudine and 

Nevirapine

N/A N/A N/A N/A

N,N-Dimethyl-ptoluidineTR-579

99-97-8

Used as a polymerization 

accelerator for the manufacture 

of bone cements & dental 

materials. Found in industrial 

glues & artificial fingernail

preps. Intermediate in dye & 

pesticide synthesis.

Clear 

Evidence

Clear 

Evidence

Clear 

Evidence

Clear 

Evidence

Ginkgo biloba

extract

TR-578

90045-36-6 Herbal supplement Some 

Evidence

Some 

Evidence

Clear 

Evidence

Clear 

Evidence

β-Picoline TR-580

108-99-6

Used as a solvent in the 

synthesis of pharmaceuticals, 

resins, dyes & rubber 

accelerators and as a lab 

reagent. Also used as an 

intermediate in the manufacture 

of insecticides, waterproofing

agents, niacin & niacinamide.

No 

Evidence

Some 

Evidence

Equivocal 

Evidence

Clear 

Evidence
5. Testing and Research 29

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 & 

colors. Also used as developer 

in photography; a mordant

for dying wool; a reagent for

antimony & bismuth; and as

a reducer for gold, silver & 

mercury salts. Used for process 

engraving & for making colloidal

solutions of metals. Used in the 

manufacture of pharmaceuticals 

& 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, & polymers 

& resins; as a component of

photopolymer & flexographic

printing plates & photoresists;

& as an ingredient in acrylic 

glues & anaerobic sealants. 

Also used in paper & wood 

impregnates, wire & cable 

extrusion, polymer-impregnated 

concrete, & polymer concrete 

structural composites.

Some 

Evidence

No 

Evidence

No 

Evidence

Some 

Evidence

*	 Indicates study conducted using genetically-modified model.

© Can Stock Photo Inc. / aremac
30 National Toxicology Program Annual Report 2012

Table 10. Technical Reports Published During FY 2012

Chemical/

Exposure – 

Study Type

Technical 

Report 

Number/ 

CASRN

Use

Levels of Evidence of 

Carcinogenic Activity

Male 

Rats

Female 

Rats

Male 

Mice

Female 

Mice

Acrylamide TR-575

79-06-1

Polyacrylamides and dye 

intermediate Flocculants. Waste 

and water treatment. Paper and 

pulp industry. 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.

Clear 

Evidence

Clear 

Evidence

Clear 

Evidence

Clear 

Evidence

alpha/beta 

Thujone mixture

TR-570

76231-76-0

The essential oils derived from 

natural oil of cedar leaves 

in which thujone occurs are

used in herbal medicine and 

as flavorings, fragrances, and

rodent and mite repellents. 

Thujone is banned as a direct

food additive in the Unites 

States Thujone-containing

plant oils are used as flavoring

substances in the alcoholic 

drink industry.

Some 

Evidence

No 

Evidence

No 

Evidence

No 

Evidence

Diethylamine TR-566

109-89-7

Used in the production of 

the corrosion inhibitor n,ndiethylethanolamine, and 

in the production of some 

pesticides and insect repellents, 

pharmaceuticals and rubber 

processing chemicals 

(Executive Summary). 

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

No 

Evidence

No 

Evidence
5. Testing and Research 31

Chemical/

Exposure – 

Study Type

Technical 

Report 

Number/ 

CASRN

Use

Levels of Evidence of 

Carcinogenic Activity

Male 

Rats

Female 

Rats

Male 

Mice

Female 

Mice

N,N-Dimethyl-ptoluidine

TR-579

99-97-8

Used as a polymerization 

accelerator for the manufacture 

of bone cements & dental 

materials. Found in industrial 

glues & artificial fingernail

preps. Intermediate in dye & 

pesticide synthesis.

Clear 

Evidence

Clear 

Evidence

Clear 

Evidence

Clear 

Evidence

Kava kava extract TR-571

9000-38-8

Herbal supplement alternative 

to anti-anxiety drugs, used to 

help children with hyperactivity 

and as a skin-conditioning

agent in cosmetics.

Equivocal 

Evidence

No 

Evidence

Clear 

Evidence

Clear 

Evidence

Methyl trans-styryl

ketone

TR-572

1896-62-4

Reactive carbonyl 

compound used in organic 

synthetic reactions such 

as intermediate in organic 

synthesis, electroplating 

chemical; pharmaceutical

intermediate, biochemical 

reagent, agricultural chemical 

intermediate, proposed 

sunscreen intermediate. Used 

as flavoring and fragrance

additive. 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

No 

Evidence

No 

Evidence

Retinoic acid and 

retinyl palmitate

TR-568

79-81-2

Retinol compound (vitamin A) 

used in skin products. N/A N/A N/A N/A

Senna* GMM-15

8013-11-4

Herbal product, extract of 

senna fruit, used in laxative 

preparations.

N/A N/A N/A N/A

Styreneacrylonitrile trimer TR-573

SAN Trimer is a by-product of 

the production of acrylonitrile 

styrene plastics and is created 

in specific manufacturing

processes for polymers of 

acrylonitrile and styrene.

No 

Evidence

No 

Evidence N/A N/A

*	 Indicates study conducted using genetically-modified model.
32 National Toxicology Program Annual Report 2012

Table 11. Technical Reports Expected to Undergo Peer Review in FY 2013

Chemical

Technical 

Report 

Number/ 

CASRN

Use

Bromodichloroacetic 

acid (water disinfection 

byproducts)

TR-583

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 fluids) 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 vinychloride or acrylonitrile for saran and saran fibers. A

widely used chemical intermediate and monomer.

B. NTP Testing

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

models for study. NTP staff designs protocols 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 (e.g., grant, contract). The following NIEHS DNTP branches are involved 

in the testing program: Biomolecular Screening Branch (led by Raymond Tice, PhD), Cellular and Molecular

Pathology Branch (led by Robert Sills, DVM, PhD), NTP Laboratory (led by Mike Waalkes, PhD), Program

Operations Branch (led by Michelle Hooth, PhD, DABT), and Toxicology Branch (led by Paul Foster, PhD).
5. Testing and Research 33

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 2012 are listed in Table 12,

and studies planned for FY 2013 are listed in Table 13. More information can be found at http://ntp.niehs.nih.

gov/go/sp. 

Table 12. Ongoing and Completed Disposition, Metabolism and Toxicokinetic Studies

During FY 2012

Chemical CASRN Species/Strain Route Study 

Scientist

Anatase (TiO2) 1317-70-0 Mice: Tg.AC (FVB/N)

Mice: FVB/N

Topical 

Application Howard

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

Bisphenol AF 1478-61-1 Mice: B6C3F1

Rats: Harlan Sprague Dawley Intravenous MercadoFeliciano

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

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

1,3-Dichloro-2-propanol 96-23-1 Mice: B6C3F1

Rats: Harlan Sprague Dawley Gavage Waidyanatha

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

Dimethylamine borane 74-94-2 Rats: Harlan Sprague Dawley

Human Gavage Germolec

Dimethylethanolamine 108-01-0 Mice: B6C3F1

Rats: Wistar Han Gavage Waidyanatha

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

Mice: B6C3F1/N Intravenous Waidyanatha

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

Mice: B6C3F1/N In-vitro DeVito

2-Ethylhexyl-pmethoxycinnamate 5466-77-3 Rats: Harlan Sprague Dawley

Mice: B6C3F1 Dermal McIntyre

Fullerene C60 (nanoscale 

material) 99685-96-8 Rats: F344/N Insufflation Waidyanatha

Furan 110-00-9 Rats: Tg.Lac1/C57BL/6 (Big Blue)

Rats: F344 (NCTR) Gavage Walker

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Mice: B6C3F1Rats: Sprague Dawley Gavage Auerbach
34 National Toxicology Program Annual Report 2012

Chemical CASRN Species/Strain Route Study 

Scientist

2-Methoxy-4-nitroaniline 97-52-9 Mice: B6C3F1

Rats: Harlan Sprague Dawley

Topical 

application Auerbach

L-beta-Methylaminoalanine 15920-93-1 Mice: B6C3F1/N

Rats: Harlan Sprague Dawley Gavage Ryan

Rutile titanium dioxide 

(nanoscale material) 1317-80-2 Mice: SKH-1 hairless Topical Application Howard

tris(2-Chloroisopropyl)

phosphate 13674-84-5 Rats: Harlan Sprague DawleyMice: B6C3F1/N Intravenous Stout

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

Mice: B6C3F1 Gavage Surh

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

Table 13: Disposition, Metabolism, and Toxicokinetic Studies Planned for FY 2013

Chemical CASRN Species/Strain Route

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

Mice: B6C3F1 Intravenous

*	 Known test articles as of 10/1/2012. Others may be scheduled as protocols are finalized.

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 2012 

are listed in Table 14. More information can be found at http://ntp.niehs.nih.gov/go/gt. 

Table 14. Ongoing and Completed Genetic Toxicity Studies during FY 2012

Chemical CASRN Testing Battery

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

Black cohosh 84776-26-1 Micronucleus

Bromodichloroacetic acid 71133-14-7 Salmonella

N-Butylbenzenesulfonamide 3622-84-2 Salmonella

Chlorhexidine 55-56-1 Salmonella

Furan 110-00-9 Micronucleus

Garcinia cambogia extract 90045-23-1 Salmonella

2-methyl-6-ethylanline 24549-06-2 Salmonella

Nelfinavir 159989-64-7 Salmonella
5. Testing and Research 35

Chemical CASRN Testing Battery

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

2-Nitro-2-methyl-1,3-propanediol 77-49-6 Salmonella

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

2,3-Pentanedione 600-14-6 Micronucleus

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 (PFOS) 1763-23-1 Micronucleus

Perfluorooctanoic acid (PFOA) 335-67-1 Micronucleus

Potassium hydroxycitrate tribasic monohydrate 232281-44-6 Salmonella

Valerian (root extract, root oil) Salmonella

Wyeth 14,643 50892-23-4 Micronucleus

Zinc carbonate, basic 5263-02-5 Micronucleus

iii. Organ System Toxicity

The NTP studies toxicity of environmental substances on organ systems, such as it relates to the immune 

system, development, and reproduction. Table 15 lists ongoing and completed organ systems toxicity studies 

during FY 2012. More information can be found at http://ntp.niehs.nih.gov/go/type. 

Table 15. Ongoing and Completed Neurotoxicity, Developmental Toxicity and Reproductive 

Toxicity Studies During FY 2012

Chemical CASRN Species/Strain Route Study 

Scientist Testing Battery

Acrylamide 79-06-1 Rats: F344 (NCTR) Gavage Beland Neurotoxicity 

assessment

Black cohosh 84776-26-1 Rats: Harlan Sprague

Dawley Gavage MercadoFeliciano

Continuous 

breeding

Butyl paraben (n-Butyl-phydroxybenzoate) 94-26-8 Rats: Harlan Sprague

Dawley Feed Blystone Continuous breeding

Dimethylethanolamine 108-01-0 Rats: Harlan Sprague

Dawley Gavage Teratology pilot study

2-Hydroxy-4-

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

Teratology 

pilot study, 

Developmental 

toxicity

4-Methylimidazole 822-36-6 Rats: Harlan Sprague

Dawley Feed Behl

Continuous 

breeding: Rangefinding
36 National Toxicology Program Annual Report 2012

Chemical CASRN Species/Strain Route Study 

Scientist Testing Battery

Nonylphenol 84852-15-3 Rats: Sprague Dawley

(NCTR) Feed Newbold, Delclos Multigeneration

3,3’,4,4’- 

Tetrachloroazobenzene 14047-09-7 Rats: Crl: CD (SpragueDawley) Gavage Hooth

Developmental–

pre-implantation, 

Postnatal 

developmental 

toxicity

Tris (2-Chloroisopropyl)

phosphate 13674-84-5 Rats: Harlan SpragueDawley Gavage Stout Developmental toxicity

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 16 lists ongoing and completed immunotoxicity studies during FY 2012. More

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

Table 16. Ongoing and Completed Immunotoxicity Studies During FY 2012

Chemical CASRN Species/

Strain Route Study Scientist Testing Battery

Autumn sunset true color 

concentrate

Mice: CBA/ Ca

Jackson

Subcutaneous 

injection Howard Hypersensitivity

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

Immunosuppression–

Range Finding*;

Developmental

Black cohosh 84776-26-1

Mice: B6C3F1 Gavage MercadoFeliciano Immunosuppression

Rats: Harlan

Sprague Dawley Gavage MercadoFeliciano Range-finding*

Blasting sand (abrasive 

blasting agents)

Rats: Harlan

Sprague Dawley Inhalation Gwinn Immunosuppression

2,3-Butanedione (Diacetyl) 431-03-8

Mice: BALB/c Topical 

application Morgan Hypersensitivity

Mice: BALB/c Inhalation Morgan Immunosuppression–

Range-finding*

Dermal lotion vehicle 

development Mice: BALB/c Topical application Germolec Hypersensitivity

2,3-Dibromo-7,8-

dichlorodibenzo-p-dioxin 50585-40-5 Mice: B6C3F1 Gavage DeVito Immunosuppression
5. Testing and Research 37

Chemical CASRN Species/

Strain Route Study Scientist Testing Battery

Dibenz(a,h)anthracene 53-70-3 Mice: B6C3F1 Gavage Germolec

Immunosuppression–

Range finding*;

Developmental;

Immunosuppression–

Full protocol**

Dibromoacetic acid (water 

disinfection byproducts) 631-64-1 Rats: HarlanSprague Dawley Dosed-Water Germolec Immunosuppression–Range finding*

1,3-Dichloropropene (Telone II) 542-75-6 Mice: B6C3F1 Dosed-Water Germolec Immunosuppression–

Full protocol**

Dimethylamine borane 74-94-2 Mice: BALB/c Topical 

application Germolec Hypersensitivity

Double dark fudge true color

concentrate

Mice: CBA/ Ca

Jackson

Subcutaneous 

injection Howard Hypersensitivity

Double fudge concentrate Mice: CBA/ Ca

Jackson

Subcutaneous 

injection Howard Hypersensitivity

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

Full protocol**

Elmiron (sodium 

pentosanpolysulfate) 37319-17-8 Mice: B6C3F1 Gavage Germolec

Immunosuppression–

Range finding;

Immunosuppression–

Full protocol**

2-Ethylhexyl 

p-methoxycinnamate 5466-77-3 Mice: BALB/c Topical Application McIntyre Hypersensitivity

Fullerene-C60 (1 micron) 

(nanoscale material) 99685-96-8 Rats: Wistar HanMice: 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

Lovastatin 75330-75-5 Mice: B6C3F1 Gavage Germolec Immunosuppression–

Range finding*

2-Methoxy-4-nitroaniline 97-52-9 Mice: BALB/c Dermal Surh Hypersensitivity

Monoclonal antibody protein

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

Monoclonal antibody protein

therapeutics (CD-8) Mice: B6C3F1 Intraperitoneal injection Germolec Immunosuppression–Full protocol**

Nelfinavir mesylate 159989-65-8 Mice: B6C3F1 Gavage Germolec

Immunosuppression –

Range finding*;

Developmental
38 National Toxicology Program Annual Report 2012

Chemical CASRN Species/

Strain Route Study Scientist Testing Battery

Nevirapine 129618-40-2 Mice: B6C3F1 Gavage Germolec

Developmental;

Immunosuppression–

Full protocol**

1,2,3,7,8- 

Pentabromodibenzofuran 107555-93-1 Mice: B6C3F1 Gavage DeVito Immunosuppression

2,3,4,7,8- 

Pentabromodibenzofuran 13116-92-2 Mice: B6C3F1 Gavage DeVito Immunosuppression

1,2,3,7,8- 

Pentachlorodibenzofuran 57117-41-6 Mice: B6C3F1 Gavage DeVito Immunosuppression

2,3,4,7,8- 

Pentachlorodibenzofuran 57117-31-4 Mice: B6C3F1 Gavage DeVito Immunosuppression

2,3-Pentanedione 600-14-6 Mice: BALB/c Topical 

application Germolec Hypersensitivity

Perfluorodecanoic acid 335-76-2 Rats: Harlan

Sprague Dawley Gavage Blystone Immunosuppression

Phenol 108-95-2 Mice: B6C3F1 Dosed-Water Germolec Immunosuppression–

Full protocol**

Resveratrol 501-36-0 Mice: NOD/

MrKTac Gavage Germolec Autoimmunity

Rosewood true color 

concentrate

Mice: CBA/ Ca

Jackson

Subcutaneous 

Injection Howard Hypersensitivity

Sodium tungstate, dihydrate 10213-10-2 Mice: B6C3F1 Dosed-Water Hooth Immunosuppression–

Full protocol**

Specular hematite (abrasive 

blasting agents)

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

2,3,7,8- 

Tetrabromodibenzofuran 67733-57-7 Mice: B6C3F1 Gavage DeVito Immunosuppression

2,3,7,8- Tetrachlorodibenzop-dioxin 1746-01-6 Mice: B6C3F1 Gavage DeVito Immunosuppression

2,3,7,8- 

Tetrachlorodibenzofuran 51207-31-9 Mice: B6C3F1 Gavage DeVito Immunosuppression

2,3,7- Tribromodibenzo-pdioxin 51974-40-4 Mice: B6C3F1 Gavage DeVito Immunosuppression

*	 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.
5. Testing and Research 39

Modified One-generation Reproduction Studies

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

design. The 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 17 lists planned or ongoing modified

one-generation studies. More information can be found at http://ntp.niehs.nih.gov/go/MG. 

Table 17. 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

Neurotoxicity assessment5

MercadoFelliciano

N-Butylbenznensulfonamide 3622-84-2 Rats: Harlan

Sprague Dawley Feed

Dose range finding1

Maternal transfer2

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

Subchronic toxicity6

Rider

Butyl paraben 94-26-8 Rats: Harlan

Sprague Dawley Feed Dose range finding1 Blystone

Dong quai (Angelica sinesis) 

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: HarlanSprague Dawley Feed

Dose range finding1

Developmental toxicity3

Fertility assessment4

Subchronic toxicity6

McIntyre

Garcinia cambogia extract 90045-23-1 Rats: Harlan

Sprague Dawley Feed

Dose range finding1

Maternal transfer2

Developmental toxicity3

Fertility assessment4

Subchronic toxicity6

Rider

Hydroquinone 123-31-9 Rats: Harlan

Sprague Dawley Gavage

Dose range finding1

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

DeVito
40 National Toxicology Program Annual Report 2012

Chemical CASRN Species/

Strain

Study 

Route Planned Cohorts Study Scientist

2-Hydroxy-4-

methoxybenzophenone 131-57-7 Rats: HarlanSprague Dawley Feed

Dose range finding1

Developmental toxicity3

Fertility assessment4

Neurotoxicity assessment5

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

(PFOS) 1763-23-1 Rats: HarlanSprague Dawley Gavage Dose range finding1 Blystone

Perfluorooctanoic acid (PFOA) 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

Dosed 

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: HarlanSprague Dawley Gavage Dose range finding1 DeVito

Wyeth 14,643 (WY) 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 and/or peripheral nervous

system.

6

	 Subchronic toxicity: 90-day study of adverse effects in the exposed rodent.
5. Testing and Research 41

iv. Toxicology/Carcinogenicity Studies

The 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 (NTP) January 2011. Additional information can be found at http://ntp.niehs.nih.gov/go/ba. 

Tables 18, 19, and 20 list the toxicity studies that were ongoing, initiated, and completed, respectively, during 

FY 2012. Table 21 lists toxicity studies planned for FY 2013. Chronic toxicology/carcinogenicity studies ongoing 

and initiated in FY 2012 are listed in Tables 22 and 23, respectively.

Table 18. Prechronic Ongoing Toxicology/Carcinogenicity Studies during FY 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

Bisphenol A 80-05-7 Rats: Harlan Sprague Dawley Gavage 90 days Delclos

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

Mice: B6C3F1 Gavage 14 days MercadoFeliciano

Cell phone radiation 

(CDMA)

Rats: Harlan Sprague Dawley

Mice: B6C3F1

Whole body 

exposure

5 days

49 days

28 days

Wyde

Cell phone radiation 

(GSM)

Rats: Harlan Sprague Dawley

Mice: B6C3F1

Whole body 

exposure

5 days

49 days

28 days

Wyde

bis(2-Chloroethoxy)

methane 111-91-1

Mice: C57BL/6J (Jackson)

Mice: C3H/HeJ

Mice: B6C3F1 (Jackson)

Gavage 3 days

10 days Dunnick

Diet evaluation study Mice: CD-1 Reg.[Crl: CD1(ICR)] Feed 90 days Delclos

Ephedrine + caffeine 

combination

Mice: C57BL/6J (Jackson)

Mice: C3H/HeJ

Mice: B6C3F1 (Jackson)

Gavage 3 days

10 days Dunnick

Formaldehyde* 50-00-0

Mice: C57BL/6

Mice: C3B6F1-+/TRP53<TM1BRD>

(NCTR)

Mice: B6C3F1

Inhalation 2 weeks

8 weeks Morgan

Melamine + Cyanuric

acid combination

Rats: F344 (NCTR)

Rats: Sprague Dawley (NCTR) Gavage 90 days Gamboa de Costa

Nanoscale silver 7440-22-4 Rats: Harlan Sprague Dawley Gavage 13 weeks Walker

Perfluorobutane

sulfonate 375-73-5 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorodecanoic acid 335-76-2 Rats: Harlan Sprague Dawley Gavage 28 days Blystone
42 National Toxicology Program Annual Report 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

Perfluorohexane

sulfonate potassium salt 3871-99-6 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorohexanoic acid 307-24-4 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorononanoic acid 375-95-1 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorooctane

Sulfonate (PFOS) 1763-23-1 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorooctanoic acid

(PFOA) 335-67-1 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

Triclosan 3380-34-5 Mice: B6C3F1/N Dermal 90 days Fang

Usnea lichen

Rats: F344 (NCTR)

Mice: B6C3F1/NCTR BR (C57BL/6N

x C3H/HEN MTV-)

Feed 2 weeks

90 days Leakey

(+)-Usnic Acid 7562-61-0

Rats: F344 (NCTR)

Mice: B6C3F1/NCTR BR (C57BL/6N

x C3H/HEN MTV-)

Feed 2 weeks

90 days Leakey

Wyeth 14,643 (WY) 50892-23-4 Rats: Harlan Sprague Dawley Gavage 28 days Blystone

*	 Indicates study conducted using genetically-modified model

Table 19. Prechronic Toxicology/Carcinogenicity Studies Initiated during FY 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

Black cohosh 84776-26-1 Rats:Harlan Sprague Dawley Gavage 14 days MercadoFeliciano

Diet evaluation study Mice:CD-1 Reg.[Crl:CD1(ICR)] Dosed-feed 90-days Delclos

Formaldehyde* 50-00-0

Mice:C57BL/6

Mice:C3B6F1-+/TRP53<TM1BRD>

(NCTR)

Mice:B6C3F1

Inhalation 2 weeks

8 weeks Morgan

Melamine + Cyanuric

Acid combination Rats:F344 (NCTR) Gavage 90 days Gamboa de Costa

Perfluorobutane

sulfonate 375-73-5 Rats:Harlan Sprague Dawley Gavage 28 days Blystone
5. Testing and Research 43

Study CASRN Species/Strain Study 

Route Length Study Scientist

Perfluorodecanoic acid 335-76-2 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorohexane

sulfonate potassium salt 3871-99-6 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorohexanoic acid 307-24-4 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorononanoic acid 375-95-1 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorooctane

Sulfonate (PFOS) 1763-23-1 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

Perfluorooctanoic acid

(PFOA) 335-67-1 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

Wyeth 14,643 (WY) 50892-23-4 Rats:Harlan Sprague Dawley Gavage 28 days Blystone

*	 Indicates study conducted using genetically-modified model

Table 20. Completed Prechronic Toxicology/Carcinogenicity Studies during FY 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

2-Aminopyridine 504-29-0 Rats:F344/NTac

Mice:B6C3F1 Gavage 14 days Dunnick

3-Aminopyridine 462-08-8 Rats:F344/NTac

Mice:B6C3F1 Gavage 14 days Dunnick

4-Aminopyridine 504-24-5 Rats:F344/NTac

Mice:B6C3F1 Gavage 14 days Dunnick

Comparison study of 

aminopyridines/troponin 

levels

Rats:F344/NTac

Mice:B6C3F1 Gavage 1 and 4 hours Dunnick

Glucosamine 

hydrochloride + 

Chondroitin sulfate

Rats:Zucker - Obese

(HsdHlr:ZUCKER-Leprfa)

Rats:Zucker - Lean

(HsdHlr:ZUCKER-Lepr+)

Gavage 13 weeks Leakey

1020 Long multiwalled 

carbon nanotube

Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Inhalation 30 days Walker

Pyridine 110-86-1 Mice:B6C3F1 Gavage 14 days Dunnick

Serotype 2 adenoassociated viral vector 

hAQP1 (rAAV2hAQP1)

Mice:BALB/c Intraductal 

cannulation 13 weeks Germolec

Usnea lichen

Rats:F344 (NCTR)

Mice:B6C3F1/NCTR BR (C57BL/6N

x C3H/HEN MTV-)

Dosed-feed 2 weeks

90 days Leakey
44 National Toxicology Program Annual Report 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

(+)-Usnic Acid 7562-61-0

Rats:F344 (NCTR)

Mice:B6C3F1/NCTR BR (C57BL/6N

x C3H/HEN MTV-)

Dosed-feed 2 weeks

90 days Leakey

Vincamine 1617-90-9 Rats:Harlan Sprague Dawley

Mice:B6C3F1 Gavage

2 weeks

range 

finding;

2 weeks

McIntyre

Table 21. Prechronic Toxicology/Carcinogenicity Studies Planned for FY 2013*

Study CASRN Species/Strain Study 

Route Length Study Scientist

Formaldehyde 50-00-0

Mice:C57BL/6

Mice:C3B6F1-+/TRP53<TM1BRD>

(NCTR)

Mice:B6C3F1

Inhalation 2 weeks

8 weeks Morgan

*	 Known test articles as of 10/1/2012. Others may be scheduled as protocols are finalized.

Table 22. Chronic Toxicity/Carcinogenicity Studies Ongoing During FY 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

Aging cohort study

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 French

Antimony trioxide 1309-64-4 Rats:Wistar Han

Mice:B6C3F1/N Inhalation 2 years Stout

3’-Azido-3’-

deoxythymidine 30516-87-1 Mice:P53 +/- (FVB/N) Gavage 45 weeks Leakey

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 MercadoFeliciano

Bromodichloroacetic 

acid (water disinfection 

byproducts)

71133-14-7 Rats:F344/NTac

Mice:B6C3F1 Water 2 years Hooth
5. Testing and Research 45

Study CASRN Species/Strain Study 

Route Length Study Scientist

2,3-Butanedione 

(Diacetyl) 431-03-8 Rats:Wistar HanMice:B6C3F1/N Inhalation 2 years Morgan

Cell phone radiation 

(CDMA)

Rats:Harlan Sprague Dawley

Mice:B6C3F1

Whole body 

exposure 2 years Wyde

Cell phone radiation 

(GSM)

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 fluids)

Rats:Wistar Han

Mice:B6C3F1/N Inhalation 2 years Morgan

Cobalt 7440-48-4 Rats:F344/NTac

Mice:B6C3F1 Inhalation 2 years Hooth

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 BR (C57BL/6N x

C3H/HEN MTV-)

Water 2 years Beland

Green tea extract 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

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

Mice:B6C3F1 Gavage 2 years Wyde

Insertional mutagenesis 

- definitive vector study 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

(PFOA) 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 DawleyMice:B6C3F1/N Water 2 years Hooth

Tetrabromobisphenol A 79-94-7 Rats:Wistar Han

Mice:B6C3F1/N Gavage 2 years Dunnick

Trim VX (metal working

fluids)

Rats:Wistar Han

Mice:B6C3F1/N Inhalation 2 years Morgan

Tripelennamine 

hydrochloride 154-69-8 Rats:F344/NMice:B6C3F1 Feed 11 months Jackson

tris(2-Chloroisopropyl)

phosphate 13674-84-5 Rats:Harlan Sprague DawleyMice:B6C3F1/N Feed 2 years Stout
46 National Toxicology Program Annual Report 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

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

*	 Indicates study conducted using genetically-modified model

Table 23. Chronic Toxicity/Carcinogenicity Studies Initiated during FY 2012

Study CASRN Species/Strain Study 

Route Length Study Scientist

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 MercadoFeliciano

Cell phone radiation 

(CDMA)

Rats:Harlan Sprague Dawley

Mice:B6C3F1

Whole body 

exposure 2 years Wyde

Cell phone radiation 

(GSM)

Rats:Harlan Sprague Dawley

Mice:B6C3F1

Whole body 

exposure 2 years Wyde

Resveratrol 501-36-0 Rats:Harlan Sprague Dawley

Mice:B6C3F1/N Gavage 2 years Germolec

Sodium tungstate, 

dihydrate 10213-10-2 Rats:Harlan Sprague DawleyMice:B6C3F1/N

Dosedwater 2 years Hooth

tris(2-Chloroisopropyl)

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

v. Toxicogenomic Studies

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

the way the program conducts its studies. Toxicogenomics examines how the entire genetic structure, or 

genome, is involved in an organism’s response to environmental toxicants. Microarray, NextGen sequencing,

proteomics, and metabolomics are among the advanced technologies that the 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 might 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 (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 will provide a genetic signature.
5. Testing and Research 47

The 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 2012 toxicogenomic studies have 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. 

The NTP is currently evaluating study conditions that may contribute to gene expression (e.g., animal and 

tissue variability), best methods of tissue sampling, and establishing standards for conducting toxicogenomic 

studies under laboratory conditions. Planned or ongoing NTP toxicogenomic studies are listed in Table 24. 

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

Table 24. Toxicogenomic Studies (Planned or Ongoing)

Chemical CASRN Species/

Strain

Study 

Route

Study 

Length

Study 

Scientist

Aflatoxin B1 1162-65-8 Rats: F344/N Feed 90 days Merrick

Acetachlor 34256-82-1 Rats: Harlan

Sprague Dawley Gavage 4 days DeVito

Toxicogenomic study of allylbenzene & 

propenylbenzene class flavor constituents

Anethole

Estragole

Eugenol

Isoeugenol

Isosafrole

Methyleugenol

Myristicin

Safrole

104-46-1

140-67-0

97-53-0

97-54-1

120-58-1

93-15-2

607-91-0

94-59-7

Rats: F344/N

Tac Gavage 90 days Vallant

tert-Butylphenyl diphenyl phosphate 56803-37-3 Rats: Harlan

Sprague Dawley Gavage 4 days Auerbach

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

Toxicogenomic estrous cycle study Rats: Wistar Han Not 

applicable 90 days Auerbach

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

Sprague Dawley Gavage 4 days Auerbach

Fusilazole 85509-19-9 Rats: Harlan

Sprague Dawley Gavage 4 days DeVito

Isodecyl diphenyl phosphate 29761-21-5 Rats: Harlan

Sprague Dawley Gavage 4 days Auerbach

Isopropylated phenol phosphate 68937-41-7 Rats: Harlan

Sprague Dawley Gavage 4 days Auerbach
48 National Toxicology Program Annual Report 2012

Chemical CASRN Species/

Strain

Study 

Route

Study 

Length

Study 

Scientist

Pentabromodiphenyl oxide (technical) (DE 

71) 32534-81-9 Rats: Wistar Han Gavage

Dams gestation 

day 6 through 

postnatal day 21

Pups postnatal 

day 12–90

Dunnick

Polybrominated diphenyl ether (PBDE) 

toxicogenomic study:

BDE 153

BDE 99

BDE 47

DE 71

BDE Mixture

68631-49-2

60348-60-9

5436-43-1

32534-81-9

Mixtures

Rats: Wistar Han

Mice: B6C3F1/N Gavage

Dams gestation 

day 6 through 

postnatal day 21

Pups postnatal 

day 12–90

Dunnick

Simazine 122-34-9 Rats: Harlan

Sprague Dawley Gavage 4 days DeVito

Tetrabromobisphenol A 79-94-7 Rats: Wistar Han Gavage 90 days Dunnick

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

Tricresyl phosphate 1330-78-5 Rats: Harlan

Sprague Dawley Gavage 4 days Auerbach

Triphenyl phosphate 115-86-6 Rats: Harlan

Sprague Dawley Gavage 4 days Auerbach

Microarray analysis studies:

2,3,7-Tribromodibenzo-p-dioxin

2,3,4,7,8-Pentabromodibenzofuran

1,2,3,7,8-Pentabromodibenzofuran

2,3,7,8-Tetrabromodibenzofuran

2,3-Dibromo-7,8-dichlorodibenzo-p-dioxin

2,3,4,7,8-Pentachlorodibenzofuran

1,2,3,7,8-Pentachlorodibenzofuran (PCDF)

51974-40-4

13116-92-2

107555-93-1

67733-57-7

50585-40-5

57117-31-4

57117-41-6

B6C3F1 mouse Gavage Single dose–3 

days DeVito

C. NTP Research

In addition to the testing activities, a variety of research projects within the NTP are underway at NIEHS,

NCTR, and NIOSH. The following NIEHS/Division of the NTP (DNTP) branches are actively involved with 

NTP research activities: Biomolecular Screening Branch (led by Raymond Tice, PhD), Cellular and Molecular

Pathology Branch (led by Robert Sills, DVM, PhD), NTP Laboratory (led by Mike Waalkes, PhD), Program

Operations Branch (led by Michelle Hooth, PhD, DABT), and Toxicology Branch (led by Paul Foster, PhD).

The NTP Laboratory within the NIEHS/DNTP 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,

or methods development to advance the NTP mission. The NTP Laboratory also focuses on the study of the 

developmental origins of adult diseases. Table 25 includes projects in the NTP laboratory in FY 2012.
5. Testing and Research 49

Table 25. NIEHS/NTP Laboratory Projects in FY 2012

NIEHS/NTP 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.

Isoflavones and soy formula as estrogens

[Fenton, Howdeshell] 

To define estrogenicity of soy formula isoflavones in mice.

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 ER 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 (eg., lung epithelium) in the 

other; and (2) to define various types of macrophages ability to release

different metals from different particles. 

Methods in histopathology of mammary gland

development 

[Fenton]

To develop standardized methods to quatitatively 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, 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 (miRNAs) are thought to be a key epigenetic or posttranscriptional gene express control mechanism.

Role of stem cells in cancer associated with 

formaldehyde exposure 

[Waalkes]

To define the potential role of stem cells in relocating in hematopoietic

cancers in mice. 

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. 
50 National Toxicology Program Annual Report 2012

NIEHS/NTP Project 

[Study Scientist] Objective and/or Project Summary

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 

Genetic and epigenetic differences between individuals within the human population are believed 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 NIEHS/DNTP Biomolecular Screening

Branch is conducting several in-house projects aimed at understanding individual susceptibility.

Table 26. NIEHS/NTP Host Susceptibility Projects in FY 2012

NIEHS/NTP Project

[Study Scientist] Objective and/or Project Summary

Aging cohort 

[French] 

Reference data is being collected on 10 inbred strains of mice including classical and 

wild-derived inbred strains representing the greatest degree of genetic diversity in the 

mouse genome that is known at the present. This data includes survival rates, spontaneous

disease incidence, histopathology, hematology, clinical chemistry, quantitative trait loci, 

and behavioral observations, as well as tissue/fluids collection for future genetic/genomic/

metabolomic analysis. See Table 22 for more details.

Diversity outbred mice and 

population based models for 

toxicology and disease

[French] 

Research to determine the utility of diversity outbred mice for the NTP research and testing 

program using 28-day old diversity outbred male mice exposed to inhaled benzene (0, 

1, 10, or 100 ppm; 75 mice/group) for 5 days per week for 4 weeks in two independent

studies. Differences in the clearance of the chemical and its metabolites suggest significant

population differences for toxicity.

Ionizing radiation 

[French]

Development of tumor suppressor gene deficient mice for predicting carcinogenicity,

determining the mode of action, the presumptive risk of environmental exposures to

humans, and the identification of causally related genes and the specific functional allelic

and copy number variants within inbred strains of mice that modify carcinogen potency. 

Ongoing studies are to define system level differences in toxicogenomics associated with

differences in strain susceptibilities and to better define a short-term cancer bioassay for

research and testing.

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

2012 involves completion of transcript expression levels of both parental strains and the 

resulting first generation offspring by RNA high-throughput sequencing technologies and

DNA sequencing data analyses.
5. Testing and Research 51

i. Assessing Exposure to Substances in the Workplace

In following its mandate to protect workers’ health and safety, NIOSH is carrying out research projects for the

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 or alerts, other technical reports, and information profiles on

chemical hazards. Table 27 lists NIOSH/NTP projects in FY 2012.

Calibration of air sampling pumps after 

field sample collection at Health Effects 

Laboratory Division of NIOSH.

NIOSH Industrial hygienist conducting a 

nanomaterial exposure assessment.
52 National Toxicology Program Annual Report 2012

Table 27. NIOSH/NTP Research Projects FY 2012

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 NICHD/NIH. 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 lowcost, 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.

UV 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.

Perfluorooctanoic

acid (PFOA) and 

perfluorooctane sulfonate

(PFOS)-induced oxidative 

stress 

[Qian]

To investigate whether exposure to PFOA and PFOS induces an oxidative stress response in a 

mouse model and in human cells. PFOA and PFOS are widely known man-made fluorocarbonbased acids, which have been used in various industrial processes in the aircraft, automotive, 

chemical, building material, and personal care products industries. They are non-biodegradable 

and persistent in the human body and environment. Surveillance data suggests that PFOA and 

PFOS may cause adverse health effects, but no consistent association between exposure and 

health effects has been proven. 

Industry-wide studies 

of workers exposed to

carbon nanotubes and 

nanofibers

[Dahm]

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.

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.
5. Testing and Research 53

NIOSH/NTP Projects

[Project Officer] Objective and/or Summary

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 Bisphenol A (BPA).

These chemicals have been nominated by various groups to the National Toxicology Program. 

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.

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.

Evaluation of perfluoralkyl

acids immunotoxicity

[Franko]

To investigate the immunotoxicity of PFOA, no longer used in manufacturing, but still persistent in 

the environment, which has been shown in a murine model to be both immunosuppressive and 

to have a potential role in asthma and allergy. Due to the potential health effects linked to PFOA

exposure, replacement perfluoralkyl acids are now being used in the manufacturing process but

little is known about what effects of these compounds will have on immune function. This project

will evaluate the immunotoxic effects associated with individual perfluoralkyl acids that are still

in use, and to investigate the mechanism mediating the identified immunological alterations

associated with PFOA exposure. 
54 National Toxicology Program Annual Report 2012

NIOSH/NTP Projects

[Project Officer] Objective and/or Summary

Airway fungal exposure 

and allergic sensitization 

in mice

[Templeton]

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 will have two major areas of study; (1)

determination of the health effects following aspiration of hyphal fragments from Stachybotrys 

chartarum and Alternaria alternata in the absence of intact spores in mice, and (2) comparison of 

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

alternata to exacerbate respiratory allergy to ovalbumin.

Immune and 

inflammatory aspects of

occupational rhinitis

[Johnson]

To investigate occupational asthma in workers who often exhibit concurrent occupational

rhinitis with the likelihood that rhinitis developed first. Thus, understanding the mechanisms of

occupational rhinitis is an important area of research in occupational safety, health and medicine. 

A combined study design utilizing human and animal research will be employed to identify 

the orthologously conserved pathways and gene networks that characterize the pathobiology

of occupational rhinitis induced by diisocyanates. The outcomes of this research will benefit

occupational safety and health through improved diagnosis and prevention of allergic airways 

disease caused by diisocyanates. 

Cutaneous bioactivation 

of xenobiotics: hapten vs.

prohapten 

[Siegel]

To develop an in vivo model of allergic skin sensitization that can discriminate between chemicals

requiring metabolic activation for sensitization (prohaptens) and those that can sensitize 

without biological activation (haptens). The model will involve the dermal application of various 

pharmacological inhibitors of the cytochrome P450 pathway prior to performing either the local 

lymph node assay (LLNA) or/and mouse ear swelling test. Selective inhibition of the cytochrome 

P450 pathway should distinguish between direct acting haptens and metabolically activated prohaptens. Validation of the models will be done using known direct acting haptens and pro-haptens.

Successful development of these models will produce data that strengthens in silico hazard 

predictive models and allows for substitution or modification of allergenic chemicals and drugs.

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 that 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.
5. Testing and Research 55

ii. Comprehensive Assessment of Occupationally-Relevant Exposures

The 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 28 address worker exposures to welding fumes, nano-sized

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

Table 28. NIEHS/NIOSH Interagency Agreement on Occupationally Relevant Exposures in 

FY 2012

NIEHS/NIOSH 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, DC.

Assess the feasibility of 

an occupational exposure 

assessment of welding 

fume with emphasis on 

manganese compounds

[Hanley]

(1) To identify industries (e.g., 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.

Exposure assessment of 

dithiobisbenzanilide in a 

manufacturing setting

[Wurzelbacher]

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

dithiobisbenzanilide during a manufacturing process; (2) to develop a NIOSH analytical

method for quantitatively assessing dithiobisbenzanilide airborne particulate and surface 

exposures; and (3) to characterize industry-wide occupational exposures, including total

number of workers, and evaluate patterns of exposure to dithiobisbenzanilide.

A pilot exposure assessment 

for 2-methoxy-4-nitroaniline 

in a manufacturing setting 

[Wurzelbacher]

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

2-methoxy-4-nitroaniline during a manufacturing process; (2) to develop a NIOSH analytical

method for quantitatively assessing 2-methoxy-4-nitroaniline airborne particulate and surface 

exposures; and (3) to characterize industry-wide occupational exposures, including total

number of workers, and evaluate patterns of exposure to 2-methoxy-4-nitroaniline.

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-chloro-4-(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.
56 National Toxicology Program Annual Report 2012

NIEHS/NIOSH Study

[Study Scientist] Objective and/or Rationale

A pilot exposure assessment 

for ethylene glycol 

2-ethylhexyl ether (EGEHE) 

in a manufacturing setting 

[Harper]

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

EGEHE during manufacturing processes; (2) to develop an analytical method for quantitatively

assessing EGEHE airborne aerosols, vapors, and surface contamination; and (3) to

characterize industry-wide occupational exposures, including total number of workers, and

evaluate patterns of exposure to EGEHE.

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 in the United States 

[Hines]

(1) To determine bisphenol A (BPA) usage in industry, e.g., 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 UV 

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.

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 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 29, evaluate unique cohorts of 

individuals from professions associated with immune-mediated occupational diseases.
5. Testing and Research 57

Table 29. NIEHS/NIOSH Interagency Agreement on Immunotoxicology Studies, FY 2012

NIEHS/NIOSH Study

[Study Scientist] Objective and/or Rationale

NIEHS agricultural 

pesticide study 

[Beezhold]

In support of the NIEHS Agricultural Health Study, our laboratory has completed screening 

1454 farmers serum for total immunoglobulin E (IgE) and mold mix specific IgE using the

Phadia Immunocap assay. Those farmers who were mold mix positive have recently been 

tested against a 10 mold specific IgE panel (n = 126). Our laboratory has completed this

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

support of the NIEHS Agricultural Health Study, our 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 the NIEHS and prepared for publication in an 

allergy journal.

A marker for Aspergillus 

terreus exposure 

[Beezhold]

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 waterdamaged buildings or are occupationally relevant.

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 (rRNA) 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 rRNA approaches from samples 

collected in contaminated and non-contaminated 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 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. Subject recruitment and genotyping have been

completed. Data analyses have been initiated.
58 National Toxicology Program Annual Report 2012

NIEHS/NIOSH Study

[Study Scientist] Objective and/or Rationale

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. Subject

enrollment and sample processing are currently underway.

The role of genetic variation 

in environmental and 

occupational diseases – 

occupational asthma

[Yucesoy]

(1) To investigate whether genetic variations in specific candidate genes (e.g., 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) 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) complete both in vitro and in vivo assays to assess adverse

health effects caused by indoor air reaction products; and (4) further investigate the role of

structurally similar indoor air chemicals present in mixtures in the indoor environment.

Diisocyanate monoclonal 

antibody production and 

characterization (improved 

methods)

[Siegel]

(1) To develop monoclonal antibodies that recognize methylene diphenyldiisocyanate 

conjugated protein as a potential biomonitoring and research tool; (2) to test the specificity of

the methylene diphenyldiisocyanate monoclonal antibodies; and (3) to evaluate the monoclonal

antibodies for potential use in immunoassay-based biomonitoring.

Alternative methods 

for chemical allergen 

identification and

assessment

[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; and (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). 

Analysis of mycotoxins in 

dust samples from a waterdamaged building

[Park]

To apply and modify newer, chemical-based methods, including the use of gas chromatographytandem mass spectrometry (GC-MSMS) and ultra-performance liquid chromatography-tandem

mass spectrometry (UPLC-MSMS), for measuring microbial cell-wall components and toxins,

the methods of which are still being developed and are not readily available. This will quantify 

fungal toxins in environmental samples from water damaged buildings for which we have 

existing data on other fungal and bacterial components as well as health questionnaire data on 

building occupants. Especially, the UPLC-MSMS 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. Specific aims of the study are: (1) develop robust, rapid, and

cost-effective methods for simultaneous determination of multiple mycotoxins in environmental 

samples in a single analysis using UPLC-MSMS; and (2) apply the developed UPLC-MSMS

methods to analyze archived dust samples from water-damaged buildings collected in previous 

projects for characterizing exposure profiles to multiple mycotoxins.
5. Testing and Research 59

iv. NTP Research in Partnership with NCTR

The National Center for Toxicological Research (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 30. Table 31 lists projects that are funded through FDA and NTP interagency agreements.

Table 30. NCTR/NTP Projects in FY 2012

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Global and locus-specific

DNA hypomethylation: a

common mechanism involved 

in genotoxic and non-genotoxic 

rat hepatocarcinogenesis 

[Pogribny]

(1) To determine if the temporal alterations in genomic methylation profile in preneoplastic

liver tissue observed in the folate/methyl-deficient model of rat endogenous

hepatocarcinogenesis also occur in other carcinogenesis models, (2) to identify genes that 

are consistently up-regulated or down-regulated in target tissue during the promotion stage 

of carcinogenesis, and (3) to evaluate whether or not the global and locus-specific DNA

hypomethylation, along with aberrant expression of related genes and changes in chromatin 

conformation, is specific only to target tissues and may be used for early detection of

chemicals with carcinogenic potential.

Relationship between liver 

epigenetic phenotype and 

susceptibility to nonalcoholic 

steatohepatitis (NASH)-

induced hepatocarcinogenesis 

in mice 

[Pogribny]

(1) To determine the role of epigenetic dysregulation in the etiology and pathogenesis 

of dietary NASH-induced hepatocarcinogenesis in mice, (2) to determine whether or 

not interstrain-specific susceptibility of mice to NASH-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 NASH-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 NASHinduced hepatocarcinogenesis in mice.

Biologically-based, doseresponse (BBDR) modeling for 

the thyroid axis in the fetus and 

neonate 

[Fisher]

(1) To develop biologically-based, dose-response models for the hypothalamic-pituitarythyroid axis in the developing rat and human as a function of iodide status; (2) to

interface the BBDR hypothalamic-pituitary-thyroid 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.

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 preclinical-hazard identification

and genotoxicity testing of food ingredients and chemicals for regulatory purposes.

Biomarkers of liver toxicity

[Mendrick]

(1) To discover biomarkers of hepatotoxicity in preclinical studies that are predictive

of adverse effects in humans, for eventual evaluation of predictivity in rodent assays 

(preclinical studies); and (2) qualification of the discovered biomarkers.

Development of predictive 

mitochondrial biomarkers for

drug-induced 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).
60 National Toxicology Program Annual Report 2012

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

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 speciesspecific transcriptional profiles to predict risk of drug toxicity or disease onset in different

mammalian species.

Validation of recently 

established gpt-delta 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 (spiselection), and (2) 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. 

Physiologically-based 

pharmacokinetic models for

BPA

[Fisher]

1) To create physiologically-based pharmacokinetic models for BPA in three species

of adult, neonatal, and pregnant (mother and fetus) and lactating (mother and neonate) 

laboratory animals (mouse, rat, and rhesus monkey); (2) to use physiologically-based

pharmacokinetic models to calculate internal measures of dose for both aglycone (i.e.

active) and conjugated (i.e. inactive) forms of BPA; (3) to create physiologically-based

pharmacokinetic models for BPA exposure in humans (adult, child, and pregnant mother

and fetus, and lactating mother and infant); (4) to extrapolate the internal doses of

BPA associated with toxicity in laboratory animals to humans using the human suite of 

physiologically-based pharmacokinetic models; and (5) to extrapolate dosimetry from

regions of observation to low levels of exposure to BPA for which no experimental data 

exist. 

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.

Human biomonitoring for BPA 

[Doerge]

(1) To develop and implement sensitive and selective analytical methodology to measure 

BPA from blood and urine samples collected under controlled conditions from children and 

adults with known exposures, reducing the current level of uncertainty about exposure of

affected individuals and populations to BPA for use in risk assessment; and (2) to integrate

human biomonitoring data with pharmacokinetic data from animals and humans to produce

a physiologically-based pharmacokinetic model for BPA.

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 61

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

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 hypoxanthineguanine phosphoribosyltransferase lymphocyte gene mutation assays.

Detection of DNA adducts in 

mice treated with benzo[a]

pyrene at low-exposure levels 

[Fu]

To determine the dose-response curves for benzo[a]pyrene DNA adducts in the A/J mouse 

lung utilizing application of high performance liquid chromatography electrospray ionisation 

with tandem mass spectrometry methodologies developed at NCTR.

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. 

Methods development for

high-resolution dedicated 

positron emission tomography 

(microPET) to rodent 

neuroplasticity and toxicity 

during development 

[Wang]

To use microPET 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, using three different age groups of developing rats (gestational day 18 

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

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 allelespecific 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 (miRNA) expression 

analysis as a biomarker for

detecting carcinogens 

[Chen]

(1) To determine miRNA 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

miRNAs, and to use the polymerase chain reaction array to conduct time-course and 

dose-response studies for miRNA expression alterations in tissues of rats treated with 

carcinogens; and (3) to define the miRNA biomarker genes that are associated with

carcinogen exposure by predicting their target genes and determining their biological 

functions.
62 National Toxicology Program Annual Report 2012

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

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 

blood-brain barrier, brain, or other visceral organs of the rat.

Assessment of gaseous 

anesthetics in the developing 

nonhuman primate 

[Wang]

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

isofluorane results in increased in 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 

(microPET) 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 anestheticinduced neuronal cell death. 

Method development for study

of antioxidant properties in 

dietary supplements 

[Fu]

(1) To determine whether or not herbal dietary supplements can enhance or inhibit 

free radical formation or lipid peroxidation, mediated by microsomal metabolism, in a 

dose-dependent manner; (2) to determine the toxic effects, including mitochondrial

dehydrogenase activity, intracellular reactive oxygen species concentration, and 

mitochondrial membrane potential; and (3) to use of the electron spin resonance oximetry

technique to determine the inhibition/induction of lipid peroxidation of selected herbal 

dietary supplements in cells, including A549 human lung carcinoma cells and rabbit brain 

rBCECs cells.

Use of electron spin resonance 

spectroscopy to characterize 

the interactions between 

nanoscale materials and model 

biological systems

[Fu]

(1) To determine whether nanomaterials can catalyze Fenton reaction to initiate hydroxyl 

radical formation in a nanoparticle-size- dependent manner, or reduction by natural 

reducing agents, such as ascorbic acid and glutathione, leading to the formation of 

reactive oxygen species; (2) to determine whether nanomaterials enhance or inhibit free

radical formation mediated by microsomal metabolism or inhibit microsomal metabolism 

mediated lipid peroxidation, in a nanoparticle size dependent manner; (3) to determine the

toxic effects, including mitochondrial dehydrogenase activity, intracellular reactive oxygen 

species concentration, and mitochondrial membrane potential; and (4) to use electron spin

resonance oximetry technique to determine the inhibition/induction of lipid peroxidation by 

nanomaterials of different particle size in cells (A549 human lung carcinoma cells and rabbit 

brain rBCECs cells).

Development of an FDA 

resource and knowledge base

for sex difference in druginduced liver injury

[Tong]

(1) To develop a knowledge base for the sex differences in drug-induced liver injury through

analysis and modeling molecular data in the public domain, through development and 

fostering of the collection of genomic data from public resources, and through development 

of a standard data curation model for the sex-biased drug-induced liver injury in ArrayTrack

to manage the collected data and (2) to conduct meta-analysis, text mining, and network

analysis to develop a relationship between drugs, molecular signatures, liver-specific

biomarkers, genes/proteins functions, pathways and sex-biased liver toxicity.

QT interval correction via 

mixed-effects 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.
5. Testing and Research 63

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

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.

Methods development for

toxicity assays using the 

zebrafish embryo as a model

system: whole animal highthroughput 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 neurondevelopment; (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 motorneuron development

and the mechanism of action.

Effect of pediatric anesthetics 

on zebrafish embryos:

neurotoxicity verses gene 

expression changes and 

neuronal kinase cyclindependant 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).

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).

Assessing acetaminopheninduced 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.

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-D-aspartate 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) 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.
64 National Toxicology Program Annual Report 2012

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Interaction of nanoparticles 

with the gastrointestinal tract 

[Khare]

To investigate the role of various cellular components of the intestine involved in the 

uptake of nanoparticles and their accumulation in various cell types and potential effects

on biodistribution by: (1) determining the effect of nanomaterials on the permeability

of epithelial cells and establish immune correlates; (2) delineating the interaction of

nanomaterials with gastro-intestinal tract and gut-associated microbiota using ex vivo

models (intestinal explants); and (3) establishing the effect of nanoparticles on the

developmental stage of intestine and assess biodistribution of nanoparticles using a 

zebrafish model.

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.

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 (PND) 21 by examining morphology of the reproductive organs and mRNA 

expression of genes related to the endocrine system.

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.

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 namomaterials 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.

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.

Evaluation of growth, 

pubertal development and 

cardiovascular function in male 

rhesus monkeys (Macaca 

mulatta) chronically exposed to 

methylphenidate hydrochloride 

[Salminen]

(1) To determine whether cardiac biomarker levels are associated with echocardiographic

measures of abnormal cardiac structure and function in non-human primates exposed to 

methylphenidate hydrochloride and (2) to determine whether panels of cardiac biomarker

levels will more accurately predict echocardiographic and clinical status than do individual 

biomarkers.
5. Testing and Research 65

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Assessment of ketamine

toxicity in the developing nonhuman primate 

[Wang]

(1) To determine, using neurohistochemical approaches, if, and at what developmental 

stages, ketamine exposure increases neuronal apoptosis/proliferation; (2) to determine,

using neurohistochemical approaches, the dose-response for ketamine to produce

apoptosis at the most sensitive developmental stage; (3) to determine the reversibility

or permanence of the response using behavioral, imaging, and neurohistochemical 

approaches; and (4) to determine, at the most sensitive stage and dose, genomic and

proteomic responses to ketamine treatment.

Effect of p53 genotype on 

gene-expression profiles

in mice exposed to the 

model mutagen, N-ethyl-N’-

nitrosourea 

[Morris]

(1) To determine the effect of mutation in the p53 tumor suppressor gene on geneexpression profiles in young and aged mice and (2) to determine the effect of mutation in

p53 tumor-suppressor gene on gene-expression profiles in young and aged mice exposed

to the model mutagen N-ethyl-N’-nitrosourea.

Quality control for focused 

and unfocused liquid 

chromatography-mass 

spectrometry based 

metabolomic profiling of blood

samples 

[Beger]

To develop and test a quality control protocol for liquid chromatography-mass spectrometry 

based metabolomic profiling of blood samples on NCTR preclinical hepatotoxicity protocols.

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 food-contact 

material and (2) to characterize and quantify silver nanoparticles in food simulants.

3D- and 4D- quantitative 

spectrometric data-activity 

relationship (QSDAR) modeling 

applied to various toxicological 

endpoints 

[Beger]

(1) To develop 3D- and 4D- QSDAR models for endocrine disruptors, lowest-observedadverse-effects level and no observed-adverse-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-QSDAR modeling. 

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.
66 National Toxicology Program Annual Report 2012

NCTR/NTP Project 

[Study Scientist] Objective and/or Project Summary

Evaluation of the applicability 

of in vivo micronucleus assays 

for assessing genotoxicity of 

engineered nanomaterials 

[Chen]

(1) To assess the genotoxicity of four types of nanoscale materials (carbon nanotubes, 

nanoscale titanium dioxide, nanoscale gold, and nanoscale silver) in three standard tests 

used for genotoxicity assessment by 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.

Characterizing the 

amphetamine-induced 

changes in vascular tone, 

vasotrauma and alterations in 

angiogenesis in rodent brain 

[Bowyer]

To use acute and repeated dose models in the rat: (1) to evaluate the effects of both acute

and chronic amphetamine exposure on the vasculature of the rat brain, examining the 

vasculature within the parenchyma of three brain regions (striatum, parietal cortex, and 

combined piriform and amygdaloid nuclear cortices) and (2) to determine the effects of 

amphetamine on the vasculature associated with meninges pial and arachnoid membranes 

and vasculature of the choroid plexus. 

Table 31. NIEHS/NCTR Interagency Agreement Studies in FY 2012

Study 

CASRN

[Study Scientist]

Objective and/or Rationale

Assessment of molecular changes in male and 

female Sprague Dawley rats orally exposed to 

bisphenol A (BPA) from GD 6 through PND 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 GD 6 through PND 90.

Evaluation of toxicity of BPA in male and female 

Sprague Dawley (NCTR) Rats exposed orally 

from GD 6 through PND 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 (e.g., 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.

The role of perinatal development on 

pharmacokinetics of BPA

80-05-7 

[Doerge]

To develop additional data from rat and monkey exposures that

will provide data to be used in the creation and validation of a 

physiologically-based pharmacokinetic model to predict internal

exposures to free BPA in the appropriate target tissues of fetuses and 

babies that are derived from food contact and medical device exposures.

Two year chronic toxicology study of BPA 

administered by gavage to Sprague Dawley 

(NCTR) Rats from GD 6 until birth and directly 

to pups from PND1; 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. 
5. Testing and Research 67

Study 

CASRN

[Study Scientist]

Objective and/or Rationale

Evaluation of molecular, morphological, and 

functional endpoints in Sprague Dawley (NCTR) 

rats treated with BPA administered by gavage 

from GD 6 until birth and directly to F1 pups 

from PND 1; continuous and stop dose (PND 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 the 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 (PND 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. 

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.

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 PND 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.

Thirteen-week study to evaluate the toxicology of

silver nanoparticles in 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.

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.
68 National Toxicology Program Annual Report 2012

Study 

CASRN

[Study Scientist]

Objective and/or Rationale

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 to 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 onegeneration 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]

To determine the potential toxicity of oxybenzone on pre and early 

postnatal development in male and female rats 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.

Neurotoxicity assessment of cell phone radio 

frequency radiation using rat and bovine brain 

microvascular endothelial cells as model blood 

brain barrier systems, PC-12 cultured cells, and 

whole animal models

[Ali]

(1) To determine whether power levels of radio frequency radiation 

that are emitted from mobile phones produce any changes in the 

central nervous system of mice and rats and (2) to determine if there 

are disruptions in the blood brain barrier after being subjected to radio

frequency radiation.

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.

Subchronic toxicity of Usnea lichen in male and 

female Fischer 344 rats and B6C3F1 mice 

84696-53-7 

[Leakey]

To evaluate the subchronic toxicity of Usnea lichen in a 90-day 

toxicology study with dose administered in chow. 

Subchronic studies of usnic acid in Fischer 344 

rats and B6C3F1 mice 

125-56-2

[Leakey]

To evaluate the subchronic toxicity of usnic acid in male and female 

Fischer 344 rats and B6C3F1 mice using 90-day toxicology studies.

Toxicity studies of glucosamine and glucosamine/

chondroitin sulfate combination in obese and lean 

Zucker rats

3416-24-8, 9007-28-7

[Leakey]

To investigate the potential toxicity of glucosamine and glucosamine/

chondroitin sulfate combinations administered by oral gavage in male 

rats.

Perinatal carcinogenicity of drug combinations 

used to prevent mother-to-child transmission of HIV 

30616-87-1, 134678-17-4

[Beland]

To determine the carcinogenicity, genotoxicity, and metabolism of 

antiretroviral drug combinations administered to mice transplacentally 

and perinatally. The studies include 14-day range-finding and twoyearchronic in pregnant C57BL6N females and in B6C3F1 hybrid 

offspring.
5. Testing and Research 69

Study 

CASRN

[Study Scientist]

Objective and/or Rationale

Toxicity studies of combination of AIDS drugs in 

p53(+/-) transgenic mice

30616-87-1, 134678-17-4

[Leakey]

(1) To evaluate the potential toxicity and carcinogenicity of perinatal and 

chronic exposures to AIDS drugs, 3’-Azido-3’-Deoxythymidine (AZT) 

and lamivudine (3TC) in C3B6F1trp53(+/-) haplodeficient F1 transgenic

mice in a range-finding and a 6-9 month chronic phase, and (2) to

evaluate the potential toxicity and carcinogenicity of AZT/3TC/Nevirapine 

(NVP) combinations in C3B6F1trp53(+/-) in a 9-month exposure in mice.

Developmental neurotoxicity assessment of 

acrylamide in rats: long-term exposure

79-06-01

[Paule]

To determine the consequences of long-term exposure to acrylamide on 

a variety of developmental milestones and measures of nervous system 

integrity throughout life.

Genotoxicity and carcinogenicity of acrylamide 

and its metabolite, glycidamide, in rodents 

79-06-1, 5694-00-8

[Beland]

To determine the genotoxicity and carcinogenicity of acrylamide and 

its metabolite, glycidamide, in male and female Fischer 344 rats and 

B6C3F1 mice using range-finding, subchronic, and two-yearchronic

carcinogenicity studies.
70 National Toxicology Program Annual Report 2012

6. Alternative Methods Development

A. Tox 21

In FY 2008, the NTP established a high-throughput screening initiative, 

representing a new paradigm in toxicological testing. On February 14, 2008, a 

Memorandum of Understanding (MOU) was signed for High Throughput Screening,

Toxicity Pathway Profiling and Biological Interpretation of Findings (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 the NIH Chemical Genomics 

Center at the National Human Genome Research Institute (now the NIH Center 

for Advancing Translational Sciences or NCATS) and the U.S. EPA’s National 

Center for Computational Toxicology located within the Office of Research and

Development. The MOU was amended in 2010 to include the FDA. FDA’s 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 U.S. 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 Tox 21 Program was developed in response to the National Research Council1s 2007 report Toxicity 

Testing in the 21st Century: A Vision and a Strategy. A central component of this MOU is exploration of

quantitative high-throughput screening (qHTS) assays using phylogenetically lower animal species (e.g., fish,

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 to

protect human health and the environment. 

The goals of Tox21 and the NTP high-throughput screening program are the same, i.e., 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,

that inform adverse outcome pathway analyses, and that 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 (FY 2006 through FY 2010), a library of approximately 2,800 compounds provided by the 

NIEHS/NTP and EPA to the NCGC was tested in approximately 100 qHTSs. These assays broadly evaluated 

the ability of compounds (1) to induce cytotoxicity, apoptosis, DNA damage, changes in methylation status, 

mitochondrial toxicity, and up-regulation of various stress response pathways (e.g., antioxidant, hypoxia, heat 

shock) in a variety of cell types or (2) 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 12,000 substance library (8307 unique; informally known

as the “10K” library), with approximately 1/3 of the total substances assembled by each partner (NTP, EPA, and 
6. Alternative Methods Development 71

NCGC). The compounds cover a wide variety of classifications, and include consumer products, food additives,

chemicals found in industrial processes, and human and veterinary drugs. A complete list of the compounds is 

publicly available at http://www.epa.gov/ncct/dsstox/. Chemical analyses to determine the identity, purity, and 

stability of all compounds in this library were initiated in FY 2012 and will continue through FY 2013.

Also in FY 2012, screening of the 10K library began as well as screening on a subset of these compounds 

(~700) in Phase II of EPA’s ToxCastTM 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 that are present on each compound plate as well as 

approximately 1,900 compounds that are present in the library in multiple copies and were obtained from 

different sources. This compound replication provides a means of evaluating inter- and intra-plate variability 

as well as cross-assay variability in compound activity as a monitor of the technical quality of a screen. 

The emphasis during the first 18–24 months of screening in Phase II is on assays measuring nuclear

receptor signaling in agonist and antagonist mode (e.g., aryl hydrocarbon receptor, androgen receptor 

(partial, full), estrogen receptor alpha (partial, full), farnesoid x receptor, glucocorticoid receptor (full), liver 

x receptor, peroxisome proliferator-activated receptor delta and gamma, pregnane x receptor, retinoic acid 

receptor-related orphan receptor, retinoid x receptor, thyroid receptor, Vitamin D receptor), induction of stress 

response pathways (e.g., antioxidant, DNA damage, heat shock, hypoxia), and assays measuring specific

toxicity endpoints such as mitochondrial membrane changes, caspase activation, aromatase inhibition, and 

upregulation of NFkB (nuclear factor kappa beta), a transcription factor that has an important role in the

immune system.

By the end of FY 2012, 87 qHTS assays measuring 29 endpoints were completed and 12 assays were 

optimized for screening the 10K library. 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. 

Further, autofluorescence of compounds within the library was evaluated in two screens to identify potential

confounding issues in data interpretation. 

The Tox21 Phase II screening is being conducted in a new, high-speed robotics screening facility dedicated 

at the NCGC in mid FY2011; it was built with funds provided by the NIEHS/NTP. This robotics facility

was used to screen the 10K compound library in triplicate (30K compounds per screen). In FY 2011 the 

differential cytotoxicity response of cells from humans was evaluated by screening 1089 densely sequenced 

lymphoblastoid cell lines representing nine racial groups against 179 toxic chemicals. This study was designed 

to measure inter-individual differences in sensitivity to environmental toxicants, based on genotype. Evaluation 

of the data generated by this study occurred throughout FY 2012, with completion scheduled for FY 2013. 

Table 32 describes the NTP Tox 21 projects in FY 2012 that are being carried out by NIEHS/DNTP staff. This

includes the 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 NTP’s 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’s ToxCast Phase II chemical 

library of 700 compounds. The data from this screen are currently being analyzed and will be ready for 

publication in FY 2013.
72 National Toxicology Program Annual Report 2012

Table 32: NTP Tox 21 Projects in FY 2012

NTP Project Title 

[Study Scientist] Objective and/or Summary

Screening for aromatase 

inhibitors 

[Teng]

To screen aromatase inhibitors from the Tox21 10K library. In collaboration with Dr. Shiuan 

Chen at Beckman Institute of City of Hope, CA, NTP developed an “AroER Tri-screen” to

detect compounds that inhibits aromatase and estrogen receptor activities. The data are 

being analyzed.

Develop stable cell line to screen 

estrogen-related receptor/

peroxisome proliferator-activated 

receptor coactivator pathway 

[Teng]

To screen compounds which interfere with 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 PGC has been accomplished.

Modeling mixtures of androgen

receptor- and estrogen receptoractive compounds 

[Parham]

To determine (1) which mathematical models can 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 University of NC

involves providing data for their structure–activity relationship mixture model. 

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 ranking/calling procedure takes into account compound

potency, efficacy, and data reproducibility.

Prioritization of Tox21 

compounds 

[Hsieh]

To prioritize compounds that show clear evidence of activity in qHTS genotoxic assays, 

some weakly active compounds could be prioritized based on chemical structure–activity

relationship analysis.

Low-dose extrapolation 

[Parham]

To determine points of departure for low-dose extrapolation by using signal-to-noise ratios 

and a benchmark-dose method.

High content screening with 

HepaRG cells 

[Ferguson]

To establish metabolically functional human HepaRG liver cells in 384-well format to 

multiplex high content screening assays in collaboration with the NCGC. 

Polycyclic aromatic hydrocarbon 

mixture project

[Ferguson]

To evaluate ~20 poly-aromatic hydrocarbons (Gulf Oil Spill) in metabolism-competent 

HepaRG cells (derived from a human hepatic progenitor cell line) using multiplexed 

high content screening assays. Multiple measures of cellular health, damage, and gene

expression will be used in collaboration with the NCGC. 

Stem cells 

[Ferguson]

To screen for chemical toxicity in stem cell lines by qHTS at the NCGC. 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.

In silico prediction of metabolism 

[Ferguson]

To evaluate various in silico methods of predicting the extent of xenobiotic metabolism, 

identity metabolites, and prioritize chemical 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. 

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.

Genomic signatures forecasting 

chemical carcinogenicity 

[Auerbach]

To develop transcriptomic signatures related to carcinogen treatment using DrugMatrix

data in combination with a machine learning approach. Signatures are being validated 

using an independent set of data from the Japanese Toxicogenomics Project.
6. Alternative Methods Development 73

NTP Project Title 

[Study Scientist] Objective and/or Summary

Unsupervised, data-driven 

analysis of Tox21 assay data 

[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.

NextGen sequencing in 

toxicology 

[Merrick]

To develop pipelines for genomic and transcriptomic gene expression and mutational 

analysis on a genome-wide level using NextGen sequencing technologies.

Epigenetic changes in chemical 

toxicity 

[Merrick]

To determine methylation 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.

Testing for gene signatures and 

profiles in NTP archival tissues

[Merrick]

To determine if RNA can be extracted from fixed tissue blocks from the NTP Archives to

measure gene signatures and profiles. Data will show future usefulness of NTP Archives

for molecular measurements for chemical hazard assessment. 

NTP WormTox Facility: assay

development

[Freedman]

Reproduction, growth, feeding, and locomotion assays measure the effects of toxicant 

exposure on complex biological phenotypes including development and neuron function. 

An in vivo stress-inducible gene expression assay that uses transgenic Caenorhabditis 

elegans (C. elegans) expressing fluorescence reporters 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 realtime 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.

NTP WormTox Facility:

ToxCAST phase 2

[Freedman]

To determine the effects of a 700-compound library on C. elegans development and 

compare the relative toxicities to those in higher organisms. 

NTP WormTox Facility: ionic

liquids 

[Freedman]

(1) To describe the effects of four ionic liquids commonly used as starting materials for 

other ionic liquids on C. elegans feeding, growth, and reproduction and (2) to compare the 

results in C. elegans to results in rodents. 

NTP WormTox Facility: flame

retardants

[Freedman]

(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.

NTP WormTox Facility:

mitochondrial toxicants

[Freedman]

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.

NTP WormTox Facility: fluorides

[Freedman]

To compare the toxicities of three fluoride compounds commonly used in drinking water

treatment processes on C. elegans feeding, growth, and reproduction. 

Validation of high-throughput 

screening ER qHTS 

transactivation assay

[Casey]

(1) To compare the quality and accuracy of the qHTS assay that evaluates transcriptional 

activation of the estrogen receptor in human ovarian cancer cells (BG1 ER TA assay) 

relative to the manual method that has been validated for regulatory use (U.S. EPA and 

OECD). (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. (Once the quality of the data is deemed to be sufficient, results can be used to

assess and further develop quantitative structure–activity relationship models for estrogen 

receptor binding.
74 National Toxicology Program Annual Report 2012

NTP Project Title 

[Study Scientist] Objective and/or Summary

Analysis of 52 compounds in 

EPA’s ToxCast program 

[Casey]

The NTP selected compounds 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.

Development of a database 

related to skin sensitization

[Casey]

NICEATM, in concert with U.S. EPA and L’Oreal, aims to develop the world’s largest public

database for skin sensitization. This database will serve as a valuable source for reference

data to help develop alternative toxicological methods, including quantitative structure–

activity relationship models that can be used to eliminate the use of animals. 

B. ICCVAM

The purpose of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)

is to advance the regulatory acceptance of scientifically valid alternative test methods, those methods that

replace, reduce, or refine (enhance animal well-being and lessen or avoid pain and distress) the use of animals

(see page 11). The NICEATM was established in 1998. NICEATM administers ICCVAM, provides scientific

support for ICCVAM activities, and conducts independent validation studies on promising test methods.

NICEATM received contract support from Integrated Laboratory Systems, Inc.

Dr. William Stokes (Rear Admiral, U.S. Public Health Service) served as the NICEATM Director and Executive

Director of ICCVAM during FY 2012. Dr. Stokes retired from the U.S. Public Health Service Commissioned

Corps in December after over 30 years of dedicated federal service including over 20 years at NIEHS. 

Information about NICEATM and ICCVAM is available on the ICCVAM website at http://iccvam.niehs.nih.gov. 

ICCVAM has contributed to the approval or endorsement of over 60 alternative safety or potency testing

methods by Federal regulatory agencies since its first test method evaluation in 1998.

i. Workshops

NICEATM and ICCVAM organized two workshops in FY 2012. Presentations and summaries of the workshops

are available on the ICCVAM website (http://iccvam.niehs.nih.gov/meetings/schedule.htm).

The International Workshop on Alternative Methods for Human and Veterinary Rabies 

Vaccine Testing was held on October 11–13, 2011, at the USDA National Centers for Animal 

Health in Ames, Iowa. The International Cooperation on Alternative Test Methods (ICATM)

and the International Alliance for Biological Standardization cosponsored this workshop with

ICCVAM and NICEATM. Over 90 experts in human and veterinary rabies vaccines from

government, industry, and academia participated in the workshop. The objectives of the

workshop included (1) to assess data gaps and research needed to allow implementation

of available alternative methods for rabies vaccine potency testing, (2) to develop an 

implementation strategy to address these data gaps, (3) to assess ways to improve the 

current rabies potency test, (4) to evaluate available process control parameters and assays 

for demonstrating batch-to-batch consistency in conjunction with in vitro rabies potency

tests, and (5) to identify best practices for minimizing the use of animals. A workshop report

was published in September 2012 in the journal Biologicals (http://dx.doi.org/10.1016/j.

biologicals.2012.07.005).
6. Alternative Methods Development 75

The International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing was held on 

September 19–21, 2012, at the USDA National Centers for Animal Health in Ames, Iowa. In addition to ICATM

and the International Alliance for Biological Standardization, the Animal Health Institute also cosponsored this 

workshop. Over 80 scientific experts from government, industry, and academia participated in the workshop.

The objectives of the workshop were to (1) assess data gaps and research needed for implementation of

available alternative methods for Leptospira vaccine potency testing and (2) develop an implementation 

strategy to address these data gaps and achieve global regulatory acceptance of these alternatives. A 

workshop report is in preparation for publication in 2013 in the journal Biologicals.

ii. Publications

Recent NICEATM and ICCVAM publications, test methods currently under review, and project status are

presented in Tables 33, 34, and 35, respectively. A list of all ICCVAM publications is available at http://ntpapps.niehs.nih.gov/iccvampb/searchDoc.cfm, and the list of articles published by NICEATM staff members

and ICCVAM members in scientific journals is available at http://iccvam.niehs.nih.gov/articles/publications.htm. 

Further information on the status of nominations and submissions is available at http://iccvam.niehs.nih.gov/

SuppDocs/submission.htm.

The following are NICEATM and ICCVAM publications from FY 2012.

● Report on the International Workshop on Alternative Methods to Reduce, Refine, and Replace the

Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions

(dedicated issue of Procedia in Vaccinology), January 13, 2012.

● ICCVAM Test Method Evaluation Report - The LUMI-CELL® ER (BG1Luc ER TA) Test Method: An In

Vitro Assay for Identifying Human Estrogen Receptor Agonist and Antagonist Activity of Chemicals, 

February 1, 2012.

● Validation of the 21st Century Toxicology Toolbox: Challenges, Opportunities, and the Way Forward

(published in ALTEX Proceedings), March 1, 2012.

● BG1Luc ER TA Test Method: Results of an International Validation Study and Proposed Performance

Standards (published in ALTEX Proceedings), March 1, 2012.

● Informational Brochure: “NICEATM–ICCVAM: Advancing Public Health and Animal Welfare”, March 6,

2012.

● Informational Brochure: “Nominations and Submissions to ICCVAM: A Guide for Test Method

Developers and Sponsors”, March 6, 2012.

● ICCVAM Biennial Progress Report 2010-2011, June 13, 2012.

● Validation and regulatory acceptance of dermatotoxicology methods: Recent progress and the role of

NICEATM and ICCVAM. Book chapter: Dermatotoxicology, 8th Edition, July 1, 2012.

● Report on The International Workshop on Alternative Methods for Human and Veterinary Rabies

Vaccine Testing: State of the Science and Planning the Way Forward (published in Biologicals), 

September 1, 2012.
76 National Toxicology Program Annual Report 2012

Table 33. Nominations or Submissions to NICEATM-ICCVAM in FY 2012

Test Method Nomination 

or Submission Nominator or Sponsor/Activity Status

Electrophilic allergic screening assay for the 

detection of substances causing allergic contact 

dermatitis

NIOSH /under consideration

In vitro sensitivity assay for detection of 

substances that cause dermal sensitization

MB Research Labs/evaluation suspended; the assay is covered under

recent FDA guidance on in vitro pyrogen testing and a separate evaluation 

is thus unnecessary

Table 34. ICCVAM Recommendations in FY 2012

Test Method ICCVAM Recommendations/Agency Status

Use of the BG1Luc ER TA test 

method for endocrine disruptor 

chemical screening

ICCVAM evaluated the scientific validity of the BG1Luc estrogen receptor (ER)

transactivation (TA) agonist and antagonist assays and recommended how they could 

be used to identify substances that induce or inhibit human ER activity in vitro. Agency 

responses received in FY 2012. http://iccvam.niehs.nih.gov/methods/endocrine/end_eval.

htm#agencyresponses

Recommendations on the 

usefulness and limitations of 

the murine local lymph node 

assay for potency categorization 

of chemicals causing allergic 

contact dermatitis in humans

ICCVAM concluded that the murine local lymph node assay (LLNA) can be used to

categorize substances as strong sensitizers. However, substances that are not identified

as strong sensitizers using the LLNA require additional information to categorize them as 

other than strong sensitizers (Globally Harmonized System of Classification and Labeling

of Chemicals Subcategory 1B). Agency responses received in FY 2012. http://iccvam.

niehs.nih.gov/methods/immunotox/LLNApotency.htm

iii. International Validation Activities

NICEATM and ICCVAM prepared, commented on, or otherwise contributed to the development of new test

guidelines, revisions of existing test guidelines, and guidance documents considered by the Organisation for 

Economic Co-operation and Development (OECD). 

● ICCVAM recommendations on the use of anesthetics, analgesics, and humane endpoints were

incorporated into a 2012 update of the OECD test guideline describing animal tests to identify potential 

eye irritants.

● Results of a NICEATM-sponsored international validation study of the BG1Luc ER TA test method

were the basis for a new OECD test guideline describing the BG1Luc ER TA test method and a 

revision of an existing test method for stably transfected transactivation in vitro assays to detect 

estrogen receptor agonists.

● NICEATM staff and ICCVAM committee members participated in expert meetings and provided

comments on ongoing revisions of test guidelines for in vitro methods for identification of dermal

corrosives.

● NICEATM staff and ICCVAM members participated as liaisons to the scientific advisory committee of

the European Union Reference Laboratory for Alternatives to Animal Testing, European Centre for the 

Validation of Alternative Methods (EURL, ECVAM) for the meeting held October 2011 and March 2012.
6. Alternative Methods Development 77

● NICEATM staff attended the November 2011 meeting of the advisory council to the Japanese Center

for the Validation of Alternative Methods (JaCVAM).

Ongoing International Cooperation on Alternative Test Methods (ICATM) (http://iccvam.niehs.nih.gov/about/

icatm.htm) collaborations on validation studies are summarized in Table 35. NICEATM hosted a meeting

of representatives of the ICATM partner organizations in September 2012, and NICEATM and ICCVAM

representatives attended ICATM coordination meetings in March 2012.

Table 35. NICEATM Participation in International Validation Studies

Test Method Type of 

Test

Lead 

Organization NICEATM-ICCVAM Involvement

CertiChem MCF-7 cell

proliferation test method

Endocrine 

disruption NICEATM

NICEATM coordinated an international validation study

that included laboratories in the United States, Korea, 

and Japan

EpiOcularTM (MatTek) and

SkinEthicTM (L’Oreal)

Ocular 

irritation 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 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

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

In vitro crystal violet staining 

method using the rabbit corneaderived cell line

Ocular 

irritation JaCVAM NICEATM and ICCVAM members served on thevalidation management team
78 National Toxicology Program Annual Report 2012

7. Public Tools

The NTP provides a variety of resources for public use; those available in FY 2012 are described below. In FY

2012, the NTP Non-Neoplastic Lesion Atlas was in development and is scheduled for release in FY 2014. This 

is a database for non-neoplastic lesions organized by organ system employing a set of guidelines developed 

for consistent terminology.

A. 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 that accepts studies from many 

sources. Governmental, academic, and 

pharmaceutical company laboratories contribute 

peer-reviewed studies to CEBS. 

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 in CEBS 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—all viewed together underneath one study name.

The investigator does not have to access different databases, each housing one specific type of data.

The NIEHS is continuing to add legacy NTP data to CEBS. Phase II Tox21 data will 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 the study’s design and execution plus the biological responses of study subjects

(i.e. animals, 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.

B. NTP Archives

The NTP Archives is an important NIEHS-supported resource comprised of stained histopathology slides, 

paraffin tissue blocks, formalin-fixed tissues and organs, and selected frozen tissue from over 2,000

studies including toxicity, carcinogenicity, immunotoxicity, reproductive, and developmental studies. There 

are ongoing efforts to organize the NTP 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
7. Public Tools 79

of archival materials for various types of molecular analysis including gene expression, epigenetics, and 

mutational analysis.

The first archival gene expression study by NIEHS/DNTP, published in FY 2012, 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 and kidney 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. FFPE RNA from lung performed less satisfactorily probably because of this organ’s sensitivity to 

fixation conditions that degraded RNA into small fragments. This study also showed that the quality of FFPE

RNA and its successful application to molecular analysis could be predicted by measuring the size range 

of the fragmented FFPE RNA. Secondly, very exciting results were obtained with FFPE RNA from these 

same rat livers that show accurate whole-genome transcript profiling could be successfully performed using

transcriptome sequencing. Third, a different transcript profiling technique–qNPA–was used to compare

transcripts from RNA isolated from fresh or FFPE brain and liver in mice. These results again demonstrated 

the usefulness of archival tissue blocks for gene profiling. A more systematic query of NTP archival materials

from different organs and storage times is planned for the future to more precisely determine the value in 

transcript profiling.

C. The NCGC BioPlanet

No one, comprehensive, and 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, the NCGC (with support from the NIEHS through NTP, see page 16) built 

an integrated pathway resource that hosts information on ~1100 human pathways from manually curated and 

publicly available resources. The NCGC BioPlanet (http://www.ncgc.nih.gov/pub/bioplanet/) complements this 

pathway warehouse by allowing easy browsing, visualization, and analysis of the universe of pathways. 

D. DrugMatrix® and ToxFX®

Related to the goal of developing analysis tools and approaches to allow an integrated assessment of highthroughput screening endpoints and associations with findings from traditional toxicology and cancer models,

the NTP acquired DrugMatrix®-a toxicogenomics reference database and the accompanying extensive frozen

tissue archives, and the informatics system. This resource expands the NTP’s ability to develop predictive 

models for toxicological effects based on gene signatures, to provide additional tools for linking in vitro data to

in vivo gene signatures and disease outcomes, and to 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 (available at https://ntp.niehs.nih.gov/drugmatrix and https://

ntp.niehs.nih.gov/toxfx/). To date, over 300 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 DNTP scientists and research groups from the FDA’s National Center for Toxicological Research, the 

City of Hope, Stanford University, Boston University, U.S. EPA, Health Canada, Abbott Laboratories, Eli Lilly 

and Company, SAS, Maastrich 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) and in collaboration with researchers at Harvard University. 

Finally all the transcriptomics data from DrugMatrix® has been integrated in the NextBio, which is a database
80 National Toxicology Program Annual Report 2012

and analysis tool allowing analysis of the data within the context of all existing genomic and transcriptomic 

data. 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.

E. MetaDataViewer

Staff within the Office of Health Assessment and Translation, DNTP at NIEHS 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 for use in publications. Meta Data Viewer is available at http://ntp.niehs.nih.gov/go/

tools_metadataviewer. 
8. Appendices 81

8. Appendices

A. Frequently Used Abbreviations

3TC lamivudine

ADME	 absorption, distribution, metabolism, and

excretion

ATSDR Agency for Toxic Substances and 

Disease Registry

AZT zidovudine, 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 

CPSC U.S. Consumer Product Safety 

Commission

CYP cytochrome P450

ECVAM	 European Centre for the Validation of

Alternative Methods

EGEHE ethylene glycol 2-ethylhexyl ether

EPA U.S. Environmental Protection Agency

ER estrogen receptor 

FDA U.S. Food and Drug Administration

FFPE 	 formalin fixed, paraffin embedded

FY	 fiscal year

GABA gamma-amino butyric acid

GC-MSMS	 gas chromatography-tandem mass

spectrometry

GD gestational day

GMM	 genetically modified model

GSM	 global system for mobile communication

HHS U.S. Department of Health and Human 

Services

HPRT hypoxanthine-guanine 

phosphoribosyltransferase

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

IRIS	 EPA’s Integrated Risk Information

System 

JaCVAM	 Japanese Center for the Validation of

Alternative Methods

KoCVAM	 Korean Center for the Validation of

Alternative Methods

LIFE Longitudinal Investigation of Fertility and 

the Environment

LLNA Local Lymph Node Assay

miRNA microRNA

MOU	 Memorandum of Understanding

N/A not applicable

NASH nonalcoholic steatohepatitis

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

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

NTP National Toxicology Program

NVP Nevirapine 

OECD Organisation for Economic Co-operation 

and Development

OSHA Occupational Safety and Health 

Administration

PCB polychlorinated biphenyl

PCBTF	 chloro-4-(trifluoromethyl) benzene

PET Positron Emission Tomography 

PFOA	 perfluorooctanoic acid
82 National Toxicology Program Annual Report 2012

PFOS	 perfluorooctane sulfonate

Pig-A phosphatidylinositol glycan 

complementation group A 

PND postnatal day

qHTS quantitative high throughput screening

qNPA quantitiative nuclease protection assay

QSDAR quantitative spectrometric data-activity 

relationship 

RoC Report on Carcinogens

SACATM	 Scientific Advisory Committee on

Alternative Toxicological Methods

SOT Society of Toxicology

TCAB Tetrachloroazobenzene

TiO2 titanium dioxide

TOX	 NTP Toxicity Report

TR NTP Technical Report

UPLC-MSMS	 ultra-performance liquid

chromatography-tandem mass 

spectrometry

UV ultraviolet

U.S. United States

VOC volatile organic compound
8. Appendices 83

Babb, Amy 870-543-7697 amy.babb@fda.hhs.gov

Beland, Frederick 870-543-7205 frederick.beland@fda.hhs.gov

Boudreau, Mary 870-543-7526 mary.boudreau@fda.hhs.gov

Bryant, Matthew 870-543-7416 matthew.bryant@fda.hhs.gov

Camacho, Luisa 870-543-7588 luisa.camacho@fda.hhs.gov

Carroll, Kathy 870-543-7626 kathy.carroll@fda.hhs.gov

Chidambaram, Mani 870-543-7368 mani.chidambaram@fda.hhs.gov

Churchwell ,Mona 870-543-7218 mona.churchwell@fda.hhs.gov

Couch ,Letha 870-543-7137 latha.couch@fda.hhs.gov

Cozart, Christy 870-543-7602 christy.cozart@fda.hhs.gov

Cunningham, Candice 870-543-7886 candice.cunningham@fda.hhs.gov

Delclos, Barry 870-543-7372 barry.delclos@fda.hhs.gov

Doerge,Daniel 870-543-7943 daniel.doerge@fda.hhs.gov

Fang, Jia-Long 870-543-7612 jia-long.fang@fda.hhs.gov

Felton, Paul 870-543-7504 robert.felton@fda.hhs.gov

Ferguson, Sherry 870-543-7589 sherry.ferguson@fda.hhs.gov

Fisher, Jeff 870-543-7703 jeffrey.fisher@fda.hhs.gov

Gamboa Da Costa, Goncalo 870-543-7400 goncalo.gamboa@fda.hhs.gov

Hansen, Deborah 870-543-7480 deborah.hansen@fda.hhs.gov

Howard, Paul 870-543-7672 paul.howard@fda.hhs.gov

Imam, Mohammed 870-543-7127 mohammed.imam@fda.hhs.gov

Inselman, Amy 870-543-7623 amy.inselman@fda.hhs.gov

Jones, Yvonne 870-543-7508 yvonne.jones@fda.hhs.gov

Juliar, Beth 870-543-7789 beth.juliar@fda.hhs.gov

Law, Delbert 870-543-7013 charles.law@fda.hhs.gov

Leakey, Julian 870-543-7916 julian.leakey@fda.hhs.gov

Lewis, Sherry 870-543-7627 sherrym.lewis@fda.hhs.gov

Loukotkova, Lucie 870-543-7406 lucie.loukotkova@fda.hhs.gov

Matson, Sandra 870-543-7941 sandra.matson@fda.hhs.gov

Mclellen, Michelle 870-543-7647 florence.mclellen@fda.hhs.gov

Miller, Barbara 870-543-7226 barbara.miller@fda.hhs.gov

Moon, Steven 870-543-7124 steven.moon@fda.hhs.gov

Myhand, Amy 870-543-7644 amy.myhand@fda.hhs.gov

Nakamura, Noriko 870-543-7175 noriko.nakamura@fda.hhs.gov

Nichols, Jasyl 870-543-7210 jasyl.nichols@fda.hhs.gov

Pack, Lindsay 870-543-7681 lindsay.pack@fda.hhs.gov

Paredes, Angel 870-543-7949 angel.paredes@fda.hhs.gov

Paule, Merle 870-543-7147 merle.paule@fda.hhs.gov

Pogribna, Marta 870-543-7731 marta.pobribna@fda.hhs.gov

Pogribny, Igor 870-543-7096 igor.pogribny@fda.hhs.gov

Shelby, Rebecca 870-543-7958 rebecca.shelby@fda.hhs.gov

Shores, Patricia 870-543-7805 patricia.shores@fda.hhs.gov

Slikker, William 870-543-7517 william.slikker@fda.hhs.gov

Smith, Sherry 870-543-7242 sherryd.smith@fda.hhs.gov

Stingley, Robin 870-543-7792 robin.stingley@fda.hhs.gov

Thorn, Brett 870-543-7338 brett.thorn@fda.hhs.gov

Trbojevich, Raul 870-543-7490 raul.trbojevich@fda.hhs.gov

Twaddle, Nathan 870-543-7361 nathan.twaddle@fda.hhs.gov

Vanlandingham, Michelle 870-543-7458 michelle.vanlandingham@fda.hhs.gov

Von Tungeln, Suzy 870-543-7620 linda.vontungeln@fda.hhs.gov

Woodling, Kellie 870-543-7491 kellie.woodling@fda.hhs.gov

Wu, Yuanfeng 870-543-7596 yuanfeng.wu@fda.hhs.gov

NIEHS/NTP

Alden, Charles (919) 541-5722 alden@niehs.nih.gov

Allen, Bonnie (919) 541-3449 allenb@niehs.nih.gov

Andrews, Danica (919) 541-2595 andrewsda@niehs.nih.gov

Auerbach, Scott (919) 541-4505 auerbachs@mail.nih.gov

Bhusari, Sachin (919) 541-3069 bhusaris@mail.nih.gov

Birnbaum, Linda (919) 541-3201 birnbaumls@niehs.nih.gov

Bishop, Jack (919) 541-1876 bishop@mail.nih.gov

Blatchford, Kate (919) 541-3814 kate.blatchford@nih.gov

Blystone, Chad (919) 541-2741 blystonecr@mail.nih.gov

Bowden, Beth (919) 541-3355 bowden1@niehs.nih.gov

Boyd, Windy (919) 541-9810 boydw@niehs.nih.gov

Boyle, Mike (919) 541-1676 boylemc@mail.nih.gov

B. Agency Staff and Contact Information

NTP staff is listed alphabetically by agency.

NCTR/NTP 
84 National Toxicology Program Annual Report 2012

Boyles, Abee (919) 541-7886 boylesa@mail.nih.gov

Brown, Quashana (919) 541-4069 brownqd@mail.nih.gov

Bucher, John (919) 541-4532 bucher@niehs.nih.gov

Casey, Warren (919) 316-4729 caseywm@mail.nih.gov

Castro Marquez, Lysandra (919) 541-3373 castro@niehs.nih.gov

Cesta, Mark (919) 541-3229 cesta@niehs.nih.gov

Chan, Po (919) 541-7992 chanp1@niehs.nih.gov

Chhabra, Rajendra (919) 541-3386 chhabrar@niehs.nih.gov

Churchill, Sheba (919) 541-2919 churchillsj@mail.nih.gov

Clayton, Natasha (919) 541-7843 clayton2@niehs.nih.gov

Collins, Brad (919) 541-4666 collin10@niehs.nih.gov

Cora, Michelle (919) 316-4717 coramc@mail.nih.gov

Corniffe, Glenda (919) 541-4388 corniffe@niehs.nih.gov

Cunningham, Michael (919) 541-4613 cunningham@mail.nih.gov

Cunny, Helen (919) 541-5717 cunnyh@mail.nih.gov

Devito, Michael (919) 541-4142 devitom@mail.nih.gov

Dixon, Darlene (919) 541-3814 dixon@niehs.nih.gov

Dunlap, Paul (919) 541-2666 dunlapp@niehs.nih.gov

Dunnick, June (919) 541-4811 dunnickj@niehs.nih.gov

Elmore, Susan (919) 541-3474 elmore@niehs.nih.gov

Fenton, Sue (919) 541-4141 fentonse@mail.nih.gov

Ferguson, Stephen (919) 541-3799 ferguso4@niehs.nih.gov

Fields, Sally (919) 541-3971 fields3@niehs.nih.gov

Filgo, Adam (919) 541-4672 filgoaj@mail.nih.gov

Flagler, Norris (919) 541-2397 flagler@niehs.nih.gov

Flake, Gordon (919) 541-0037 flake@niehs.nih.gov

Foley, Julie (919) 541-3772 foley1@niehs.nih.gov

Fostel, Jennifer (919) 541-5055 fostel@mail.nih.gov

Foster, Paul (919) 541-2513 foster2@niehs.nih.gov

Frawley, Rachel (919) 541-2151 frawleyr@niehs.nih.gov

French, Jef (919) 541-2569 french@niehs.nih.gov

Freedman, Jonathan (919) 541-7899 freedma1@niehs.nih.gov

Fulbright, Camille (919) 541-3814 fulbrightcn@mail.nih.gov

Gao, Xiaohua (919) 316-4611 xiaohua.gao@nih.gov

Germolec, Dori (919) 541-3230 germolec@niehs.nih.gov

Goldsmith, Rachel (919) 541-2522 goldsmithre@mail.nih.gov

Guy, Robbin (919) 541-4363 guyr2@niehs.nih.gov

Gwinn, William (919) 541-0598 gwinnwm@niehs.nih.gov

Hall, Carolyn (919) 541-4995 hall7@niehs.nih.gov

Hall, Julie (919) 541-2679 julie.hall2@nih.gov

Hall, Laura (919) 541-7931 hall@niehs.nih.gov

Harry, Jean (919) 541-0927 harry@niehs.nih.gov

Herbert, Ron (919) 541-4613 herbert1@niehs.nih.gov

Hoenerhoff, Mark (919) 541-3440 hoenerhm@mail.nih.gov

Hong, Hue-Hua (919) 541-2141 hong5@niehs.nih.gov

Hooth, Michelle (919) 316-4643 hooth@niehs.nih.gov

Howdeshell, Kembra (919) 316-4708 howdeshellkl@mail.nih.gov

Hsieh, Jui-Hua (919) 316-4879 hsiehj2@mail.nih.gov

Irwin, Richard (919) 541-3340 irwin@niehs.nih.gov

Jahnke, Gloria (919) 541-3376 jahnke@mail.nih.gov

Jayaram, Beby (919) 541-1480 jayaram1@niehs.nih.gov

Jensen, Heather (919) 541-4075 jensen1@mail.nih.gov

Jeter, Shawn (919) 541-3476 jeter@niehs.nih.gov

Jones, Tina (919) 541-2836 jones30@niehs.nih.gov

King, Debra (919) 541-0698 kingdh@mail.nih.gov

King-Herbert, Angela (919) 541-3464 kingher1@mail.nih.gov

Kissling, Grace (919) 541-1756 kissling@niehs.nih.gov

Lasko, Denise (919) 541-0255 lasko@niehs.nih.gov

Lekwuwa, Michael (919) 541-5042 michael.lekwuwa@nih.gov

Litton, Dan (919) 541-2100 dan.litton@nih.gov

Lunn, Ruth M. (919) 316-4637 lunn@niehs.nih.gov

Macon, Madisa (919) 541-4141 maconmb@mail.nih.gov

Malarkey, David (919) 541-1745 malarkey@niehs.nih.gov

Masinde, Tiwanda (919) 541-4069 marsh2@mail.nih.gov

Masten, Scott (919) 541-5710 masten@niehs.nih.gov

Maull, Elizabeth (919) 316-4668 maull@niehs.nih.gov

McCarley, Debbie (919) 541-2384 mccarley@niehs.nih.gov

McIntyre, Barry (919) 316-4747 mcintyrebs@mail.nih.gov

McPherson, Christopher (919) 541-0167 mcphers1@niehs.nih.gov

Mercado-Feliciano, Minerva (919) 541-2756 mercadofelicim@niehs.nih.gov

Merrick, Alex (919) 541-1531 merrick1@niehs.nih.gov

Moore, Alicia (919) 541-7914 moore5@niehs.nih.gov

Morgan, Dan (919) 541-2264 morgan3@niehs.nih.gov

Ney, Eli (919) 541-5182 ney@niehs.nih.gov
8. Appendices 85

Ngalame, Olive (919) 541-5436 ngalamenn@mail.nih.gov

Nichols, Sharon (919) 541-7992 nicholss2@mail.nih.gov

Orihuela Garcia, Ruben (919) 541-3986 orihuelarg@mail.nih.gov

Ovwigho, Pamela (919) 541-3278 ovwighope@mail.nih.gov

Oyelowo, Olutoyosi (919) 541-0927 olutoyosi.oyelowo@nih.gov

Paragone, Ashley (919) 541-3278 paragonear@mail.nih.gov

Parham, Fred (919) 541-0760 fp20b@nih.gov

Pelch, Katie (919) 541-5564 pelchke@mail.nih.gov

Person, Rachel (919) 541-0685 personrj@mail.nih.gov

Plair, Jennifer (919) 541-3814 jennifer.plair@nih.gov

Qu, Wei (919) 541-0540 qu@niehs.nih.gov

Quist, Erin (919) 541-3036 quiste@mail.nih.gov

Rebolloso, Yvette (919) 541-4209 rebollo1@mail.nih.gov

Rice, Julie (919) 541-2533 ricej2@mail.nih.gov

Rider, Cynthia (919) 541-7638 cynthia.rider@nih.gov

Roach, Donna (919) 541-3023 roachd@mail.nih.gov

Robinson, Deirdre (919) 316-4660 robinsondk@mail.nih.gov

Robinson, Veronica (919) 541-2238 godfrey@niehs.nih.gov

Rooney, Andrew (919) 541-2999 rooneyaa@mail.nih.gov

Ryan, Kristen (919) 541-9468 ryankr@mail.nih.gov

Shines, Cassandra (919) 541-0959 shines1@niehs.nih.gov

Shockley, Keith (919) 541-3033 shockleykr@niehs.nih.gov

Sills, Robert (919) 541-0180 sills@niehs.nih.gov

Smith-Roe, Stephanie (919) 541-4253 stephanie.smith-roe@nih.gov

Smith, Cynthia (919) 541-3473 smith19@niehs.nih.gov

Soward, Sharon (919) 541-5132 soward@niehs.nih.gov

Spencer, Diane (919) 541-2759 spencer2@niehs.nih.gov

Stanko, Jason (919) 541-0461 stankojp@niehs.nih.gov

Stockton, Patricia (919) 541-4471 stockton@niehs.nih.gov

Stokes, William (919) 541-7997 stokes@niehs.nih.gov

Stout, Matt (919) 541-3489 stoutm@mail.nih.gov

Sun, Yang (919) 541-2679 suny2@mail.nih.gov

Surh, In Ok (919) 541-3862 surhi@mail.nih.gov

Sutton, Deloris (919) 541-5112 sutton2@niehs.nih.gov

Taylor, Kyla (919) 316-4707 taylorkw@mail.nih.gov

Teng, Tina (919) 541-0344 teng1@niehs.nih.gov

Thakur, Sheetal (919) 541-2784 thakursa@mail.nih.gov

Thayer, Kris (919) 541-5021 thayer@mail.nih.gov

Tice, Ray (919) 541-4482 tice@mail.nih.gov

Tokar, Erik (919) 541-3951 tokare@mail.nih.gov

Ton, Thai-Vu (919) 541-2118 ton@niehs.nih.gov

Travlos, Greg (919) 541-0653 travlos@niehs.nih.gov

Tubbs, Jacquelyn (708) 903-0135 tubbsj@mail.nih.gov

Vallant, Molly Kutty (919) 541-5234 vallant@niehs.nih.gov

Waalkes, Mike (919) 541-2328 waalkes@niehs.nih.gov

Waidyanatha, Suramya (919) 541-2144 waidyanathas@mail.nih.gov

Walker, Nigel (919) 541-4893 walker3@niehs.nih.gov

Walker, Vickie (919) 541-4514 ray3@niehs.nih.gov

White, Lori (919) 541-9834 whiteld@mail.nih.gov

White, Sally (919) 541-4388 whitess@mail.nih.gov

Wilson Sr, Ralph (919) 541-4140 wilson@niehs.nih.gov

Withers, Sheila (919) 541-5066 withersg@niehs.nih.gov

Witt, Kristine (919) 541-2761 witt@niehs.nih.gov

Wolfe, Mary (919) 541-7539 wolfe@niehs.nih.gov

Wyde, Michael (919) 316-4640 wyde@mail.nih.gov

Xu, Yuanyuan (919) 541-3415 xuy5@mail.nih.gov

Yu, Linda (919) 316-4599 yu1@niehs.nih.gov

NIOSH/NTP

Anderson, Stacey (304) 285-6174 dbx7@cdc.gov

Beezhold, Don (304) 285-5963 zec1@cdc.gov

Brown, Kenneth (513) 841-4364 krb1@cdc.gov

Curwin, Brian (513) 841-4432 bic4@cdc.gov

Dahm, Matt (513) 458-7136 Iwa6@cdc.gov

DeBord, D. Gayle (513) 841-4256 ded4@cdc.gov 

Erdely, Aaron (304) 285-5903 efi4@cdc.gov

Geraci, Chuck (513) 533-8339 ciu9@cdc.gov

Green, Brett (304) 285-5884 dox6@cdc.gov

Hanley, Kevin (513) 841-4113 kwh0@cdc.gov

Harper, Martin (304) 285-6034 zzg7@cdc.gov

Hines, Cynthia (513) 841-4453 cjh8@cdc.gov

Hodson, Laura (513) 533-8526 gey7@cdc.gov

Luster, Michael (304) 285-5940 myl6@cdc.gov 

Martinez, Kenneth (513) 533-8640 kfm1@cdc.gov
86 National Toxicology Program Annual Report 2012

Munson, Albert E. (304) 285-6121 akm5@cdc.gov 

Park, Ju-Hyeong (304) 285-5967

Qian, Yong (304) 285-6286 yaq2@cdc.gov

Reynolds, Steve (304) 285-5806 sir6@cdc.gov

Schubauer-Berigan, Mary (513-841-4251 zcg3@cdc.gov

Siegel, Paul (304) 285-5855 pds3@cdc.gov 

Snawder, John (513) 533-8496 jts5@cdc.gov 

Stefaniak, Aleksandr (304) 285-6302 boq9@cdc.gov

Streicher, Robert (513) 841-4296 rps3@cdc.gov

Striley, Cynthia (513) 533-8123 chs3@cdc.gov 

Wells, Ray (304) 285-6341 ozw0@cdc.gov

Whelan, Elizabeth (513) 841-4437 eaw0@cdc.gov

Wurzelbacher, Steve (513) 841-4322 srw3@cdc.gov

Yucesoy, Berran (304) 285-5993 yab7@cdc.gov
8. Appendices 87

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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

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5 Funded by the NTP/NHGRI/EPA/FDA Interagency MOU

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