TOXICOLOGICAL PROFILE FOR
 

DINITROTOLUENES
 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
 

Public Health Service
 

Agency for Toxic Substances and Disease Registry
 

February 2016 
DISCLAIMER 

Use of trade names is for identification only and does not imply endorsement by the Agency for Toxic 

Substances and Disease Registry, the Public Health Service, or the U.S. Department of Health and Human 

Services. 

DINITROTOLUENES ii 
UPDATE STATEMENT
 

A Toxicological Profile for Dinitrotoluenes, Draft for Public Comment was released in April 2013. This 

edition supersedes any previously released draft or final profile. 

Toxicological profiles are revised and republished as necessary. For information regarding the update 

status of previously released profiles, contact ATSDR at: 

Agency for Toxic Substances and Disease Registry
 

Division of Toxicology and Human Health Sciences
 

Environmental Toxicology Branch
 

1600 Clifton Road NE
 

Mailstop F-57
 

Atlanta, Georgia 30329-4027
 

DINITROTOLUENES iii 
DINITROTOLUENES iv 

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DINITROTOLUENES v 

FOREWORD 

This toxicological profile is prepared in accordance with guidelines* developed by the Agency for Toxic 

Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA). The 

original guidelines were published in the Federal Register on April 17, 1987. Each profile will be revised 

and republished as necessary. 

The ATSDR toxicological profile succinctly characterizes the toxicologic and adverse health effects 

information for these toxic substances described therein. Each peer-reviewed profile identifies and 

reviews the key literature that describes a substance's toxicologic properties. Other pertinent literature is 

also presented, but is described in less detail than the key studies. The profile is not intended to be an 

exhaustive document; however, more comprehensive sources of specialty information are referenced. 

The focus of the profiles is on health and toxicologic information; therefore, each toxicological profile 

begins with a public health statement that describes, in nontechnical language, a substance's relevant 

toxicological properties. Following the public health statement is information concerning levels of 

significant human exposure and, where known, significant health effects. The adequacy of information to 

determine a substance's health effects is described in a health effects summary. Data needs that are of 

significance to protection of public health are identified by ATSDR. 

Each profile includes the following: 

(A) The examination, summary, and interpretation of available toxicologic information and 

epidemiologic evaluations on a toxic substance to ascertain the levels of significant human 

exposure for the substance and the associated acute, subacute, and chronic health effects; 

(B) A determination of whether adequate information on the health effects of each substance 

is available or in the process of development to determine levels of exposure that present a 

significant risk to human health of acute, subacute, and chronic health effects; and 

(C) Where appropriate, identification of toxicologic testing needed to identify the types or 

levels of exposure that may present significant risk of adverse health effects in humans. 

The principal audiences for the toxicological profiles are health professionals at the Federal, State, and 

local levels; interested private sector organizations and groups; and members of the public. 

This profile reflects ATSDR’s assessment of all relevant toxicologic testing and information that has been 

peer-reviewed. Staffs of the Centers for Disease Control and Prevention and other Federal scientists have 

also reviewed the profile. In addition, this profile has been peer-reviewed by a nongovernmental panel 

and was made available for public review. Final responsibility for the contents and views expressed in 

this toxicological profile resides with ATSDR. 

Patrick N. Breysse, Ph.D., CIH
 

Director, National Center for Environmental Health and
 

Agency for Toxic Substances and Disease Registry
 

Centers for Disease Control and Prevention
 
*Legislative Background 

The toxicological profiles are developed under the Comprehensive Environmental Response, 

Compensation, and Liability Act of 1980, as amended (CERCLA or Superfund). CERCLA section 

104(i)(1) directs the Administrator of ATSDR to “...effectuate and implement the health related 

authorities” of the statute. This includes the preparation of toxicological profiles for hazardous 

substances most commonly found at facilities on the CERCLA National Priorities List and that pose the 

most significant potential threat to human health, as determined by ATSDR and the EPA. Section 

104(i)(3) of CERCLA, as amended, directs the Administrator of ATSDR to prepare a toxicological profile 

for each substance on the list. In addition, ATSDR has the authority to prepare toxicological profiles for 

substances not found at sites on the National Priorities List, in an effort to “...establish and maintain 

inventory of literature, research, and studies on the health effects of toxic substances” under CERCLA 

Section 104(i)(1)(B), to respond to requests for consultation under section 104(i)(4), and as otherwise 

necessary to support the site-specific response actions conducted by ATSDR. 

DINITROTOLUENES vi 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES vii 

QUICK REFERENCE FOR HEALTH CARE PROVIDERS 

Toxicological Profiles are a unique compilation of toxicological information on a given hazardous 

substance. Each profile reflects a comprehensive and extensive evaluation, summary, and interpretation 

of available toxicologic and epidemiologic information on a substance. Health care providers treating 

patients potentially exposed to hazardous substances will find the following information helpful for fast 

answers to often-asked questions. 

Primary Chapters/Sections of Interest 

Chapter 1: Public Health Statement: The Public Health Statement can be a useful tool for educating 

patients about possible exposure to a hazardous substance. It explains a substance’s relevant 

toxicologic properties in a nontechnical, question-and-answer format, and it includes a review of 

the general health effects observed following exposure. 

Chapter 2: Relevance to Public Health: The Relevance to Public Health Section evaluates, interprets, 

and assesses the significance of toxicity data to human health. 

Chapter 3: Health Effects: Specific health effects of a given hazardous compound are reported by type 

of health effect (death, systemic, immunologic, reproductive), by route of exposure, and by length 

of exposure (acute, intermediate, and chronic). In addition, both human and animal studies are 

reported in this section. 

NOTE: Not all health effects reported in this section are necessarily observed in the clinical 

setting. Please refer to the Public Health Statement to identify general health effects observed 

following exposure. 

Pediatrics: Four new sections have been added to each Toxicological Profile to address child health 

issues: 

Chapter 1 How Can (Chemical X) Affect Children?
 

Chapter 1 How Can Families Reduce the Risk of Exposure to (Chemical X)?
 

Section 3.7 Children’s Susceptibility
 

Section 6.6 Exposures of Children
 

Other Sections of Interest: 

Section 3.8 Biomarkers of Exposure and Effect 

Section 3.11 Methods for Reducing Toxic Effects 

ATSDR Information Center 

Phone: 1-800-CDC-INFO (800-232-4636) or 1-888-232-6348 (TTY) 

Internet: http://www.atsdr.cdc.gov 

The following additional material is available online at www.atsdr.cdc.gov: 

Case Studies in Environmental Medicine—Case Studies are self-instructional publications designed to 

increase primary care provider’s knowledge of a hazardous substance in the environment and to 

aid in the evaluation of potentially exposed patients. 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES viii 

Managing Hazardous Materials Incidents is a three-volume set of recommendations for on-scene 

(prehospital) and hospital medical management of patients exposed during a hazardous materials 

incident. Volumes I and II are planning guides to assist first responders and hospital emergency 

department personnel in planning for incidents that involve hazardous materials. Volume III— 

Medical Management Guidelines for Acute Chemical Exposures—is a guide for health care 

professionals treating patients exposed to hazardous materials. 

Fact Sheets (ToxFAQsTM) provide answers to frequently asked questions about toxic substances. 

Other Agencies and Organizations 

The National Center for Environmental Health (NCEH) focuses on preventing or controlling disease, 

injury, and disability related to the interactions between people and their environment outside the 

workplace. Contact: NCEH, Mailstop F-29, 4770 Buford Highway, NE, Atlanta, 

GA 30341-3724 • Phone: 770-488-7000 • FAX: 770-488-7015. 

The National Institute for Occupational Safety and Health (NIOSH) conducts research on occupational 

diseases and injuries, responds to requests for assistance by investigating problems of health and 

safety in the workplace, recommends standards to the Occupational Safety and Health 

Administration (OSHA) and the Mine Safety and Health Administration (MSHA), and trains 

professionals in occupational safety and health. Contact: NIOSH, 395 E Street, S.W., Suite 9200, 

Patriots Plaza Building, Washington, DC 20201 • Phone: (202) 245-0625 or 1-800-CDC-INFO 

(800-232-4636). 

The National Institute of Environmental Health Sciences (NIEHS) is the principal federal agency for 

biomedical research on the effects of chemical, physical, and biologic environmental agents on 

human health and well-being. Contact: NIEHS, PO Box 12233, 104 T.W. Alexander Drive, 

Research Triangle Park, NC 27709 • Phone: 919-541-3212. 

Clinical Resources 

The Association of Occupational and Environmental Clinics (AOEC) has developed a network of clinics 

in the United States to provide expertise in occupational and environmental issues. Contact: 

AOEC, 1010 Vermont Avenue, NW, #513, Washington, DC 20005 • Phone: 202-347-4976 

• FAX: 202-347-4950 • e-mail: AOEC@AOEC.ORG • Web Page: http://www.aoec.org/. 

The American College of Occupational and Environmental Medicine (ACOEM) is an association of 

physicians and other health care providers specializing in the field of occupational and 

environmental medicine. Contact: ACOEM, 25 Northwest Point Boulevard, Suite 700, Elk 

Grove Village, IL 60007-1030 • Phone: 847-818-1800 • FAX: 847-818-9266. 
 

 

 

 

 

DINITROTOLUENES ix 

CONTRIBUTORS 

CHEMICAL MANAGER(S)/AUTHOR(S): 

Carolyn Harper, Ph.D.
 

Nickolette Roney, M.P.H.
 

Mike Fay, Ph.D.
 

Selene Chou, Ph.D.
 

ATSDR, Division of Toxicology and Human Health Sciences, Atlanta, GA
 

Heather Carlson-Lynch, M.S., DABT
 

Julie M. Klotzbach, Ph.D.
 

Kelly Salinas, Ph.D.
 

H. Danielle Johnson, B.S.
 

Mario Citra, Ph.D.
 

SRC, Inc., North Syracuse, NY
 

THE PROFILE HAS UNDERGONE THE FOLLOWING ATSDR INTERNAL REVIEWS: 

1.	 Health Effects Review. The Health Effects Review Committee examines the health effects 

chapter of each profile for consistency and accuracy in interpreting health effects and classifying 

end points. 

2.	 Minimal Risk Level Review. The Minimal Risk Level Workgroup considers issues relevant to 

substance-specific Minimal Risk Levels (MRLs), reviews the health effects database of each 

profile, and makes recommendations for derivation of MRLs. 

3.	 Data Needs Review. The Environmental Toxicology Branch reviews data needs sections to 

assure consistency across profiles and adherence to instructions in the Guidance. 

4.	 Green Border Review. Green Border review assures the consistency with ATSDR policy. 
DINITROTOLUENES x 

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DINITROTOLUENES xi 

PEER REVIEW
 

A peer review panel was assembled for dinitrotoluenes. The panel consisted of the following members: 

1.	 William J. George, Ph.D., Department of Pharmacology SL83, Tulane University School of 

Medicine, New Orleans, Louisiana; 

2.	 James Klaunig, Ph.D., Professor and Director of Toxicology, Department of Pharmacology and 

Toxicology, Division of Toxicology, School of Medicine, Indiana University, Indianapolis, 

Indiana; 

3.	 Volker Harth, MD, MPH, Professor of Occupational Medicine, University Medical Center 

Hamburg-Eppendorf (UKE), Director Institute for Occupational and Maritime Medicine (ZfAM), 

Hamburg, Germany. 

These experts collectively have knowledge of dinitrotoluene’s physical and chemical properties, 

toxicokinetics, key health end points, mechanisms of action, human and animal exposure, and 

quantification of risk to humans. All reviewers were selected in conformity with the conditions for peer 

review specified in Section 104(I)(13) of the Comprehensive Environmental Response, Compensation, 

and Liability Act, as amended. 

Scientists from the Agency for Toxic Substances and Disease Registry (ATSDR) have reviewed the peer 

reviewers' comments and determined which comments will be included in the profile. A listing of the 

peer reviewers' comments not incorporated in the profile, with a brief explanation of the rationale for their 

exclusion, exists as part of the administrative record for this compound. 

The citation of the peer review panel should not be understood to imply its approval of the profile's final 

content. The responsibility for the content of this profile lies with the ATSDR. 
DINITROTOLUENES xii 

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CONTENTS
 

DISCLAIMER ..............................................................................................................................................ii
 

UPDATE STATEMENT .............................................................................................................................iii
 

FOREWORD ................................................................................................................................................v
 

QUICK REFERENCE FOR HEALTH CARE PROVIDERS....................................................................vii
 

CONTRIBUTORS.......................................................................................................................................ix
 

PEER REVIEW ...........................................................................................................................................xi
 

CONTENTS...............................................................................................................................................xiii
 

LIST OF FIGURES ..................................................................................................................................xvii
 

LIST OF TABLES.....................................................................................................................................xix
 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES (DNTs)..............................................1
 

2. RELEVANCE TO PUBLIC HEALTH ...................................................................................................7
 

2.1 BACKGROUND AND ENVIRONMENTAL EXPOSURES TO DNT IN THE UNITED 

STATES...........................................................................................................................................7
 

2.2 SUMMARY OF HEALTH EFFECTS...........................................................................................8
 

2.3 MINIMAL RISK LEVELS (MRLs) ............................................................................................12
 

3. HEALTH EFFECTS..............................................................................................................................35
 

3.1 INTRODUCTION........................................................................................................................35
 

3.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE .....................................35
 

3.2.1 Inhalation Exposure ..............................................................................................................36
 

3.2.1.1 Death..............................................................................................................................37
 

3.2.1.2 Systemic Effects.............................................................................................................38
 

3.2.1.3 Immunological and Lymphoreticular Effects................................................................41
 

3.2.1.4 Neurological Effects ......................................................................................................41
 

3.2.1.5 Reproductive Effects......................................................................................................42
 

3.2.1.6 Developmental Effects................................................................................................... 43
 

3.2.1.7 Cancer............................................................................................................................43
 

3.2.2 Oral Exposure........................................................................................................................44
 

3.2.2.1 Death..............................................................................................................................45
 

3.2.2.2 Systemic Effects............................................................................................................48
 

3.2.2.3 Immunological and Lymphoreticular Effects..............................................................111
 

3.2.2.4 Neurological Effects ....................................................................................................112
 

3.2.2.5 Reproductive Effects....................................................................................................115
 

3.2.2.6 Developmental Effects.................................................................................................119
 

3.2.2.7 Cancer..........................................................................................................................119
 

3.2.3 Dermal Exposure.................................................................................................................122
 

3.2.3.1 Death............................................................................................................................122
 

3.2.3.2 Systemic Effects...........................................................................................................123
 

3.2.3.3 Immunological and Lymphoreticular Effects..............................................................125
 

3.2.3.4 Neurological Effects ....................................................................................................126
 

3.2.3.5 Reproductive Effects....................................................................................................126
 

3.2.3.6 Developmental Effects.................................................................................................126
 

3.2.3.7 Cancer..........................................................................................................................126
 

3.3 GENOTOXICITY......................................................................................................................127
 

3.4 TOXICOKINETICS...................................................................................................................137
 

3.4.1 Absorption...........................................................................................................................137
 

3.4.1.1 Inhalation Exposure .....................................................................................................137
 

DINITROTOLUENES xiii 
3.4.1.2 Oral Exposure ..............................................................................................................137
 

3.4.1.3 Dermal Exposure .........................................................................................................138
 

3.4.2 Distribution .........................................................................................................................138
 

3.4.2.1 Inhalation Exposure .....................................................................................................138
 

3.4.2.2 Oral Exposure ..............................................................................................................138
 

3.4.2.3 Dermal Exposure .........................................................................................................139
 

3.4.3 Metabolism..........................................................................................................................139
 

3.4.4 Elimination and Excretion...................................................................................................147
 

3.4.4.1 Inhalation Exposure .....................................................................................................147
 

3.4.4.2 Oral Exposure ..............................................................................................................147
 

3.4.4.3 Dermal Exposure .........................................................................................................148
 

3.4.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models...........149
 

3.5 MECHANISMS OF ACTION ...................................................................................................150
 

3.5.1 Pharmacokinetic Mechanisms.............................................................................................150
 

3.5.2 Mechanisms of Toxicity......................................................................................................152
 

3.5.3 Animal-to-Human Extrapolations.......................................................................................155
 

3.6 TOXICITIES MEDIATED THROUGH THE NEUROENDOCRINE AXIS...........................156
 

3.7 CHILDREN’S SUSCEPTIBILITY............................................................................................157
 

3.8 BIOMARKERS OF EXPOSURE AND EFFECT .....................................................................160
 

3.8.1 Biomarkers Used to Identify or Quantify Exposure to DNTs.............................................161
 

3.8.2 Biomarkers Used to Characterize Effects Caused by DNTs...............................................162
 

3.9 INTERACTIONS WITH OTHER CHEMICALS .....................................................................163
 

3.10 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE ............................................163
 

3.11 METHODS FOR REDUCING TOXIC EFFECTS................................................................ 164
 

3.11.1 Reducing Peak Absorption Following Exposure.............................................................165
 

3.11.2 Reducing Body Burden ...................................................................................................165
 

3.11.3 Interfering with the Mechanism of Action for Toxic Effects..........................................165
 

3.12 ADEQUACY OF THE DATABASE.....................................................................................166
 

3.12.1 Existing Information on Health Effects of DNTs............................................................166
 

3.12.2 Identification of Data Needs............................................................................................171
 

3.12.3 Ongoing Studies ..............................................................................................................179
 

4. CHEMICAL AND PHYSICAL INFORMATION..............................................................................181
 

4.1 CHEMICAL IDENTITY............................................................................................................181
 

4.2 PHYSICAL AND CHEMICAL PROPERTIES.........................................................................181
 

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL..........................................................187
 

5.1 PRODUCTION ..........................................................................................................................187
 

5.2 IMPORT/EXPORT ....................................................................................................................187
 

5.3 USE ............................................................................................................................................190
 

5.4 DISPOSAL.................................................................................................................................190
 

6. POTENTIAL FOR HUMAN EXPOSURE.........................................................................................191
 

6.1 OVERVIEW...............................................................................................................................191
 

6.2 RELEASES TO THE ENVIRONMENT...................................................................................193
 

6.2.1 Air .......................................................................................................................................194
 

6.2.2 Water...................................................................................................................................194
 

6.2.3 Soil ......................................................................................................................................197
 

6.3 ENVIRONMENTAL FATE ......................................................................................................197
 

6.3.1 Transport and Partitioning...................................................................................................197
 

6.3.2 Transformation and Degradation ........................................................................................ 199
 

DINITROTOLUENES xiv 
6.3.2.1 Air................................................................................................................................199
 

6.3.2.2 Water............................................................................................................................200
 

6.3.2.3 Sediment and Soil ........................................................................................................203
 

6.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT..................................205
 

6.4.1 Air .......................................................................................................................................205
 

6.4.2 Water...................................................................................................................................206
 

6.4.3 Sediment and Soil ...............................................................................................................207
 

6.4.4 Other Environmental Media................................................................................................208
 

6.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE........................................208
 

6.6 EXPOSURES OF CHILDREN..................................................................................................209
 

6.7 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES ..............................................211
 

6.8 ADEQUACY OF THE DATABASE ........................................................................................211
 

6.8.1 Identification of Data Needs ...............................................................................................212
 

6.8.2 Ongoing Studies..................................................................................................................215
 

7. ANALYTICAL METHODS................................................................................................................217
 

7.1 BIOLOGICAL MATERIALS....................................................................................................217
 

7.2 ENVIRONMENTAL SAMPLES ..............................................................................................218
 

7.3 ADEQUACY OF THE DATABASE ........................................................................................222
 

7.3.1 Identification of Data Needs ...............................................................................................227
 

7.3.2 Ongoing Studies..................................................................................................................227
 

8. REGULATIONS, ADVISORIES, AND GUIDELINES.....................................................................229
 

9. REFERENCES ....................................................................................................................................237
 

10. GLOSSARY ......................................................................................................................................261
 

APPENDICES 

A. ATSDR MINIMAL RISK LEVELS AND WORKSHEETS .............................................................A-1
 

B. USER’S GUIDE.................................................................................................................................. B-1
 

C. ACRONYMS, ABBREVIATIONS, AND SYMBOLS...................................................................... C-1
 

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DINITROTOLUENES xvi 

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LIST OF FIGURES
 

2-1. Health Effects for Ingesting 2,3-Dinitrotoluene .................................................................................13
 

2-2. Health Effects for Ingesting 2,4-Dinitrotoluene .................................................................................14
 

2-3. Health Effects for Ingesting 2,5-Dinitrotoluene .................................................................................15
 

2-4. Health Effects for Ingesting 2,6-Dinitrotoluene .................................................................................16
 

2-5. Health Effects for Ingesting 3,4-Dinitrotoluene .................................................................................17
 

2-6. Health Effects for Ingesting 3,5-Dinitrotoluene .................................................................................18
 

3-1. Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral.............................................................. 50
 

3-2. Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral.............................................................. 70
 

3-3. Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral .............................................................77
 

3-4. Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral..............................................................86
 

3-5. Levels of Significant Exposure to 3,4-Dinitrotoluene - Oral.............................................................. 90
 

3-6. Levels of Significant Exposure to 3,5-Dinitrotoluene - Oral..............................................................92
 

3-7. Proposed Metabolic Pathways for the Hepatic Metabolism of 2,4-Dinitrotoluene ..........................141
 

3-8. Proposed Pathways for the Anaerobic Metabolism of 2,4-Dinitrotoluene in Rat Intestinal 

Microflora.........................................................................................................................................142
 

3-9. Proposed Pathways for Metabolism of 2,6-Dinitrotoluene ..............................................................143
 

3-10. Conceptual Representation of a Physiologically Based Pharmacokinetic (PBPK) Model for a
 

Hypothetical Chemical Substance..................................................................................................151
 

3-11. Existing Information on Health Effects of 2,4-Dinitrotoluene .......................................................167
 

3-12. Existing Information on Health Effects of 2,6-Dinitrotoluene .......................................................168
 

3-13. Existing Information on Health Effects of 2,3-, 2,5-, 3,4-, and 3,5-Dinitrotoluene ....................... 169
 

3-14. Existing Information on Health Effects of Technical-Grade Dinitrotoluene..................................170
 

6-1. Frequency of NPL Sites with 2,4- and 2,6-Dinitrotoluene Contamination ......................................192
 

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LIST OF TABLES
 

2-1. Summary of Oral MRLs (mg/kg/day) for Dinitrotoluenes.................................................................20
 

3-1. Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral.............................................................. 48
 

3-2. Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral.............................................................. 51
 

3-3. Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral .............................................................75
 

3-4. Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral..............................................................78
 

3-5. Levels of Significant Exposure to 3,4-Dinitrotoluene - Oral.............................................................. 89
 

3-6. Levels of Significant Exposure to 3,5-Dinitrotoluene - Oral..............................................................91
 

3-7. NOAELs and LOAELs for Hematological Effects Following 14-Day Gavage Dosing of Male 

Sprague-Dawley Rats with Individual DNT Isomers.......................................................................101
 

3-8. NOAELs and LOAELs for Hepatic Effects Following 14-Day Gavage Dosing of Male 

Sprague-Dawley Rats with Individual DNT Isomers.......................................................................105
 

3-9. Genotoxicity of Dinitrotoluene Isomers In Vitro..............................................................................128
 

3-10. Genotoxicity of Dinitrotoluene Isomers In Vivo.............................................................................133
 

4-1. Chemical Identity of Dinitrotoluenes ...............................................................................................182
 

4-2. Physical and Chemical Properties of Dinitrotoluenes ......................................................................184
 

5-1. Facilities that Produce, Process, or Use 2,4-Dinitrotoluene .............................................................188
 

5-2. Facilities that Produce, Process, or Use 2,6-Dinitrotoluene .............................................................189
 

6-1. Releases to the Environment from Facilities that Produce, Process, or Use 2,4-Dinitrotoluene......195
 

6-2. Releases to the Environment from Facilities that Produce, Process, or Use 2,6-Dinitrotoluene......196
 

7-1. Analytical Methods for Determining Dinitrotoluenes in Biological Materials................................219
 

7-2. Analytical Methods for Determining Dinitrotoluenes in Environmental Samples...........................223
 

8-1. Oral Minimal Risk Levels (MRLs) for Dinitrotoluenes...................................................................231
 

8-2. Regulations, Advisories, and Guidelines Applicable to Dinitrotoluenes .........................................232
 

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DINITROTOLUENES 1 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES (DNTs) 

This Public Health Statement summarizes the Division of Toxicology and Human Health Science’s 

findings on DNTs, tells you about them, the effects of exposure, and describes what you can do to limit 

that exposure. 

WHAT ARE DNTs? 

DNTs consist of a mixture of isomers (forms). These particular isomers have the same molecular weight 

and molecular formula and the same nitro functional groups. However, the functional groups are at 

different positions on the chemical structure. Two of the isomers of DNT, 2,4-DNT and 2,6-DNT, make 

up 95% of DNTs. The other 5% is mostly four other isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNTs are 

not natural substances and are commercially produced by reacting concentrated sulfuric and nitric acid 

with toluene. 

The U.S. Environmental Protection Agency (EPA) identifies the most serious hazardous waste sites in the 

nation. These sites make up the National Priorities List (NPL) and are sites targeted for long-term federal 

clean-up activities. U.S. EPA has found DNTs in at least 98 of the 1,699 current or former NPL sites. 

The total number of NPL sites evaluated for DNTs is not known. But the possibility remains that as more 

sites are evaluated, the sites at which DNTs are found may increase. This information is important 

because these future sites may be sources of exposure, and exposure to DNTs may be harmful. 

If you are exposed to DNTs many factors determine whether you’ll be harmed. These include how much 

you are exposed to (dose), how long you are exposed (duration), and how you are exposed (route of 

exposure). You must also consider the other chemicals you are exposed to and your age, sex, diet, family 

traits, lifestyle, and state of health. 

Most DNTs are used in the production of toluene diisocyanate (TDI), which is used in the manufacture of 

polyurethane foam. They are also used to make the explosive, TNT, and in the production of dyes and 

smokeless powders in the munitions industry. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 2 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES 

WHERE ARE DNTs FOUND? 

DNTs can be released into the air, water, and soil at places where they are produced or used. They are not 

commonly found outside of source areas such as DNT manufacturing facilities or contaminated waste 

sites. 

HOW MIGHT I BE EXPOSED TO DNTs? 

Workers in plants that use DNTs may be exposed by inhalation and skin contact. Other people are not 

likely to be exposed to DNTs unless they live near facilities involved in production, use, storage, and/or 

disposal of DNTs. People living near such facilities may be exposed by skin contact with contaminated 

soil, by accidentally eating contaminated soil, by drinking contaminated water, or by skin contact with 

contaminated water. Most people are not likely to be exposed to DNTs by inhalation; however, one 

might be exposed by breathing in DNT released to the air from DNT-contaminated bath or shower water. 

HOW CAN DNTs ENTER AND LEAVE MY BODY? 

If you breathe air containing DNTs, they will enter your body through your lungs. If present in drinking 

water, DNTs will rapidly enter your body through the digestive tract. Some soil samples may contain a 

high level of DNTs. People can be exposed by skin contact with DNT-contaminated soil or by 

swallowing DNT-contaminated soil or dust. Such exposure would be more likely among young children 

playing in DNT-contaminated soil and/or placing it in their mouth. DNTs leave your body rapidly. They 

DNTs are rarely detected in outside air, but are detected in workplace air where they are manufactured or 

used. Military and industrial activities have led to reported release of DNTs into soil, groundwater, or 

surface water. DNT levels as high as 10 mg/L were reported in potable groundwater at the Joliet Army 

Ammunition Plant located in Illinois. It was unspecified as to what years these levels were reported; 

levels were reported to be as high as 3.2 mg/L and 2.7 mg/L in 1989. Current levels of DNTs in the 

groundwater may be significantly lower. DNTs are slowly broken down in water by microbial (bacterial 

or other microscopic) organisms a can be broken down by sunlight in surface water. DNTs have been 

detected in soil at levels of ~100 mg/kg in areas like ammunition manufacturing sites and military firing 

ranges. DNTs do not adsorb strongly to soil. Therefore, they can move from soil into groundwater, 

where they can contaminate drinking water. DNTs are not used extensively in consumer products and are 

not often detected in food samples. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 3 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES 

break down into other chemicals that leave your body in the urine within 24 hours. Small amounts of the 

DNTs may also be present in the feces. 

HOW CAN DNTs AFFECT MY HEALTH? 

The health effects of DNTs depend on several factors, including how much DNTs you are exposed to and 

the length of that exposure. Environmental monitoring data are limited, but they do suggest that any DNT 

levels the public might encounter by contact through air, water, or soil are generally much lower than 

levels used in animal studies. 

See Chapters 2 and 3 for more information on health effects of DNTs. 

HOW CAN DNTs AFFECT CHILDREN? 

This section discusses potential health effects of DNT exposure in humans from when they’re first 

conceived to 18 years of age. 

In one study of 457 munitions workers exposed to DNTs for periods ranging from 30 days to >5 years, 

there was some evidence for increased death from heart disease; however, the study was limited by the 

small number of workers evaluated. Animal studies have shown that breathing vapors or aerosols of 

DNTs can cause damage to the lungs. Animal studies have also shown that ingesting DNTs over short or 

long periods causes anemia, and damage to the nervous system, male reproductive system, and liver. 

Several studies in workers have looked for possible associations between DNT exposure and cancer. No 

association was found between DNT exposure and liver cancer. One study in mine workers reported an 

association between DNT exposure and kidney cancer. Three studies reported associations between DNT 

exposure and bladder cancers in workers. However, there is no clear evidence that DNT causes cancer in 

people. Laboratory animals ingesting DNTs during most of their lives developed cancer of the liver and 

tumors in the kidneys. The U.S. EPA states that a mixture of 2,4- and 2,6-DNT is a probable human 

carcinogen, based on findings of cancer in animal studies. The International Agency for Research on 

Cancer states that 2,4- and 2,6-DNT are possibly carcinogenic to humans, but that carcinogenicity for 

3,5-DNT in humans cannot be determined due to a lack of information. 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 4 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES 

No data describe the effects of exposure to DNTs on children or young animals. Although we think that 

children would likely show the same health effects as adults, we don’t know whether children are more 

susceptible than adults to DNT effects. 

We don’t know whether DNTs can harm an unborn child. Results of animal studies show that newborns 

of mothers exposed to DNTs during pregnancy can have anemia and nervous system damage at birth. 

These effects are similar to those seen in adult animals. 

HOW CAN FAMILIES REDUCE THE RISK OF EXPOSURE TO DNTs? 

If your doctor finds that you have been exposed to significant amounts of DNTs, ask whether your 

children might also be exposed. Your doctor might need to ask your state health department to 

investigate. 

DNTs are not often detected in drinking water supplies. However, if you live close to areas where DNTs 

may contaminate soil or drinking water (for example near facilities where DNTs are produced or disposed 

of), limit your exposure to contaminated drinking water and soil. 

ARE THERE MEDICAL TESTS TO DETERMINE WHETHER I HAVE BEEN EXPOSED TO 

DNTs? 

DNTs and their breakdown products (metabolites) can be measured in urine. However, the detection of 

DNTs or metabolites cannot predict the kind of health effects that might develop from that exposure. 

Because DNTs and their metabolites leave the body fairly rapidly, the tests need to be conducted within 

days after exposure. 

For more information on the different substances formed by DNT breakdown and on tests to detect these 

substances in the body, see Chapters 3 and 7. 

WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT 

HUMAN HEALTH? 

The federal government develops regulations and recommendations to protect public health. Regulations 

can be enforced by law. Federal agencies that develop regulations for toxic substances include the 

Environmental Protection Agency (EPA), the Occupational Safety and Health Administration (OSHA), 

and the Food and Drug Administration (FDA). Recommendations provide valuable guidelines to protect 

public health but cannot be enforced by law. Federal organizations that develop recommendations for 
 

 

 

 

 

 

 

 

DINITROTOLUENES 5 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES 

toxic substances include the Agency for Toxic Substances and Disease Registry (ATSDR) and the 

National Institute for Occupational Safety and Health (NIOSH). 

Regulations and recommendations can be expressed as “not-to-exceed” levels; that is, levels of a toxic 

substance in air, water, soil, or food that do not exceed a critical value usually based on levels that affect 

animals; levels are then adjusted to help protect humans. Sometimes these not-to-exceed levels differ 

among federal organizations. Different organizations use different exposure times (an 8-hour workday or 

a 24-hour day), different animal studies, or emphasize some factors over others, depending on their 

mission. 

Recommendations and regulations are also updated periodically as more information becomes available. 

For the most current information, check with the federal agency or organization that issued the regulation 

or recommendation. 

The U.S. EPA has determined that exposure to 2,4-DNT in drinking water at concentrations of 1 mg/L for 

1 or 10 days is not expected to cause any adverse effects in a child. OSHA set a legal limit of 1.5 mg/m3 

DNT in workplace air averaged over an 8-hour work day. NIOSH recommends a limit of 1.5 mg/m3 

DNT in workplace air averaged over a 10-hour work day. 

WHERE CAN I GET MORE INFORMATION? 

If you have any questions or concerns, please contact your community or state health or environmental 

quality department, or contact ATSDR at the address and phone number below. ATSDR can also provide 

publically available information regarding medical specialists with expertise and experience recognizing, 

evaluating, treating, and managing patients exposed to hazardous substances. 

•	 Call the toll-free information and technical assistance number at
 

1-800-CDCINFO (1-800-232-4636) or
 

•	 Write to:
 

Agency for Toxic Substances and Disease Registry
 

Division of Toxicology and Human Health Sciences
 

1600 Clifton Road NE
 

Mailstop F-57
 

Atlanta, GA 30329-4027
 

Toxicological profiles and other information are available on ATSDR’s web site: 

http://www.atsdr.cdc.gov. 
 

DINITROTOLUENES 6 

1. PUBLIC HEALTH STATEMENT FOR DINITROTOLUENES 

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DINITROTOLUENES 7 

2. RELEVANCE TO PUBLIC HEALTH
 

2.1 BACKGROUND AND ENVIRONMENTAL EXPOSURES TO DNT IN THE UNITED 

STATES 

Dinitrotoluene (DNTs) are generally produced as a mixture called technical-grade DNT (Tg-DNT), which 

contains approximately 76.5% 2,4-DNT and 18.8% 2,6-DNT (with the remainder consisting of other 

isomers and minor contaminants such as TNT and mononitrotoluenes). It is primarily used as a chemical 

intermediate for the production of toluene diisocyanate (TDI). DNTs are also used in the production of 

2,4,6-TNT, dyes, and polyurethane foams. 

When released to the environment, DNTs have the potential to leach into groundwater or contaminate 

surface waters through soil runoff and erosion. Volatilization from water bodies or soil surfaces occurs 

slowly for DNTs and as a consequence, they are not frequently detected in ambient air. DNTs generally 

biodegrade slowly under environmental conditions, but are rapidly degraded by photolysis in natural 

water. Measured bioconcentration factors in fish suggest that the potential for bioaccumulation is low in 

fish and other aquatic organisms. 

DNTs are very infrequently detected in drinking water. There were no detections of 2,4-DNT in 

3,251 samples taken from small public water systems and there was only 1 detection out of 

30,513 samples obtained from large systems throughout the United States. 2,6-DNT was not detected in 

any of the 33,765 samples (both large and small systems) for which it was tested. DNTs have been 

detected in surface water and groundwater near source locations such as munitions sites. Concentrations 

of 2,4- and 2,6-DNT obtained from a small brook and a river adjacent to a former ammunition plant were 

0.5–13.0 and 0.1–7.6 μg/L, respectively. DNT levels as high as 10,000 μg/L were reported in potable 

groundwater at the Joliet Army Ammunition Plant located in Will County, Illinois. 2,3-, 2,5-, 3,4-, and 

3,5-DNT isomers were identified in both monitoring wells and a few private water supply wells near an 

Army ammunition site in Wisconsin. 2,4-DNT was measured in soil samples obtained from the Joliet 

Army Ammunition at levels of <0.1 mg/kg (detection limit) to 117 mg/kg. 2,6-DNT was detected on this 

site at concentrations ranging from <0.1 to 8 mg/kg. 

The major route of exposure to DNTs for populations residing near hazardous waste sites or munitions 

facilities is via ingestion of contaminated water or dermal contact with contaminated soil. Inhalation 

and/or dermal exposure to DNTs could also occur when washing or bathing with DNT-contaminated 

water or when cooking with DNT-contaminated water. Occupational exposure by inhalation or dermal 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 8 

2. RELEVANCE TO PUBLIC HEALTH 

contact may occur at workplaces where DNT is manufactured or used. Monitoring data suggest that 

populations that do not live near source areas are not exposed to significant levels of DNTs. 

2.2 SUMMARY OF HEALTH EFFECTS 

Data on health effects of DNTs are available from occupational exposure studies and studies in laboratory 

animals. Most occupational exposure studies evaluate health effects in workers exposed to 2,4-, 2,6-, or 

Tg-DNT. However, most of these studies were conducted before 1950 and provide very little information 

on exposure concentrations. Furthermore, interpretation of study results is limited due to the absence of 

appropriate control groups, mixed exposures to other chemicals, and small number of workers studied. 

Nearly all studies in laboratory animals are oral exposure studies of 2,4-, 2,6-, or Tg-DNT, and include 

data for acute, intermediate, and chronic exposure durations. Very little information is available 

regarding health effects of inhaled DNTs; however, systemic effects of DNTs are expected to be similar 

for all routes of exposure. 

Results of occupational exposure studies and studies in laboratory animals identify the hematological 

(methemoglobinemia, anemia, and compensatory hematopoiesis) and nervous systems (clinical signs of 

neurotoxicity, ataxia, tremors, leg weakness, and convulsions) as the most sensitive targets of 

DNT-induced toxicity. In addition to hematological and neurological effects, results of animal studies 

provide evidence that the liver, respiratory tract, and reproductive system also are targets for 

DNT-induced toxicity. However, effects to these other organ systems occur at levels that are higher than 

those producing hematological effects or neurotoxicity under similar exposure conditions. 

In occupational exposure studies, findings of anemia and cyanosis in workers exposed to 2,4- or Tg-DNT 

are consistent with the DNT-induced hematological effects observed in laboratory animals; however, 

available human data provide only limited evidence, as studies did not include adequate control groups or 

report exposure concentrations. Adverse hematological effects, especially methemoglobinemia and 

anemia, have been reported in laboratory animals exposed to oral 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, or 

Tg-DNT for acute, intermediate, and chronic durations. Hematological effects of DNTs are initiated by 

methemoglobin production, which occurs when the ferrous iron in complex with the heme groups of 

hemoglobin is oxidized to ferric iron. Ferric iron does not bind oxygen and, therefore, reduces the 

oxygen-carrying capacity of the blood. Ferric iron also contributes to the denaturation of hemoglobin and 

subsequent removal of erythrocytes from the blood, resulting in anemia. Heinz bodies (granules of 

denatured hemoglobin) form and can be detected in erythrocytes. Increased hematopoiesis is often 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 9 

2. RELEVANCE TO PUBLIC HEALTH 

observed as a compensatory response to decreased erythrocyte count. Hematological parameters typically 

affected by DNT exposure include increased blood methemoglobin levels, increased reticulocyte count, 

decreased erythrocyte count, decreased hematocrit, and decreased blood hemoglobin levels, and the 

presence of Heinz bodies; the severity of effects increases with dose. In addition, extramedullary 

erythropoiesis of the spleen, a compensatory response, may also occur. In acute-duration (14-day) oral 

studies of rats, this effect was seen after exposure to each of the six isomers of DNT. Results of a singledose oral exposure study in rats indicate that the onset of hematological effects is rapid, with effects 

observed 24 hours after administration of 99 mg/kg of 2,4- or 2,6-DNT. For acute exposure durations, the 

lowest daily oral dose associated with hematological effects is 4 mg/kg/day in dogs exposed to 2,6-DNT 

for 14 consecutive days. Similar hematological effects have been observed following intermediateduration exposure, with the development of methemoglobinemia, anemia, and compensatory 

erythropoiesis in dogs administered daily oral doses of 1.5 mg/kg of 2,4-DNT for 3–9 months. 

Extramedullary erythropoiesis of the spleen was observed in dogs administered ≥4 mg/kg/day of 2,6-DNT 

for 4–13 weeks. For chronic-duration exposure, hematological effects were observed following exposure 

of dogs to 2,4-DNT at 1.5 mg/kg/day for 24 months. Following cessation of exposure, hematological 

effects showed complete reversal, although time to reversal increased with severity of effects. 

In occupational exposure studies, neurological effects, including headache, dizziness, insomnia, 

unpleasant taste in the mouth, and pain, numbness, and tingling in the extremities, have also been reported 

in workers exposed to 2,4- or Tg-DNT; however, available human data provide only limited evidence, as 

studies did not include adequate control groups or report exposure concentrations. The nervous system is 

also identified as a sensitive target for DNT-induced toxicity, with symptoms of neurotoxicity reported in 

laboratory animals exposed to oral 2,4-, 2,6-, 3,4-, 3,5-, and Tg-DNT for acute, intermediate, and chronic 

durations. However, results of these studies indicate that, in general, the nervous system is a less 

sensitive target than the hematological system. Symptoms of neurotoxicity observed in laboratory 

animals include weakness, stiffness, or rigid paralysis of the hind legs, abnormal gait, tremors, ataxia, and 

convulsions, with severity increasing with dose. For acute- and intermediate-duration exposures, the 

lowest doses producing symptoms of neurotoxicity were 25 mg/kg/day in dogs exposed to 2,4-DNT for 

12 days to 13 weeks and 20 mg/kg/day in dogs exposed to 2,6-DNT for 13 weeks. Demyelinization in the 

cerebellum and brain stem was observed in rats exposed to dietary exposures of 93 mg/kg/day 2,4-DNT 

for 13 weeks. In chronic-duration exposure studies, the lowest dose producing neurotoxicity was 

1.5 mg/kg/day 2,4-DNT in a 2-year study in dogs; however, this effect (loss of hindquarter control) was 

only observed intermittently in one of six dogs. More severe signs of neurotoxicity and central nervous 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 10 

2. RELEVANCE TO PUBLIC HEALTH 

system lesions (vacuolization, hypertrophy, endothelial mitosis, focal gliosis in the cerebellum, and 

perivascular hemorrhage in the cerebellum and brain stem) occurred at 10 mg/kg/day. 

Occupational exposure studies do not provide conclusive evidence of DNT-induced hepatotoxicity due to 

lack of appropriate controls. Hepatic effects have been observed in animals exposed to 2,3-, 2,4-, 2,6-, 

3,4-, or Tg-DNT in laboratory animals. However, as discussed below, 2,4-, 2,6-, and Tg-DNT have been 

shown to induce hepatocellular carcinoma following chronic-duration oral exposure. Thus, hepatic 

effects observed at less-than-chronic exposure durations may represent early stages of progressive 

development to hepatic cancer. The potential for DNTs to induce liver cancer complicates interpretation 

of study results showing possible effects in the liver, as hepatotoxicity may be a precursor to or a result 

from the development of hepatic neoplasms. Hepatic effects, including liver discoloration, inflammation, 

increased liver weights, hepatocellular hyperplasia and hypertrophy, degeneration of hepatocytes, 

hepatocellular necrosis, karyocytomegaly, proliferation of bile duct epithelium, and elevated blood levels 

of hepatic enzymes, have been reported in oral exposure studies of 2,3-, 2,4-, 2,6-, 3,4-, or Tg-DNT in 

laboratory animals. Hepatic effects occurred after exposure to 2,3-DNT doses ≥275 mg/kg/day, to 

2,4-DNT doses ≥10 mg/kg/day, to 2,6-DNT doses ≥7 mg/kg/day, and to 3,4-DNT doses ≥113 mg/kg/day, 

with severity related to dose and exposure duration. At higher oral doses (≥50 mg/kg/day), onset of 

hepatic toxicity appears to be rapid; congested sinusoids with sloughed hepatocytes, infiltration of 

segmented neutrophils, pyknotic nuclei, microvesiculated cytoplasm, and apoptosis of hepatocytes were 

observed 48 hours after administration of 2,6-DNT. Following chronic dietary exposure to 2,4-DNT for 

1 year, hepatocellular alterations were observed in male rats at a dose of 0.6 mg/kg/day; however, 

neoplastic nodules of the liver were also observed in these animals at doses ≥0.6 mg/kg/day. Biliary 

hyperplasia was observed in dogs exposed to 2,4-DNT for 24 months, with no-observed-adverse-effect 

level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) values of 1.5 and 10 mg/kg/day, 

respectively. 

Adverse reproductive effects, including effects on male and female reproductive systems and decreased 

neonatal viability, have been observed in laboratory animals following acute, intermediate, and chronic 

oral exposure to 2,4-, 2,6-, 3,5-, or Tg-DNT. Severity of reproductive effects is dose- and exposure 

duration-related, and the male reproductive system is more sensitive than the female reproductive system 

or neonatal viability. In males, adverse effects include decreased testes and epididymides weights, 

decreased sperm production, testicular atrophy, degeneration of seminiferous tubules, multinucleated 

giant cell formation, and changes in Sertoli cell morphology. The lowest dose producing effects to the 

male reproductive system (testicular atrophy and decreased spermatogenesis) was 14 mg/kg/day in mice 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 11 

2. RELEVANCE TO PUBLIC HEALTH 

fed diets containing 2,4-DNT for up to 2 years. Effects on the female reproductive system (ovarian 

atrophy and nonfunctional follicles) were observed in mice fed 898 mg/kg/day 2,4-DNT. Decreased 

neonatal viability was observed in rats fed ≥34.5 mg/kg/day 2,4-DNT for up to 6 months in a threegeneration reproductive study. Occupational exposure studies do not provide evidence of DNT-induced 

adverse reproductive effects due to both concomitant exposure of workers to other chemicals and 

reporting insufficiencies. Dietary exposure of rats to 2,4-DNT for up 6 months in a three-generation 

reproductive study produced delayed eye opening at doses fed ≥35 mg/kg/day, but no other 

developmental effects. In neonates exposed to Tg-DNT during gestation, systemic effects similar to those 

observed in adult animals, including delayed eye opening and signs of mild neurological effects (cliff 

avoidance behavior) and effects on hematological parameters, were observed at maternal doses 

≥100 mg/kg/day; recovery from these effects was observed by postpartum day 60. Although no studies 

were located regarding developmental effects in humans after oral exposure to DNTs, developmental 

toxicity from DNTs could potentially occur because exposure to any substance that depletes the amount 

of oxygen available to developing fetal tissues may cause toxicity to the developing organism. 

Respiratory distress, pulmonary congestion, and increased relative lung weights were observed in rats 

following a single 6-hour inhalation exposure of rats to near-lethal or lethal concentrations of 2,6-DNT 

(vapor or aerosol). No effects on the respiratory system have been observed following acute, 

intermediate, or chronic oral exposure of laboratory animals to DNTs. Occupational studies do not 

include reports of respiratory effects in workers. 

The carcinogenic activity of DNTs has been extensively studied in typical chronic bioassays and in some 

less-than-lifetime studies. Results show the development of renal cancer in mice fed 2,4-DNT at 

95 mg/kg/day and hepatocellular carcinoma in male and female rats fed 2,4-DNT at doses of 34.5 and 

45.3 mg/kg/day, respectively. Hepatocellular carcinomas were observed in rats administered 2,6-DNT at 

doses ≥7 mg/kg/day. 2,4-DNT was a hepatic tumor promoter, but not a tumor initiator, using in vivo 

hepatic initiation-promotion protocols. Both tumor-initiating and -promoting activities of 2,6- and 

Tg-DNT in rat liver were reported. A retrospective cohort mortality study at two army ammunition plants 

that used Tg-DNT and/or 2,4-DNT showed no significant increases in mortality from malignant 

neoplasms as a whole or from particular cancers (liver, lung, gallbladder, kidney, and connective tissues), 

although interpretation of study results is limited by a small cohort size. Three studies of German copper 

miners exposed to high levels of Tg-DNT from explosives found increases in the incidence of urothelial 

cancers. An association between renal cancer and duration of Tg-DNT exposure has also been reported in 

this cohort. The National Toxicology Program (NTP) has not evaluated DNTs. EPA has classified 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 12 

2. RELEVANCE TO PUBLIC HEALTH 

2,4-DNT/2,6-DNT mixture as a probable human carcinogen based on sufficient evidence of 

carcinogenicity in animals. The International Agency for Research on Cancer (IARC) has classified 

2,4- and 2,6-DNT as possibly carcinogenic to humans, and 3,5-DNT as not classifiable as to its 

carcinogenicity to humans. 

Heath effects of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT ingestion in laboratory animals and the dose 

ranges at which these effects occur are shown in Figures 2-1 through 2-6, respectively. Estimates of oral 

doses posing minimal risk to humans (MRLs) are also presented in these figures. 

2.3 MINIMAL RISK LEVELS (MRLs) 

Estimates of exposure levels posing minimal risk to humans (MRLs) have been made for 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, and 3,5-DNT. An MRL is defined as an estimate of daily human exposure to a substance that 

is likely to be without an appreciable risk of adverse effects (noncarcinogenic) over a specified duration 

of exposure. MRLs are derived when reliable and sufficient data exist to identify the target organ(s) of 

effect or the most sensitive health effect(s) for a specific duration within a given route of exposure. 

MRLs are based on noncancerous health effects only and do not consider carcinogenic effects. MRLs can 

be derived for acute, intermediate, and chronic duration exposures for inhalation and oral routes. 

Appropriate methodology does not exist to develop MRLs for dermal exposure. 

Inhalation MRLs 

Although methods have been established to derive these levels (Barnes and Dourson 1988; EPA 1990), 

uncertainties are associated with these techniques. Furthermore, ATSDR acknowledges additional 

uncertainties inherent in the application of the procedures to derive less than lifetime MRLs. As an 

example, acute inhalation MRLs may not be protective for health effects that are delayed in development 

or are acquired following repeated acute insults, such as hypersensitivity reactions, asthma, or chronic 

bronchitis. As these kinds of health effects data become available and methods to assess levels of 

significant human exposure improve, these MRLs will be revised as appropriate. 

Most of the data on health effects associated with inhalation exposure of humans to DNTs are from 

studies of workers exposed to 2,4-, 2,6-, or Tg-DNT. However, most of these studies were conducted 

before 1950 and provide very little information on exposure concentrations. Furthermore, interpretation 

of study results is limited due to the absence of appropriate control groups, mixed exposures to other 
 

DINITROTOLUENES 13 

2. RELEVANCE TO PUBLIC HEALTH 

Figure 2-1. Health Effects for Ingesting 2,3-Dinitrotoluene 
 

DINITROTOLUENES 14 

2. RELEVANCE TO PUBLIC HEALTH 

Figure 2-2. Health Effects for Ingesting 2,4-Dinitrotoluene 
 

DINITROTOLUENES 15 

2. RELEVANCE TO PUBLIC HEALTH 

Figure 2-3. Health Effects for Ingesting 2,5-Dinitrotoluene 
 

DINITROTOLUENES 16 

2. RELEVANCE TO PUBLIC HEALTH 

Figure 2-4. Health Effects for Ingesting 2,6-Dinitrotoluene 
 

DINITROTOLUENES 17 

2. RELEVANCE TO PUBLIC HEALTH 

Figure 2-5. Health Effects for Ingesting 3,4-Dinitrotoluene 
 

DINITROTOLUENES 18 

2. RELEVANCE TO PUBLIC HEALTH 

Figure 2-6. Health Effects for Ingesting 3,5-Dinitrotoluene 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 19 

2. RELEVANCE TO PUBLIC HEALTH 

chemicals, and small number of workers studied. Therefore, while available occupational studies provide 

supportive information for observations in animals regarding identification of target organs, they do not 

report dose-response data that can be used to derive MRLs for DNTs. Regarding available animal data, 

one study was located that examined the acute inhalation toxicity of 2,6-DNT in rats (CMA 1991). This 

study identified mortality and clinical signs of respiratory toxicity (exaggerated respiratory movements) in 

rats exposed to 2,6-DNT as an aerosol at concentrations ≥196 mg/m3

; rats that died showed increased 

relative lung weights and evidence of lung congestion. No effects were observed in rats exposed noseonly to 2,6-DNT as a vapor at 26 mg/m3 for 6 hours and observed 14 days after dosing. No studies were 

located regarding inhalation exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT in experimental animals. Thus, data 

were insufficient for the derivation of acute-, intermediate-, and chronic-duration inhalation MRLs for 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT. 

Oral MRLs 

Acute-Duration Oral MRL for 2,3-DNT 

Acute-, intermediate-, and chronic-duration oral MRLs were derived for 2,4-DNT and acute- and 

intermediate-duration oral MRLs were derived for 2,6-DNT; acute-duration oral MRLs were derived for 

2,3-, 2,5-, 3,4-, and 3,5-DNT. For all isomers, studies in humans did not provide sufficient data regarding 

exposure levels and their correlation with observed effects; thus, animal studies were used for the 

derivation of these oral MRLs. Data were insufficient for the derivation of intermediate- and chronicduration oral MRLs for 2,3-, 2,5-, 3,4-, and 3,5-DNT and for the derivation of a chronic oral MRL for 

2,6-DNT. Table 2-1 summarizes the oral MRLs for the DNT isomers. 

•	 An MRL of 0.09 mg/kg/day has been derived for acute-duration oral exposure (≤14 days) to 

2,3-DNT.
 

No human acute-duration oral toxicity studies on 2,3-DNT were identified. For 2,3-DNT, the only 

available oral exposure studies are a 14-day gavage study in rats, in which a number of toxicological end 

points were assessed (Lent et al. 2012a; USAPHC 2011a), and a single-dose gavage study, which reported 

data on lethality only (Vernot et al. 1977). Vernot et al. (1977) reported LD50 values of 1,120 and 

1,070 mg/kg in rats and mice, respectively. The 14-day study in rats (Lent et al. 2012a; USAPHC 2011a) 

identified effects on survival and the kidney and hematopoietic system. Groups of six male SpragueDawley rats were given 2,3-DNT via gavage doses of 0, 17, 34, 69, 138, 275, or 550 mg/kg/day for 
 

DINITROTOLUENES 20 

2. RELEVANCE TO PUBLIC HEALTH 

Table 2-1. Summary of Oral MRLs (mg/kg/day) for Dinitrotoluenes 

Isomer Acute Intermediate Chronic 

2,3-DNT 0.09 NA NA 

2,4-DNT 0.05 0.007 0.001 

2,5-DNT 0.007 NA NA 

2,6-DNT 0.09 0.004 NA 

3,4-DNT 0.03 NA NA 

3,5-DNT 0.03 NA NA 

DNT = dinitrotoluene; NA = not applicable; data are not adequate to derive MRL 
 

 

 

 

 

 

 

 

DINITROTOLUENES 21 

2. RELEVANCE TO PUBLIC HEALTH 

14 days; evaluations included morbidity, clinical observations, hematology, serum chemistry, gross 

necropsy, organ weights, and histopathology. At the highest dose (550 mg/kg/day), all rats died. Rats 

exposed to 275 mg/kg/day 2,3-DNT exhibited renal tubular dilatation (2/6) and lymphocytic infiltration 

(4/6), as well as trace extramedullary hematopoiesis (3/6 rats), lymphoid hyperplasia (1/6), and lymphoid 

depletion (1/6) in the spleen. Histopathology examinations of the kidney and spleen were not performed 

on lower dose groups. Although none of these incidences was statistically significantly increased in 

pairwise comparisons with controls, the lack of statistical significance likely reflects the very small group 

sizes rather than a lack of treatment-related effect, and the increases are considered to be biologically 

significant. Results of this study identify an acute-duration LOAEL value for renal and splenic effects of 

275 mg/kg/day in rats. A NOAEL cannot be determined in the absence of histopathology examinations in 

lower dose groups. 

Effects on the kidney and spleen were identified as the most sensitive effects of acute-duration oral 

exposure to 2,3-DNT, based on the LOAEL value of 275 mg/kg/day in rats administered 2,3-DNT via 

gavage for 14 days (Lent et al. 2012a; USAPHC 2011a). The LOAEL value of 275 mg/kg/day was used 

as the point of departure (POD). Available data were not suitable for benchmark dose (BMD) modeling, 

as spleen and kidney histopathology examinations were limited to the 275 mg/kg/day group and the 

control groups. The POD of 275 mg/kg/day was divided by an uncertainty factor of 3,000 (10 for use of a 

LOAEL, 10 for extrapolation from animals to humans, 10 for human variability, and 3 for database 

limitations), resulting in an acute-duration oral MRL for 2,3-DNT of 0.09 mg/kg/day. 

Acute-Duration Oral MRL for 2,4-DNT 

•	 An MRL of 0.05 mg/kg/day has been derived for acute-duration oral exposure (≤14 days) to 

2,4-DNT.
 

No human acute-duration oral toxicity studies on 2,4-DNT were identified. Acute-duration studies in 

animals identified the nervous system, hematological system, liver, and reproductive system as targets for 

DNT-induced toxicity, with neurotoxicity as the most sensitive effect. 

Dogs (4/sex/group) exposed to 2,4-DNT at 0, 1, 5, or 25 mg/kg/day in capsules for 13 weeks were 

observed for behavioral changes and clinical signs of toxicity during the first 14 days of treatment. Signs 

of neurotoxicity were evident at 25 mg/kg/day. Onset of neurotoxicity (loss of hind leg control) was 

observed in a female dog on day 12; three additional male dogs showed similar effects on day 14. Signs 

of neurotoxicity were reported in all dogs exposed to 25 mg/kg/day after treatment for 12–22 days. No 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 22 

2. RELEVANCE TO PUBLIC HEALTH 

clinical signs were observed in dogs treated with 1 or 5 mg/kg/day. Results of this study identify acuteduration no-observed-adverse-effect level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) 

values for neurotoxicity in dogs of 5 and 25 mg/kg/day, respectively. Although comprehensive end 

points were not evaluated at 14 days, data obtained following the 13-week portion of this study identify 

the same NOAEL and LOAEL values of 5 and 25 mg/kg/day, respectively, for neurotoxicity, 

hematological effects, and reproductive effects. Thus, it is unlikely that hematological or reproductive 

effects would occur at lower doses than neurotoxicity in the acute-duration portion of the study. 

Acute-duration studies in rodents identified the hematological system, liver, and reproductive system as 

targets of acute-duration exposure to 2,4-DNT. The most sensitive hematological effect (slight cyanosis) 

was observed in male Sprague-Dawley rats exposed to 2,4-DNT at 60 mg/kg/day (lowest tested dose) for 

5 days (Lane et al. 1985). The lowest LOAEL for hepatotoxicity (increased incidences of single cell 

necrosis and glycogen deposition) was reported in male Sprague-Dawley rats treated at 36 mg/kg/day for 

14 days; the NOAEL was 18 mg/kg/day (Lent et al. 2012a; USAPHC 2011b). The lowest LOAEL for 

reproductive effects (decreased thickness of spermatogenic cell layers) was 78 mg/kg/day for male 

Sprague-Dawley rats administered 2,4-DNT for 14 days (McGown et al. 1983). Lane et al. (1985) 

identified a NOAEL of 60 mg/kg/day, with a LOAEL of 180 mg/kg/day, for decreased fertility in male 

rats treated with 2,4-DNT for 5 days. 

Neurotoxicity (loss of hind limb control) was identified as the most sensitive effect of acute-duration oral 

exposure to 2,4-DNT, based on the NOAEL value of 5 mg/kg/day in dogs administered 2,4-DNT via 

capsules for 12–14 days (U.S. Army 1978b). Based on the available data, neurotoxicity was selected as 

the critical effect for the basis of the acute-duration oral MRL. The NOAEL value for 5 mg/kg/day was 

used as POD. Neurotoxicity data were not suitable for BMD modeling, since effects were observed only 

at the highest dose tested. The POD of 5 mg/kg/day was divided by an uncertainty factor of 100 (10 for 

extrapolation from animals to humans and 10 for human variability), resulting in an acute-duration oral 

MRL for 2,4-DNT of 0.05 mg/kg/day. 

Intermediate-Duration Oral MRL for 2,4-DNT 

•	 An MRL of 0.007 mg/kg/day has been derived for intermediate-duration oral exposure (15–
 

364 days) to 2,4-DNT.
 

Information on effects of intermediate-duration oral exposure of laboratory animals is available from 

studies treating animals with 2,4-DNT for intermediate durations (15–364 days) and from chronic­
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 23 

2. RELEVANCE TO PUBLIC HEALTH 

duration studies evaluating end points at intermediate-duration time points. Results of studies in dogs, 

rats, and mice showed the development of hematological, neurological, and reproductive effects 

following intermediate-duration exposure to 2,4-DNT, with the hematological effects as the most 

sensitive end point. Adverse hematological effects were observed in studies in dogs, rats, and mice, with 

the lowest LOAEL values of 1.5 and 10 mg/kg/day in females and males, respectively, observed 

following intermediate-duration oral exposure of dogs to 2,4-DNT (Ellis et al. 1985; U.S. Army 1979). 

As part of this 2-year study in dogs, hematological end points were evaluated in dogs administered 

2,4-DNT at 3, 6, and 9 months as part of a 2-year study in dogs administered 0, 0.2, 1.5, or 10 mg/kg/day. 

Hematological effects consistent with development of methemoglobinemia were observed at doses of 

1.5 and 10 mg/kg/day at all intermediate-duration time points (3, 6, and 9 months). Although effects at 

all time points were qualitatively similar, hematological changes observed after 9 months of exposure 

were more consistent and pronounced than those observed at the 3- and 6-month time periods. Male and 

female dogs exposed for 9 months at doses of 1.5 and 10 mg/kg/day showed detectable amounts of 

methemoglobin in the serum, with changes reaching statistical significance in males and females at 

10 mg/kg/day. In female dogs administered 10 mg/kg/day, statistically significant decreases in 

erythrocyte count, hematocrit, and hemoglobin, a statistically significant increase in reticulocyte count, 

and the presence of Heinz bodies in serum were observed. Similar hematological effects were observed 

in female dogs administered 0.2 and 1.5 mg/kg/day, although effects did not reach statistical significance, 

most likely because the power of the study to detect statistically significant changes was compromised by 

the small number of dogs per treatment group. However, based on a clinically significant increase in 

methemoglobin levels of 225% in female dogs administered 1.5 mg/kg/day, the NOAEL and LOAEL 

values for hematological effects in this study are 0.2 and 1.5 mg/kg/day, respectively. Similar effects 

were observed in male dogs, although changes did not reach statistical significance in the 10 mg/kg/day 

group, possibly due to the small number of male dogs evaluated. Comprehensive end points were not 

evaluated at intermediate-duration time points in this study. However, the chronic-duration portion of this 

study, in which comprehensive end points were examined, identified hematological effects as the most 

sensitive effect of chronic-duration exposure. Thus, it is unlikely that effects to other target organs would 

occur at lower doses than the hematological effects for intermediate exposure durations (see overview of 

chronic-duration oral data). 

Intermediate-duration studies in animals administered higher daily doses show the development of similar 

hematological effects, along with neurotoxicity and toxicity to the reproductive system. Beagle dogs 

administered 2,4-DNT for up to 13 weeks developed hematological effects (methemoglobinemia, anemia, 

and compensatory hematopoiesis), neurological effects (incoordination, abnormal gait, and paralysis), and 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 24 

2. RELEVANCE TO PUBLIC HEALTH 

reproductive effects (testicular degeneration and decreased spermatogenesis) at 25 mg/kg/day, with a 

NOAEL for these effects of 5 mg/kg/day (Lee et al. 1985; U.S. Army 1979). Hematological effects 

similar to those observed in dogs were reported in rats treated with 2,4-DNT at 93–371 mg/kg/day 

(Kozuka et al. 1979; U.S. Army 1978b) and in mice treated with 2,4-DNT at 413 and 468 mg/kg/day (for 

males and females, respectively) for up to 13 weeks (Hong et al. 1985; U.S. Army 1978b). The lowest 

LOAEL for neurological effects (clinical signs of neurotoxicity and demyelination of the cerebellum and 

brain stem) in rodents was 93 mg/kg/day for male CD rats administered 2,4-DNT for 4 or 13 weeks; the 

NOAEL for neurological effects in males was 34 mg/kg/day (Lee et al. 1985; U.S. Army 1978b). In 

rodents, respective NOAEL and LOAEL values for reproductive effects (testicular atrophy and decrease 

or absent spermatogenesis) were 9–34 and 35–371 mg/kg/day in rats and 137–295 and 413– 

1,032 mg/kg/day in mice treated for 13 weeks (Bloch et al. 1988; Hong et al. 1985; Kozuka et al. 1979; 

Lee et al. 1985; U.S. Army 1978b, 1979). 

Based on the available data, changes to hematological parameters in dogs exposed to oral 2,4-DNT for 

9 months (Ellis et al. 1985; U.S. Army 1979) were evaluated as possible PODs for the intermediateduration oral MRL for 2,4-DNT. To determine the POD, all available continuous-variable models in the 

EPA BMD Software (BMDS, version 2.1) were fit to the data for selected hematological values 

(methemoglobin, reticulocyte count, hemoglobin, erythrocyte count, and hematocrit) (see Appendix A for 

detailed description of BMD modeling). The BMD and the 95% lower confidence limit (BMDL) were 

estimated for doses associated with a change of 1 standard deviation from the controls. Neither the 

constant nor the non-constant variance model provided an adequate fit to the data for erythrocyte count or 

methemoglobin; therefore, these data were not considered suitable for BMD modeling. Models provided 

an adequate fit to data for hematocrit, hemoglobin, and reticulocyte count. Among all of the models 

providing adequate fit to the data for these parameters, the lowest BMDL1SD values were 0.67 mg/kg/day 

for hematocrit (exponential model 4), 3.66 mg/kg/day for hemoglobin (the exponential model 2), and 

5.64 mg/kg/day for reticulocyte count (polynomial 3-degree model); of these, the lowest BMDL1SD of 

0.67 mg 2,4-DNT/kg/day for decreased hematocrit was selected as the POD. This value was divided by 

an uncertainty factor of 100 (10 for animals to human extrapolation and 10 for human variability), 

resulting in an intermediate-duration oral MRL of 0.007 mg/kg/day. 

Chronic-Duration Oral MRL for 2,4-DNT 

•	 An MRL of 0.001 mg/kg/day has been derived for chronic-duration oral exposure (≥1 year) to 

2,4-DNT. 
 

 

 

 

 

 

 

DINITROTOLUENES 25 

2. RELEVANCE TO PUBLIC HEALTH 

Information on effects of chronic-duration oral exposure is available from studies in dogs, rats, and mice. 

Results of these studies indicate that hematological, neurological, hepatic, and reproductive effects are 

associated with chronic-duration exposure to 2,4-DNT, with hematological effects and neurotoxicity as 

the most sensitive effects. Adverse hematological effects were observed in studies in dogs, rats, and 

mice, with the lowest NOAEL and LOAEL values of 0.2 and 1.5 mg/kg/day, respectively, observed 

following chronic oral exposure of dogs to 2,4-DNT (Ellis et al. 1985; U.S. Army 1979). Beagle dogs 

treated with 2,4-DNT at 1.5 and 10 mg/kg/day after 12 months developed hematological effects consistent 

with methemoglobinemia, anemia, and compensatory hematopoiesis. Female dogs administered 2,4-DNT 

at 1.5 mg/kg/day for 12 months showed statistically significant reductions in erythrocyte count, 

hematocrit, and hemoglobin concentration. At 10 mg/kg/day, more pronounced changes in these 

hematological parameters were observed, including statistically significant reductions in erythrocyte 

count, hematocrit, and hemoglobin and a statistically significant increase in reticulocyte count. Similar 

hematological effects (decreased erythrocytes and decreased hematocrit) were observed in dogs treated 

with 0.2 mg/kg/day, but changes did not reach statistical significance. Although not statistically 

significant, effects on hematological parameters in male dogs were also similar to those seen in female 

dogs. In rodents, the lowest LOAEL for hematological effects was 3.9 mg/kg/day for decreased 

erythrocyte count (with no changes in methemoglobin or Heinz bodies) in male CD rats after treatment 

with 2,4-DNT for 12 months; the NOAEL for this effect was 0.6 mg/kg/day (Lee et al. 1985; U.S. Army 

1978b, 1979). 

Neurotoxicity was also observed in dogs, rats, and mice orally exposed to 2,4-DNT for chronic durations. 

In chronic-duration exposure studies, the lowest dose producing neurotoxicity was 1.5 mg/kg/day 

2,4-DNT in a 2-year study in dogs; however, this effect (loss of hindquarter control) was only observed 

intermittently in one of six dogs. More severe signs of neurotoxicity and central nervous system lesions 

(vacuolization, hypertrophy, endothelial mitosis, focal gliosis in the cerebellum, and perivascular 

hemorrhage in the cerebellum and brain stem) occurred at 10 mg/kg/day (Ellis et al. 1985; U.S. Army 

1979). In rodents, the lowest LOAEL values for neurotoxicity were 35 and 45 mg/kg/day for male and 

female CD rats, respectively, treated with 2,4-DNT for 1–2 years (Lee et al. 1985; U.S. Army 1978b, 

1979). In mice, neurotoxicity was reported at 898 mg/kg/day (but not at 95 mg/kg/day) (Hong et al. 

1985; U.S. Army 1979). 

Hepatic effects have also been observed in laboratory animals following chronic-duration oral exposure to 

DNT. However, these effects are often observed in conjunction with the development of hepatocellular 

carcinoma and may represent precancerous changes. For example, dietary exposure of male rats to 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 26 

2. RELEVANCE TO PUBLIC HEALTH 

0.6 mg/kg/day 2,4-DNT induced “hepatocellular” alterations; however, this exposure level also induced 

neoplastic nodules (Lee et al. 1985). Thus, interpretation of data on hepatic toxicity is complicated due to 

the potential of 2,4-DNT to induce hepatocellular carcinoma. Biliary hyperplasia was observed in dogs 

exposed to 2,4-DNT for 24 months, with NOAEL and LOAEL values of 1.5 and 10 mg/kg/day, 

respectively (U.S. Army 1979). The lowest LOAEL value for hepatic effects in mice was 14 mg/kg/day 

for hepatocellular dysplasia following exposure to 2,4-DNT for 24 months (Hong et al. 1985; U.S. Army 

1979). Hepatocellular degeneration, vacuolization, and altered hepatocellular foci were also observed in 

male F344 rats treated at 27 mg/kg/day for 52 weeks (Leonard et al. 1987). 

Chronic-duration studies in laboratory animals have also demonstrated both male and female reproductive 

effects. The lowest LOAEL reported for reproductive effects is 14 mg/kg/day for atrophy of the testes 

and decreased spermatogenesis in male CD-1 mice exposed to dietary 2,4-DNT for 12 months (Hong et 

al. 1985). Female mice fed 898 mg/kg/day 2,4-DNT had ovarian atrophy with non-functioning follicles, 

with no effects observed at 95 mg/kg/day (Hong et al. 1985). Male CD rats that received 34 mg/kg/day 

2,4-DNT in the diet for 12 month showed an increased incidence of seminiferous tubule atrophy 

compared to controls (100% affected at the high-dose versus 0% of controls) (Lee et al. 1985; U.S. Army 

1978b, 1979). No adverse reproductive effects were found in dogs fed 10 mg/kg/day 2,4-DNT for 

24 months (Ellis et al. 1985; U.S. Army 1979). 

Hematological effects and neurotoxicity were identified as the most sensitive effects of chronic-duration 

exposure to 2,4-DNT, with NOAEL and LOAEL values of 0.2 and 1.5 mg/kg/day, respectively, in dogs 

exposed to 2,4-DNT for 12 months (Ellis et al. 1985; U.S. Army 1979). For derivation of the chronicduration oral MRL, hematological effects were selected as the critical effect rather than neurotoxicity, 

which was observed only intermittently in one of six dogs exposed to 1.5 mg/kg/day. Hematological data 

are expressed as group means; therefore, these data are considered more robust than observations of 

intermittent neurotoxicity in a single animal. To determine the POD, all available continuous-variable 

models in the EPA BMDS (version 2.1) were fit to the hematological data for erythrocyte count, 

hematocrit, and hemoglobin in female dogs. Hematological data from male dogs were not considered for 

additional BMD analyses due to the low number of dogs evaluated in the 10 mg/kg/day group (data were 

available for only two dogs). The BMD and the 95% lower confidence limit (BMDL) were estimated for 

doses associated with a change of 1 standard deviation from the controls. A detailed description of BMD 

modeling is provided in Appendix A. Neither the constant nor the non-constant variance model provided 

an adequate fit to the data for hemoglobin; therefore, these data were not considered suitable for BMD 

modeling. Models provided an adequate fit to data for erythrocyte count and hematocrit. Among all of 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 27 

2. RELEVANCE TO PUBLIC HEALTH 

the models providing adequate fit to the data for these parameters, the lowest BMDL1SD values were 

0.12 mg/kg/day for decreased hematocrit (exponential model 4) and 0.13 mg/kg/day for decreased 

hemoglobin (exponential model 4); of these, the lowest BMDL1SD of 0.12 mg/kg/day for decreased 

erythrocyte count was selected as the POD. This value was divided by an uncertainty factor of 

100 (10 for animals to human extrapolation and 10 for human variability), resulting in a chronic-duration 

oral MRL of 0.001 mg/kg/day. 

Acute-Duration Oral MRL for 2,5-DNT 

•	 An MRL of 0.007 mg/kg/day has been derived for acute-duration oral exposure (≤14 days) to 

2,5-DNT.
 

No human acute-duration oral toxicity studies on 2,5-DNT were identified. For 2,5-DNT, the only 

available oral exposure studies are a 14-day repeated dose gavage study in rats, in which a number of 

toxicological end points were assessed (Lent et al. 2012a; USAPHC 2011c), and a single-dose gavage 

study, which reported data on lethality only (Vernot et al. 1977). Vernot et al. (1977) reported oral LD50 

values of 710 and 1,230 mg/kg in rats and mice, respectively. The 14-day study in rats (Lent et al. 2012a; 

USAPHC 2011c) identified effects on survival, the hematopoietic system, and the heart; the 

hematopoietic system was the most sensitive. Groups of six male Sprague-Dawley rats were given 

2,5-DNT via gavage doses of 0, 10, 19, 39, 77, 154, or 308 mg/kg/day for 14 days; evaluations included 

morbidity, clinical observations, hematology, serum chemistry, gross necropsy, organ weights, and 

histopathology. At the highest dose (308 mg/kg/day), one rat died. Of six rats exposed to 39 mg/kg/day 

2,5-DNT, three exhibited mild-to-moderate extramedullary hematopoiesis, and all rats in higher dose 

groups exhibited this lesion; histopathology examinations were not performed in animals of lower dose 

groups. Marked increases (≥90%) in absolute and relative spleen weight were observed at doses 

≥77 mg/kg/day. In addition, nonsignificant, but dose-related, increases (≥20%) in absolute and relative 

spleen weight were seen at doses ≥19 mg/kg-day. Results of this study identify an acute-duration 

LOAEL value for hematopoietic system effects (extramedullary hematopoiesis and increased absolute 

and relative spleen weight) of 39 mg/kg/day in rats. A NOAEL cannot be defined in the absence of 

spleen histopathology at lower doses, particularly in light of the spleen weight increases at lower doses. 

The hematopoietic effects were identified as the most sensitive effects of acute-duration oral exposure to 

2,5-DNT, based on the LOAEL value of 39 mg/kg/day in rats administered 2,5-DNT via gavage for 

14 days (Lent et al. 2012a; USAPHC 2011c). Data on splenic lesions and absolute and relative spleen 

weights were subjected to BMD modeling to identify a POD for derivation of the acute oral MRL. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 28 

2. RELEVANCE TO PUBLIC HEALTH 

BMDLs for the best-fitting models were the BMDL10 of 2.05 for incidence of mild-to-moderate 

extramedullary hematopoiesis, the BMDL1SD of 7.78 mg/kg/day for absolute spleen weight, and the 

BMDL1SD of 8.26 mg/kg/day for spleen weight relative to body weight. Neither the homogenous nor 

non-constant variance models within the EPA BMDS provided adequate fit to the data on spleen weight 

relative to brain weight. The lowest BMDL value (BMDL10 of 2.05 mg/kg/day for extramedullary 

hematopoiesis) was selected as the POD for derivation of the acute-duration oral MRL. The POD of 

2.1 mg/kg/day was divided by an uncertainty factor of 300 (10 for extrapolation from animals to humans, 

10 for human variability, and 3 for database limitations), resulting in an acute-duration oral MRL for 

2,5-DNT of 0.007 mg/kg/day. 

Acute-Duration Oral MRL for 2,6-DNT 

•	 An MRL of 0.09 mg/kg/day has been derived for acute-duration oral exposure (≤14 days) to 

2,6-DNT.
 

Information on effects of acute-duration oral exposure of laboratory animals to 2,6-DNT are available 

from a single-dose lethality studies in rats (Deng et al. 2011; Lee et al. 1975; U.S. Army 1978a; Vernot et 

al. 1977), a 14-day gavage study in Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011d), and from 

an intermediate-duration study in dogs that evaluated clinical signs of toxicity, hematology, and clinical 

chemistry after 14 days of exposure (U.S. Army 1976). Results of these studies show that acute exposure 

to 2,6-DNT induces hematological effects, neurotoxicity, and hepatotoxicity, with hematological effects 

(methemoglobin-induced anemia and compensatory hematopoiesis) as the most sensitive effect. Results 

of acute lethality studies provide very little information on 2,6-DNT-induced toxicity at nonlethal levels. 

When groups of six male rats were given 14 gavage doses of 2,6-DNT (0, 4, 7, 14, 35, 68, or 

134 mg/kg/day), reductions in body weight of at least 10% were seen in rats exposed to doses 

≥35 mg/kg/day, and statistically significant, dose-related reductions in hemoglobin and hematocrit were 

seen at 134 mg/kg/day. Significant alterations in the leukocyte differential occurred primarily at the 

highest dose, including increased neutrophils, increased monocytes, and increased percentages of 

neutrophils and lymphocytes. Gross necropsy showed small testes in all rats receiving 134 mg/kg/day; 

this was accompanied by marked decreases in absolute and relative testes weight at this dose. At the 

highest dose, absolute and relative (to brain weight) epididymides weights were decreased by ~30%, and 

absolute and relative (to brain weight) spleen weights were increased by ~50%. Animals treated with 

2,6-DNT exhibited microscopic lesions in the testes (at doses ≥68 mg/kg/day), liver (doses 

≥35 mg/kg/day), kidney (at 134 mg/kg/day), and spleen (≥35 mg/kg/day). A LOAEL of 35 mg/kg/day 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 29 

2. RELEVANCE TO PUBLIC HEALTH 

was identified for this study based on liver (hepatocellular hyperplasia, oval cell hyperplasia, and 

hepatocellular hypertrophy) and spleen (lymphoid hyperplasia) lesions. The NOAEL was 14 mg/kg/day. 

Administration of 2,6-DNT to dogs at 20 and 100 mg/kg/day (but not 4 mg/kg/day) for 2 weeks produced 

hematological effects consistent with the development of methemoglobin-induced anemia and 

compensatory hematopoiesis (U.S. Army 1976). Dogs treated with 20 mg/kg/day showed a statistically 

significant decrease in erythrocyte count and a significant increase in mean cell hemoglobin. At 

100 mg/kg/day, more pronounced changes in these hematological parameters were observed; dogs 

showed statistically significant reductions in erythrocyte count, hematocrit, and hemoglobin and a 

statistically significant increase in reticulocyte count. In the 4 mg/kg/day group, similar hematological 

effects (decreased erythrocyte count, hemoglobin, and hematocrit, and increased reticulocytes) were 

observed, but these changes did not achieve statistical significance. No effects on clinical chemistry 

parameters were observed at any dose. Results of this study identify a NOAEL and a LOAEL of 4 and 

20 mg/kg/day, respectively, for hematological effects in dogs treated with 2,6-DNT for 2 weeks. The 

LOAEL from this study (20 mg/kg/day; U.S. Army 1976) was lower than the LOAEL from the rat study 

(35 mg/kg/day; Lent et al. 2012a, USAPHC 2011d). Although comprehensive end points were not 

evaluated after 2 weeks in the dog study, the identification of hematological effects as the most sensitive 

effect after acute-duration exposure is consistent with the most sensitive effect identified after 

intermediate-duration exposure to 2,6-DNT. 

In addition to hematological effects, clinical signs of neurotoxicity were observed in dogs administered 

2,6-DNT at 100 mg/kg/day (U.S. Army 1976). The study authors noted that at least three dogs (gender 

not specified) developed neurotoxicity (listlessness, incoordination, lack of balance, and weakness, 

particularly of the hind limbs) and other clinical signs of toxicity (pale gums, dark urine) within the first 

2 weeks of the exposure (incidence data were not reported). At this dose, one male dog died during the 

second week of exposure. No clinical signs of toxicity were observed at doses up to 20 mg/kg/day during 

the first 14 days of exposure, although similar (but milder) symptoms were reported at this dose following 

4 weeks of treatment. 

Hepatotoxicity, including increased alanine aminotransferase (ALT) activity and histopathological 

changes to the liver (congested sinusoids with sloughed hepatocytes and segmented neutrophils, 

disorganized midzonal regions characterized by infiltration of erythrocytes and hepatocytes with pyknotic 

nuclei and microvesiculated cytoplasm, and apoptotic hepatocytes) were reported in rats administered 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 30 

2. RELEVANCE TO PUBLIC HEALTH 

doses of 2,6-DNT (Deng et al. 2011). Results of this study identify acute-duration NOAEL and LOAEL 

values for hepatotoxicity for 2,6-DNT of 25 and 50 mg/kg/day, respectively. 

Based on the available data, toxicity to the hematological system (anemia and compensatory 

hematopoiesis) was identified as the most sensitive effect of acute-duration oral exposure to 2,6-DNT. In 

dogs exposed for 14 days, a statistically significant decrease in erythrocyte count and a statistically 

significant increase in mean cell hemoglobin were observed after treatment with 2,6-DNT at 

20 mg/kg/day for 2 weeks (U.S. Army 1976). Changes to other hematological parameters only reached 

statistical significance at 100 mg/kg/day. Therefore, the most sensitive hematological parameters were 

erythrocyte count and mean cell hemoglobin. To determine the POD for derivation of the acute-duration 

oral MRL for 2,6-DNT, all available continuous-variable models in the EPA BMDS (version 2.1) were fit 

to the data for erythrocyte count and mean cell hemoglobin. The BMD and the 95% lower confidence 

limit (BMDL) were estimated for doses associated with a change of 1 standard deviation from the 

controls. A detailed description of BMD modeling is provided in Appendix A. Neither the constant nor 

the non-constant variance model provided an adequate fit to the data for mean cell hemoglobin; therefore, 

these data were not considered suitable for BMD modeling. With the nonconstant variance model 

applied, the linear, polynomial, and power models provided an adequate fit to the data for erythrocyte 

count. The polynomial and power models converged to the linear model. The BMDL1SD value of 

9.31 mg/kg/day derived from this model was selected as the POD. This value was divided by an 

uncertainty factor of 100 (10 for animals to human extrapolation and 10 for human variability), resulting 

in an acute-duration oral MRL of 0.09 mg/kg/day. 

Intermediate-Duration Oral MRL for 2,6-DNT 

•	 An MRL of 0.004 mg/kg/day has been derived for intermediate-duration oral exposure (15–
 

364 days) to 2,6-DNT.
 

Information on effects of intermediate-duration oral exposure of laboratory animals is available from a 

13-week study in beagle dogs, CD rats, and CD-1 mice (U.S. Army 1976). Results of this study show 

that 2,6-DNT produces hematological, neurological, reproductive, and hepatic toxicity, with 

histopathological changes of the spleen (extramedullary erythropoiesis) that are likely secondary to 

methemoglobinemia and anemia. 

Intermediate-duration oral exposure of beagle dogs to 2,6-DNT at 4 mg/kg/day (lowest tested dose) 

produced extramedullary erythropoiesis (formation of erythrocytes outside of the bone marrow) in the 
 

 

 

 

 

 

 

 

DINITROTOLUENES 31 

2. RELEVANCE TO PUBLIC HEALTH 

spleen secondary to methemoglobinemia and anemia (U.S. Army 1976). The incidence and severity of 

this lesion was dose-related. Changes in hematological parameters associated with anemia and 

compensatory hematopoiesis, including decreased hematocrit and hemoglobin and increased numbers of 

reticulocytes, were observed at 20 and 100 mg/kg/day. However, mortality also occurred in dogs exposed 

to these doses. Results of this study identify a LOAEL of 4 mg/kg/day for extramedullary hematopoiesis 

secondary to hematological effects in dogs treated with 2,6-DNT for 13 weeks (U.S. Army 1976). In 

rodents exposed for 13 weeks, the lowest LOAELs for hematological effects (extramedullary 

erythropoiesis) were 7 mg/kg/day for rats and 51 mg/kg/day for mice (U.S. Army 1976). 

Hepatic, neurological, and reproductive effects were also observed following a 13-week exposure of dogs, 

rats, and mice to 2,6-DNT (U.S. Army 1976); however, these effects occurred at doses higher than those 

producing extramedullary erythropoiesis (4 mg/kg/day) in dogs. Note that in dogs, lethality also occurred 

at doses of 20 and 100 mg/kg/day. The lowest NOAEL and LOAEL values for hepatic effects (bile duct 

hyperplasia and degeneration and inflammatory changes to the liver) were 4 and 20 mg/kg/day, 

respectively, in dogs treated with 2,6-DNT for 13 weeks (U.S. Army 1976). Bile duct hyperplasia was 

observed at higher doses in rats (35 mg/kg/day) and mice (51 mg/kg/day) fed 2,6-DNT for 13 weeks (U.S. 

Army 1976). Neurotoxic effects (listlessness, incoordination, and lack of balance) were observed in dogs 

treated with 20 and 100 mg/kg/day. No signs of neurotoxicity were observed in rats at doses up to 

145 and 155 mg/kg/day (for males and females, respectively) or in mice at doses up to 289 and 

299 mg/kg/day (for males or females, respectively) for 13 weeks (U.S. Army 1976). For reproductive 

effects following 13-week exposure to 2,6-DNT, the lowest NOAEL and LOAEL values of 4 and 

20 mg/kg/day, respectively, were reported in dogs for testicular degeneration (U.S. Army 1976). NOAEL 

and LOAEL values for reproductive effects were 11 and 51 mg//kg/day, respectively, in male mice for 

decreased spermatogenesis and 7 and 35 mg/kg/day, respectively, in male mice for testicular atrophy 

(U.S. Army 1976). 

The LOAEL value of 4 mg/kg/day for an increased incidence of extramedullary erythropoiesis in the 

spleens of dogs was identified as the POD for derivation of the intermediate-duration oral MRL for 

2,6-DNT (U.S. Army 1976). Histopathology data were not suitable for BMD modeling, since the number 

of animals evaluated at each dose and time was small (n=2 animals). Therefore, the LOAEL value for 

4 mg/kg/day was used as the POD. This value was divided by an uncertainty factor of 1,000 (10 for use of 

a LOAEL, 10 for extrapolation from animals to humans, and 10 for human variability), resulting in an 

intermediate-duration oral MRL for 2,6-DNT of 0.004 mg/kg/day. 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 32 

2. RELEVANCE TO PUBLIC HEALTH 

Chronic-Duration Oral MRL for 2,6-DNT 

The only chronic exposure study identified for 2,6-DNT is a 1-year study in F344 rats (Leonard et al. 

1987). In this study, oral exposure to 7 and 14 mg/kg/day 2,6-DNT produced hepatocellular carcinoma, 

cellular alterations to hepatocytes (vacuolization accompanied by acidophilic and basophilic foci), and 

increased liver weights and serum hepatic enzyme activity. Hepatocellular carcinoma was observed in 

85 and 100% of rats treated with 7 and 14 mg/kg/day, respectively. No other effects of 2,6-DNT were 

reported in this study. Data from this study are not appropriate for derivation of a chronic-duration oral 

MRL for 2,6-DNT, as no noncancer effects were reported. 

Acute-Duration Oral MRL for 3,4-DNT 

•	 An MRL of 0.03 mg/kg/day has been derived for acute-duration oral exposure (≤14 days) to 

3,4-DNT.
 

No human acute-duration oral toxicity studies on 3,4-DNT were identified. For 3,4-DNT, the only 

available oral exposure study is a 14-day gavage study in rats in which a number of toxicological end 

points were assessed (Lent et al. 2012a; USAPHC 2011e). Lent et al. (2012a; USAPHC 2011e) identified 

effects on body weight, the hematopoietic system, the kidneys, and the neurological system; the 

hematopoietic system was affected at lower doses than the other target systems. Groups of six male 

Sprague-Dawley rats were given 3,4-DNT via gavage doses of 0, 7, 14, 28, 57, 113, or 227 mg/kg/day for 

14 days; evaluations included morbidity, clinical observations, hematology, serum chemistry, gross 

necropsy, organ weights, and histopathology. All rats survived. Clinical signs in the highest dose 

animals included cyanosis, lethargy, facial twitching, and hypoactivity or staring behavior. A 10% 

reduction in body weight was seen at the highest dose (227 mg/kg/day); at this dose, trace-to-mild renal 

lesions (proximal tubular degeneration, tubular basophilia, and lymphocytic infiltration) were seen, as 

were mild fibrosis, inflammation, and necrosis in the heart of one rat. Splenic lesions, including trace-tomild extramedullary hematopoiesis and lymphoid hyperplasia, were observed at doses ≥57 mg/kg/day. 

Spleens were not examined in lower dose groups. Results of this study identify an acute-duration 

LOAEL value for hematopoietic system effects of 57 mg/kg/day in rats. A NOAEL for hematopoietic 

system effects cannot be determined in the absence of histopathology examinations in lower dose groups. 

Hematopoietic system toxicity was identified as the most sensitive effect of acute-duration oral exposure 

to 3,4-DNT, based on the LOAEL value of 57 mg/kg/day in rats administered 3,4-DNT via gavage for 

14 days (Lent et al. 2012a; USAPHC 2011e). Incidences of trace-to-mild extramedullary hematopoiesis 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 33 

2. RELEVANCE TO PUBLIC HEALTH 

and lymphoid hyperplasia were subjected to BMD modeling. The best-fitting model for both end points 

yielded the same BMDL10 of 8.05 mg/kg/day; this value was selected as the POD. The POD of 

8.05 mg/kg/day was divided by an uncertainty factor of 300 (10 for extrapolation from animals to 

humans, 10 for human variability, and 3 for database limitations), resulting in an acute-duration oral MRL 

for 3,4-DNT of 0.03 mg/kg/day. 

Acute-Duration Oral MRL for 3,5-DNT 

•	 An MRL of 0.03 mg/kg/day has been derived for acute-duration oral exposure (≤14 days) to 

3,5-DNT.
 

No human acute-duration oral toxicity studies on 3,5-DNT were identified. For 3,5-DNT, the only 

available oral exposure study is a 14-day gavage study in rats, in which a number of toxicological end 

points were assessed (Lent et al. 2012a; USAPHC 2011f). Lent et al. (2012a; USAPHC 2011f) identified 

effects on survival, the reproductive tissues, and neurological system; effects on the reproductive tissues 

were the most sensitive. Groups of six male Sprague-Dawley rats were given 3,5-DNT via gavage doses 

of 0, 5, 10, 19, 39, 77, and 155 mg/kg/day for 14 days; evaluations included morbidity, clinical 

observations, hematology, serum chemistry, gross necropsy, organ weights, and histopathology. At doses 

≥77 mg/kg/day, all rats died; in addition, one rat exposed to 39 mg/kg/day died. Animals exposed to 

39 mg/kg/day exhibited neurological signs progressing from facial twitching to paralysis of the lower 

forelimbs. Rats exposed to ≥19 mg/kg/day 3,5-DNT exhibited testicular lesions including mild-to-severe 

tubular degeneration and trace-to-severe multinucleated giant cell formation. Results of this study 

identify acute-duration NOAEL and LOAEL values for testicular lesions of 10 and 19 mg/kg/day, 

respectively, in rats. 

Testicular toxicity was identified as the most sensitive effect of acute-duration oral exposure to 3,5-DNT, 

based on the LOAEL value of 19 mg/kg/day in rats administered 3,5-DNT via gavage for 14 days (Lent et 

al. 2012a; USAPHC 2011f). The NOAEL value of 10 mg/kg/day was used as the POD. Available data 

were not suitable for BMD modeling, as the incidences of testicular lesions increased from 0/6 in the 

control group to 6/6 at the LOAEL. The POD of 10 mg/kg/day was divided by an uncertainty factor of 

300 (10 for extrapolation from animals to humans, 10 for human variability, and 3 for database 

limitations), resulting in an acute-duration oral MRL for 3,5-DNT of 0.03 mg/kg/day. 
 

DINITROTOLUENES 34 

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DINITROTOLUENES 35 

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3.1 INTRODUCTION 

The primary purpose of this chapter is to provide public health officials, physicians, toxicologists, and 

other interested individuals and groups with an overall perspective on the toxicology of 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, and 3,5-DNT. It contains descriptions and evaluations of toxicological studies and 

epidemiological investigations and provides conclusions, where possible, on the relevance of toxicity and 

toxicokinetic data to public health. 

3.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE 

To help public health professionals and others address the needs of persons living or working near 

hazardous waste sites, the information in this section is organized first by route of exposure (inhalation, 

oral, and dermal) and then by health effect (death, systemic, immunological, neurological, reproductive, 

developmental, genotoxic, and carcinogenic effects). These data are discussed in terms of three exposure 

periods: acute (14 days or less), intermediate (15–364 days), and chronic (365 days or more). 

A glossary and list of acronyms, abbreviations, and symbols can be found at the end of this profile. 

Levels of significant exposure for each route and duration are presented in tables and illustrated in 

figures. The points in the figures showing no-observed-adverse-effect levels (NOAELs) or lowestobserved-adverse-effect levels (LOAELs) reflect the actual doses (levels of exposure) used in the studies. 

LOAELs have been classified into "less serious" or "serious" effects. "Serious" effects are those that 

evoke failure in a biological system and can lead to morbidity or mortality (e.g., acute respiratory distress 

or death). "Less serious" effects are those that are not expected to cause significant dysfunction or death, 

or those whose significance to the organism is not entirely clear. ATSDR acknowledges that a 

considerable amount of judgment may be required in establishing whether an end point should be 

classified as a NOAEL, "less serious" LOAEL, or "serious" LOAEL, and that in some cases, there will be 

insufficient data to decide whether the effect is indicative of significant dysfunction. However, the 

Agency has established guidelines and policies that are used to classify these end points. ATSDR 

believes that there is sufficient merit in this approach to warrant an attempt at distinguishing between 

"less serious" and "serious" effects. The distinction between "less serious" effects and "serious" effects is 

considered to be important because it helps the users of the profiles to identify levels of exposure at which 

major health effects start to appear. LOAELs or NOAELs should also help in determining whether or not 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 36 

3. HEALTH EFFECTS 

the effects vary with dose and/or duration, and place into perspective the possible significance of these 

effects to human health. 

The significance of the exposure levels shown in the Levels of Significant Exposure (LSE) tables and 

figures may differ depending on the user's perspective. Public health officials and others concerned with 

appropriate actions to take at hazardous waste sites may want information on levels of exposure 

associated with more subtle effects in humans or animals (LOAELs) or exposure levels below which no 

adverse effects (NOAELs) have been observed. Estimates of levels posing minimal risk to humans 

(Minimal Risk Levels or MRLs) may be of interest to health professionals and citizens alike. 

A User's Guide has been provided at the end of this profile (see Appendix B). This guide should aid in 

the interpretation of the tables and figures for Levels of Significant Exposure and the MRLs. 

The primary focus of this document is information on 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT. However, 

some information on technical-grade DNT (Tg-DNT) is also provided. Tg-DNT contains approximately 

76% 2,4-DNT, 19% 2,6-DNT, and <5% 3,4-, 2,3-, and 2,5-DNT. For each route of exposure (inhalation, 

oral, and dermal), human data will be discussed first. Available data for individual isomers (2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, and 3,5-DNT, in that order) will be presented followed by data for Tg-DNT. Studies 

conducted in experimental animals will be discussed thereafter, with data on the individual isomers 

preceding data for Tg-DNT. 

3.2.1 Inhalation Exposure 

Most of the data on health effects associated with exposure of humans to DNTs are from studies of 

workers exposed to 2,4-, 2,6-, or Tg-DNT. Exposure monitoring of workers in the past has generally 

been inadequate; consequently, few dose-response data based on human exposure to DNT isomers are 

available. No data were identified regarding inhalation exposure of humans to 2,3-, 2,5-, 3,4-, or 

3,5-DNT. Regarding available animal data, one study was located that examined the acute inhalation 

toxicity of 2,6-DNT in experimental animals. No studies were located regarding inhalation exposure to 

2,3-, 2,5-, 3,4-, or 3,5-DNT in experimental animals. 

Human exposure to chemicals in an occupational setting can occur via multiple routes: inhalation, 

dermal, and inadvertent ingestion (Hamill et al. 1982). Although the low vapor pressure of DNTs makes 

inhalation of vapors unlikely, it can occur when contaminated particulate material is in the air. In 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 37 

3. HEALTH EFFECTS 

addition, some dermal exposure is probable, and some ingestion may also occur as the result of eating or 

smoking without prior handwashing. 

3.2.1.1 Death 

In a retrospective cohort mortality study of 457 munitions workers who were exposed to either 2,4-DNT 

or Tg-DNT at two geographically different U.S. manufacturing plants, significant increases in death rates 

due to ischemic heart disease and residual diseases of the circulatory system were found (standardized 

mortality ratios [SMRs] of 126 and 143; 95% confidence intervals [CIs] of 65–234 and 112–179, 

respectively) (Levine et al. 1986a). Residual diseases of the circulatory system include congestive heart 

failure, cardiac arrest, and arteriosclerosis. The workers had been exposed to unreported concentrations 

of either 2,4-DNT (98% pure) or Tg-DNT for periods ranging from 30 days to >5 years (Levine et al. 

1986a). Cigarette smoking was not taken into account in this study, but the study authors suggested that 

it may not have been a risk factor because mortality from lung cancer was less than expected. Among 

workers at both plants, there appeared to be a latency period of >15 years for a significant increase in 

mortality due to ischemic heart disease. There also appeared to be a relationship between heart disease 

and the intensity of exposure to DNTs. No statistical increase was found in death due to cancer, either 

from malignant neoplasms as a whole or from individual cancers, although the statistical power of the 

study was insufficient to detect anything but gross changes in the death rate due to cancer. 

The Levine et al. (1986a) retrospective cohort mortality study was limited by small cohort size, and thus, 

the study had diminished power to detect an effect. As a result, the finding of elevated mortality from 

heart disease among workers in two plants from different parts of the United States linked only by 

exposure to DNTs is unusual. Workers in the United States generally have lower rates of heart disease 

than the general population because of the "healthy worker effect." At both plants, mortality from 

ischemic heart disease during the first 15 years following cohort entry was less than expected, and 

mortality increased only in later years. Suggestive, but not significant, is evidence of a relationship 

between heart disease and duration and intensity of exposure, also reported by Levine et al. (1986a). No 

studies were identified with respect to mortality after inhalation exposure of humans to 2,3-, 2,5-, 2,6-, or 

3,5-DNT. 

In an acute-duration study, no mortality was observed in male or female F344 rats (5/sex/group) exposed 

nose-only to 2,6-DNT as a vapor at 26 mg/m3 for 6 hours and observed 14 days after dosing (CMA 1991). 

In the same study, groups of male and female rats exposed to 2,6-DNT at 0, 196, 473, or 694 mg/m3 as an 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 38 

3. HEALTH EFFECTS 

aerosol exhibited mortality at ≥196 mg/m3. Two of five males (and no females) died at 196 mg/m3; all 

males and three of five females died at 694 mg/m3

. Rats that died showed evidence of lung congestion 

and had increased relative lung weights compared to controls. An LC50 of 0.43 mg/L was identified in 

rats, with LC50 values for males and females of 0.24 and 0.66 mg/L, respectively. 

No studies were located regarding death in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 3,4-, or 

3,5-DNT. 

3.2.1.2 Systemic Effects 

No studies were located regarding dermal, ocular, endocrine, or body weight effects in humans or animals 

after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. No studies were located regarding 

respiratory effects in humans after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT or in 

animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 3,4-, or 3,5-DNT. 

Respiratory Effects. No studies were identified with respect to respiratory effects after inhalation 

exposure of humans to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

In an acute-duration study, groups of male and female rats were exposed to 2,6-DNT at 0, 196, 473, or 

694 mg/m3 as an aerosol for 6 hours and observed 14 days after dosing (CMA 1991). Rats exposed to 

2,6-DNT as an aerosol at ≥196 mg/m3 exhibited signs of respiratory distress (exaggerated respiratory 

movements) for several days following exposure; recovery from this effect occurred by day 5 of the 

observation period. Rats that died showed evidence of lung congestion and had increased relative lung 

weights compared to controls. 

No studies were identified with respect to respiratory effects after inhalation exposure of animals to 2,3-, 

2,4-, 2,5-, 3,4-, or 3,5-DNT. 

Cardiovascular Effects. No studies were identified with respect to cardiovascular effects after 

inhalation exposure of humans to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Levine et al. (1986a) reported a significant increase in deaths from diseases of the circulatory system in 

workers involved in the manufacture and processing of 2,4-DNT and/or Tg-DNT. The preponderance of 

circulatory disease deaths was due to ischemic heart disease. The SMR was significantly elevated for 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 39 

3. HEALTH EFFECTS 

ischemic heart disease when compared to the U.S. male population and to persons living in the area. 

When workers were divided by exposure duration, the significant increase in cardiovascular deaths was 

only observed in workers exposed for at least 5 months. Although the study did not control for cigarette 

smoking, a known risk factor for heart disease, the investigators noted that the study did not find 

significant increases in lung cancer or disease deaths. 

No studies were located regarding cardiovascular effects in animals after inhalation exposure to 2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Gastrointestinal Effects. Vomiting and nausea were mentioned as health complaints in a survey of 

male workers involved in the production of smokeless gunpowders during World War II (McGee et al. 

1947). The exposure concentrations of 2,4-DNT were not specified. The investigators noted that prior to 

improving industrial hygiene practices, a much higher incidence of gastrointestinal symptoms was found. 

Since exposure to other compounds cannot be ruled out, attribution of these symptoms to DNT cannot be 

verified. 

No studies were identified with respect to gastrointestinal effects after inhalation exposure of humans to 

2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. No studies were located regarding gastrointestinal effects in animals 

after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Hematological Effects. Several hematological effects, including anemia and cyanosis, were found in 

male workers employed by a munitions factory during World War II (McGee et al. 1947). In some cases, 

there were increases in leukocyte count, which may be related to prolonged exposure to DNT. The study 

authors presumed that the exposure concentrations to 2,4-DNT were relatively high because of the 

relatively primitive industrial hygiene practices at that time. Although 36 of 154 workers were anemic in 

the earlier study and 73 of 714 workers were anemic in the follow-up study, no control groups were used 

as a basis for comparison. Because of possible exposure to other compounds, lack of work histories, lack 

of exposure monitoring, lack of a control population, and small cohort size, the results obtained are 

equivocal and may be best used as qualitative descriptions of symptoms. Marked cyanosis and other 

incapacitating symptoms were reported after exposure to unspecified concentrations of Tg-DNT in a 

study of French workers in a DNT production plant during World War I (Perkins 1919). It is assumed 

that workers were exposed to high concentrations of Tg-DNT via both inhalation and dermal pathways, 

since the processes described involved direct handling of large amounts of Tg-DNT without protective 

equipment. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 40 

3. HEALTH EFFECTS 

No studies were identified with respect to hematological effects after inhalation exposure of humans to 

2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

In an acute-duration study, male and female F344 rats (5/sex/group) were exposed to 2,6-DNT as a vapor 

at 0 or 26 mg/m3 or as an aerosol at 0, 196, 473, or 694 mg/m3 for 6 hours and observed 14 days after 

dosing (CMA 1991). Blood was analyzed for serum methemoglobin concentration 24 hours prior to 

dosing and 1, 24, and 48 hours and 7 days after dosing. Although the study authors noted slight increases 

in serum methemoglobin in 2,6-DNT-exposed animals in the first 24 hours after exposure, these changes 

were not clearly dose-related and were not statistically significantly different from control animals. No 

other hematological end points were evaluated. 

No studies were located regarding hematological effects in animals after inhalation exposure to 2,3-, 2,4-, 

2,5-, 3,4-, or 3,5-DNT. 

Musculoskeletal Effects. Joint pain, especially in the knees, and other incapacitating symptoms 

were found in unspecified numbers of French workers in a plant that produced DNTs during World War I 

(Perkins 1919). No exposure concentrations were reported, but it is assumed that they were high because 

of the direct handling of large amounts of Tg-DNT without protective equipment, which also suggests 

that the workers were exposed dermally. However, because exposure to other compounds cannot be ruled 

out and no control data are available, caution must be used when interpreting these results. 

No studies were located regarding musculoskeletal effects humans after inhalation exposure to 2,3-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Hepatic Effects. A study of 714 workers at a munitions plant found that 29 experienced liver 

tenderness (McGee et al. 1947). Other factors, such as alcohol consumption, may account for these 

results which should be viewed with caution because of the lack of control data, lack of information on 

exposure concentrations, and possible multiple chemical exposure. Medical surveys of 52 male workers 

exposed to Tg-DNT in a chemical plant that manufactured toluenediamine (TDA) revealed no differences 

in hepatic blood chemistry profiles (NIOSH 1982). Air samples contained concentrations ranging from 

0.026 to 0.890 mg/m3 Tg-DNT (mean 0.207 mg/m3

). 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 41 

3. HEALTH EFFECTS 

No studies were located regarding hepatic effects in humans after inhalation exposure to 2,3-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT, or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Renal Effects. No effects were observed on either of the renal parameters (blood urea nitrogen 

[BUN], creatinine) monitored in blood chemistry in a medical survey of 52 male workers exposed to 

Tg-DNT in a chemical plant that manufactured TDA (NIOSH 1982). Exposure concentrations in air 

samples taken for this study ranged from 0.026 to 0.890 mg/m3 Tg-DNT (mean 0.207 mg/m3

). The study 

was limited by a small exposure population and lack of historical individual exposure monitoring. 

Evidence of tubular and/or glomerular damage (alterations in urinary protein excretion patterns) was 

found among a cohort of approximately 160 workers at a copper mine exposed to Tg-DNT explosives 

(Brüning et al. 2001). Dividing the workers into exposure categories resulted in a significant dose-related 

trend for the incidence of tubular and/or glomerular damage among the workers without renal cell cancer 

or urothelial cancer. Approximately 80% of the 25 workers in the very high exposure category had 

evidence of renal damage. Dose-related increases in the excretion of α1-microglobulins and glutathioneS-transferase-α were also observed suggesting proximal tubule damage. The lack of effect on 

glutathione-S-transferase-π levels suggested a lack of damage to the distal tubules. 

No studies were located regarding renal effects in humans or animals after inhalation exposure to 2,3-, 

2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.2.1.3 Immunological and Lymphoreticular Effects 

No studies were located regarding immunological or lymphoreticular effects in humans or animals after 

inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.2.1.4 Neurological Effects 

Dizziness and headache were reported by Perkins (1919) in a study of French workers exposed to 

Tg-DNT at a production plant during World War I. Although no exposure concentrations were reported, 

it is assumed that the workers were exposed to high concentrations of Tg-DNT via both inhalation and 

dermal pathways, since the manufacturing processes required workers to handle large amounts of 

Tg-DNT without protective equipment. Exposure to chemicals other than DNT in this environment could 

not be ruled out. Health effects of munitions workers exposed to unspecified levels of what was 

presumed to be 2,4-DNT were studied by McGee et al. (1947). Neurological signs reported by these 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 42 

3. HEALTH EFFECTS 

workers included headache, dizziness, and pain, numbness, and tingling in the extremities. The 2,4-DNT 

exposure concentrations were not specified, but were considered by these authors to be relatively high as 

a result of the lack of safety practices. 

No studies were located regarding neurological effects in humans after inhalation exposure to 2,3-, 2,5-, 

2,6-, or 3,5-DNT or in animals after inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.2.1.5 Reproductive Effects 

Studies of men occupationally exposed to Tg-DNT at DNT and TDA plants showed no significant 

differences in sperm counts or morphology, follicle stimulating hormone (FSH) levels, or incidence of 

miscarriage in their wives compared to controls (Hamill et al. 1982; NIOSH 1982). In the NIOSH (1982) 

study, Tg-DNT concentrations ranged from 0.026 to 0.890 mg/m3 (mean 0.207 mg/m3

). Interpretation of 

these studies is somewhat confounded by the lack of distinction between DNT and TDA exposure and the 

lack of information regarding exposure concentration in the Hamill et al. (1982) study. The limitations of 

these studies are similar (small exposure populations and the lack of individual exposure monitoring) and 

limit the ability of the studies to detect adverse effects. 

No significant effects on the fertility of workers occupationally exposed to Tg-DNT have been found in 

several studies (Hamill et al. 1982; Levine et al. 1985a; NIOSH 1982). However, Levine et al. (1985a) 

estimate that only a 50–70% reduction in fertility could have been detected in the worker population that 

they studied. 

One study by the CDC (1981) noted that sperm counts were decreased by >50% in workers in a Kentucky 

chemical plant exposed to DNTs and TDA compared to workers not exposed to these chemicals. The 

study was limited because of multiple chemical exposures and the small numbers of workers examined. 

Thirty workers participated in the study: 9 currently exposed, 12 previously exposed, and 9 with no 

history of exposure to DNTs/TDA. 

No studies were located regarding reproductive effects in humans or animals after inhalation exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 43 

3. HEALTH EFFECTS 

3.2.1.6 Developmental Effects 

No studies were located regarding developmental effects in humans or animals after inhalation exposure 

to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.2.1.7 Cancer 

The mortality of a cohort of 4,989 men who worked at least 5 months in a munitions facility was analyzed 

to determine whether DNT exposure was associated with an increased risk of cancer of the liver and 

biliary tract (Stayner et al. 1993). Workers were considered exposed if they had worked at least 1 day on 

a job with probable exposure to DNT. In this study, a significant increase in hepatobiliary cancer 

mortality (standard rate ratio [SRR]=3.88, 95% CI 1.04–14.41) was observed among DNT-exposed 

workers compared to non-exposed control workers. However, no significant changes were noted when 

compared to the U.S. population, the SRR for hepatobiliary cancer being 2.67 (95% CI 0.98–5.83; 

p=0.052). No quantitative data were available on the DNT exposure of these men. This study is limited 

by the small numbers of hepatobiliary cancer cases, small numbers of workers with long term exposure to 

DNT, and possible exposure of the workers to other chemicals. However, no significant increases in 

mortality from malignant neoplasms as a group or from particular cancers (liver, lung, gallbladder, 

kidney, and connective tissues) were observed in workers occupationally exposed to 2,4-DNT and/or 

Tg-DNT (Levine et al. 1986b). Exposures were not quantified and the cohort was small. The study 

authors estimated that an 8-fold increase in liver and gallbladder cancer in exposed workers would be 

necessary in order to be detected at the p=0.05 level; thus, the statistical analysis was not strong enough to 

detect small increases in cancer. 

Among 500 workers in an underground copper mine where Tg-DNT-containing explosives were 

extensively used, increases, as compared to the German national cancer registry, in the incidence of renal 

cell cancer and urothelial cancer incidence were observed (Brüning et al. 1999). However, when the 

workers were divided by exposure categories, no apparent relationship between renal cell cancer 

incidence and exposure category was found. In contrast, four of the six workers with urothelial cancer 

cases were in the high exposure group (one was in the lowest group and the other was in the highest 

exposure group). The genotype distribution of several polymorphic xenobiotic enzymes, including 

N-acetyltransferase 2 and glutathione-S-transferases M1 (GSTM1) and T1 (GSTT1), was examined in the 

14 workers with renal cell cancer and 6 workers with urothelial cancer. The genotype distribution among 

the renal cell cancer cases did not differ from the normal German population. Similarly, the distribution 

of GSTM1 and GSTT1 genotypes for the urothelial cancer cases was similar to the German population. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 44 

3. HEALTH EFFECTS 

However, all of the urothelial cancer cases were found to be “slow acetylators” compared to a 58% 

distribution in the German population. 

Harth et al. (2005) reported three cases of urothelial carcinoma of the urinary bladder among 60 workers 

exposed to Tg-DNT at a German explosive manufacturing facility. The investigators noted that this 

cancer rate was 15.9 times higher than the expected incidence in the federal state. 

Seidler et al. (2014a) examined the cancer incidence through 2005 in a cohort of 16,441 male copper 

miners who worked between 1953 and 1990 in one of two German mines that used Tg-DNT explosives. 

The mean duration of employment was 6.8 years. Compared with the cancer incidence in the general 

population of the region in Germany where the mines were located (Saxony-Anhalt), the cancer 

incidences in the miners were not significantly increased for any cancer other than lung cancer 

(standardized incidence ratio [SIR] = 1.29, 95% CI 1.13–1.46). Moderate, but not statistically significant, 

increases in the incidences of kidney and bladder cancer (SIRs of 1.25 and 1.41 for kidney and bladder 

cancer, respectively, among workers with >20 years of employment with exposure to Tg-DNT) were seen 

when the data were stratified by duration of employment with Tg-DNT exposure. A follow-up casecohort study of this group, with follow-up through 2006, compared the Tg-DNT exposure history (by job 

exposure matrix) of 109 renal cancer cases with that of 999 randomly chosen cohort members (Seidler et 

al. 2014b). The Cox proportional hazard ratio (HR) was elevated, but not significantly, for high 

inhalation exposure to Tg-DNT (HR 1.36, 95% CI 0.84–2.21). When combined across medium and high 

exposure categories for both dermal and inhalation exposure, the HR for renal cancer was significantly 

increased (HR 2.12, 95% CI 1.03–4.37). 

No studies were located regarding cancer in humans following inhalation exposure to 2,3-, 2,5-, 3,4-, or 

3,5-DNT or in animals following inhalation exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.2.2 Oral Exposure 

No studies were located regarding health effects in humans following oral exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. However, it is assumed that oral ingestion could be a secondary route for 

occupationally exposed humans. 

In one series of studies performed at the U.S. Army Public Health Command (Lent et al. 2012a; 

USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f), groups of male Sprague-Dawley rats were 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 45 

3. HEALTH EFFECTS 

administered one of the DNT isomers (2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 3,4-DNT, or 3,5-DNT) 

by gavage in corn oil vehicle for 14 days at doses ranging from 1/128th or 1/64th of the reported LD50 

value to 1/4 or 1/2 LD50. The observed isomer-specific differences in hematological, hepatic, renal, 

neurological, and reproductive effects are discussed at the end of each corresponding subsection of 

Section 3.2.2. 

3.2.2.1 Death 

No studies were located regarding death in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT. 

2,4-DNT is lethal to experimental animals after oral administration. Animals generally developed 

cyanosis and ataxia after dosing. In general, rats are more sensitive than mice to the lethal effects of 

2,4-DNT. The LD50 values that have been determined for rats after gavage dosing with 2,4-DNT range 

from 270 to 650 mg/kg (U.S. Army 1975, 1978a; Vernot et al. 1977); in mice, LD50 values were reported 

to be between 1,340 and 1,954 mg/kg after 2,4-DNT administration (U.S. Army 1975, 1978a; Vernot et 

al. 1977). In female Sprague-Dawley rats (5/group) administered a single dose of 2,4-DNT (in 5% v/v 

DMSO in corn oil) via gavage at 398 mg/kg and observed for 24 or 48 hours after dosing, two of five 

animals and one of five animals died within 24 and 48 hours, respectively (Deng et al. 2011). No deaths 

occurred in rats administered 2,4-DNT at 5, 50, 99, or 198 mg/kg. In a dominant lethal study by Lane et 

al. (1985), 8 of 15 male Sprague-Dawley rats died after receiving five daily doses of 240 mg/kg 2,4-DNT. 

No deaths were reported when male and female Sprague-Dawley rats were fed 78 or 82 mg/kg/day 

2,4-DNT, respectively, in the diet for 14 days (McGown et al. 1983). When male Sprague-Dawley rats 

were given gavage doses of 284 mg/kg/day for 14 days, all six exposed rats died (Lent et al. 2012a; 

USAPHC 2011b). 

Death has been reported after intermediate- and chronic-duration exposure to 2,4-DNT in numerous 

studies. One of eight male and eight of eight female CD rats died after 3–13 weeks of ingesting 2,4-DNT 

in the diet (Lee et al. 1985; U.S. Army 1978b). Concentrations in the feed causing these deaths were 

equivalent to doses of 93 and 145 mg/kg/day in males and females, respectively. Death has also been 

reported in rodents fed concentrations equivalent to doses of 371–413 mg/kg 2,4-DNT in the diet for up 

to 6 months (Hong et al. 1985; Kozuka et al. 1979; U.S. Army 1978b). No treatment-related deaths were 

reported in rats fed up to 16.5 mg/kg/day 2,4-DNT or mice fed up to 28.5 mg/kg/day 2,4-DNT for 

4 weeks, or in rats fed up to 22 mg/kg/day 2,4-DNT or mice fed up to 76 mg/kg/day 2,4-DNT for 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 46 

3. HEALTH EFFECTS 

78 weeks (NCI 1978). In a 3-generation reproductive study, there appeared to be an increased incidence 

of death among F0 dams during parturition after receiving 45.3 mg/kg/day 2,4-DNT in the diet for 

6 months (U.S. Army 1979). These deaths were associated with prolonged parturition, hemorrhage, and 

placental retention. However, because these effects were also seen to a lesser extent in control animals, it 

may be that the effects of 2,4-DNT simply enhanced effects caused by the advancing age of the dams 

(U.S. Army 1979). 

In a 13-week study, some dogs fed 25 mg/kg/day became moribund after ≥22 days and had to be 

terminated, whereas no treatment-related deaths were reported in dogs fed 5 mg/kg/day (Ellis et al. 1985; 

U.S. Army 1978b). In addition to severe weight loss, severe neurological effects and histopathological 

changes were found in these animals, including vacuolization and focal gliosis in the cerebellum and 

perivascular hemorrhages in the cerebellum and brain stem, as well as peripheral neuropathy, testicular 

degeneration, and biliary hyperplasia. In a 24-month study of dogs, the administration of 10 mg/kg/day 

2,4-DNT by capsule caused death within 6 months, but no deaths were reported at 1.5 mg/kg/day; clinical 

signs prior to death were similar to those reported in the 13-week study (Ellis et al. 1985; U.S. Army 

1979). Decreased longevity was reported in 1–2-year studies of CD rats at average daily intakes as low as 

3.9 mg/kg/day (males) and 5.1 mg/kg/day (females), and of CD-1 mice at 898 mg/kg/day (Hong et al. 

1985; Lee et al. 1985; U.S. Army 1978b, 1979). 

The experimental data are more limited for 2,6-DNT than for 2,4-DNT. After administration of 2,6-DNT, 

LD50 values have been reported to range from 180 to 795 mg/kg in rats and from 621 to 807 mg/kg in 

mice (U.S. Army 1975, 1978a; Vernot et al. 1977). Complete lethality was reported in female SpragueDawley rats (5/group) administered a single dose of 2,6-DNT via gavage (in 5% v/v DMSO in corn oil) at 

398 mg/kg (Deng et al. 2011). No deaths occurred in animals administered 5–199 mg/kg and observed 

for 2 days after dosing. Mortality was reported in a dose-range finding study of male rats given oral doses 

of 200 mg/kg/day 2,6-DNT in corn oil for 3 days; no rats died at 100 mg/kg/day (Rothfuss et al. 2010). 

The maximum tolerated dose (MTD) of 2,6-DNT corresponding to 100% survival of A/J mice after 

6 doses over a 2-week period was 250 mg/kg (Schut et al. 1983). No male Sprague-Dawley rats died 

when groups of six rats were given 14 consecutive daily gavage doses up to 134 mg/kg/day 2,6-DNT 

(Lent et al. 2012a; USAPHC 2011d). 

All six male rats exposed to oral doses of 33 mg/kg/day 2,6-DNT in corn oil for 29 consecutive days 

survived the treatment (Rothfuss et al. 2010). Intermediate-duration studies have shown an increase in 

mortality of mice and dogs after 2,6-DNT administration. After feeding 51 mg/kg/day 2,6-DNT to male 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 47 

3. HEALTH EFFECTS 

Swiss albino mice in the diet for up to 13 weeks, 8 of 16 of these animals died; 6 of 16 females fed 

55 mg/kg/day 2,6-DNT also died (U.S. Army 1976). No treatment-related deaths were reported when rats 

were fed up to 155 mg/kg/day 2,6-DNT for the same duration (U.S. Army 1976). Two of eight dogs 

treated with 20 mg/kg 2,6-DNT by capsule died in a 13-week study (U.S. Army 1976). Thus, dogs seem 

to be the most sensitive of the three species to intermediate-duration oral 2,6-DNT exposure. 

Vernot et al. (1977) reported LD50 values for 2,3- DNT of 1,120 mg/kg/day in male Sprague-Dawley rats 

and 1,070 mg/kg/day in male CF-1 mice. No cause of death or additional information was reported. 

Exposure to 550 mg/kg/day 2,3-DNT via gavage for 14 days resulted in the deaths of 6/6 male SpragueDawley rats; no deaths occurred at doses of ≤275 mg/kg/day (Lent et al. 2012a; USAPHC 2011a). 

For 2,5-DNT, LD50 values of 710 and 1,230 mg/kg/day, respectively, were reported in male SpragueDawley rats and male CF-1 mice (Vernot et al. 1977); no additional information was provided. One rat in 

a group of six male Sprague-Dawley rats died after 14 consecutive daily gavage doses of 308 mg/kg/day 

2,5-DNT (Lent et al. 2012a; USAPHC 2011c). 

Lent et al. (2012a; USAPHC 2011e, 2011f) provide the only acute-duration lethality data for 3,4- and 

3,5-DNT. When groups of six male Sprague-Dawley rats received gavage doses of 3,4-DNT up to 

227 mg/kg/day for 14 days, there were no deaths. In contrast, 3,5-DNT was lethal to 1/6 rats at a dose of 

39 mg/kg/day and to all rats at doses of 77 and 155 mg/kg/day. 

Administration of up to 150 mg/kg/day Tg-DNT for 14 days was lethal to 6 of 13 pregnant F344 rats 

when administered by gavage during gestation (Jones-Price et al. 1982), yet this same concentration of 

Tg-DNT fed in the diet for 30 days did not kill any of the same strain of rats in another study (Hazleton 

Laboratories 1977). Decreased survival was found in CDF rats fed 35 mg/kg/day Tg-DNT for 52 weeks 

or 14 mg/kg/day Tg-DNT for 104 weeks (Hazleton Laboratories 1982). 

All LOAEL values from each reliable study for death in each species and duration category for 2,3-, 

2,4-DNT, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, 

respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively. 
208

550

205

1120

203

1070

209

275

275

275

275

275

211

275

Table 3-1 Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

ACUTE EXPOSURE Death 

1 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

System 

NOAEL 

(mg/kg/day) 

Less Serious 

(mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

550 M (6/6 died) 

Reference Chemical Form 

Lent et al. 2012a 2,3-DNT 

2 Rat 

(SpragueDawley) 

once (G) 1120 M (LD50) Vernot et al. 1977 2,3-DNT 

3 Mouse 

(CF-1) 

Systemic 

4 Rat 

(SpragueDawley) 

once (G) 1 x/d 14 d (G) 

Cardio 275 M 

1070 M (LD50) Vernot et al. 1977 

2,3-DNT Lent et al. 2012a 2,3-DNT 

Hemato 

b

275 M (extramedullary 

hematopoiesis and lymphoid hyperplasia of spleen, lymphoid depletion) 

Hepatic 275 M Renal 275 M (trace tubular dilatation 

and lymphocytic infiltration) 

Bd Wt 275 M 

Neurological 

5 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

275 M Lent et al. 2012a 

2,3-DNT 
210

275

Table 3-1 Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

Reproductive 

6 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

275 M Lent et al. 2012a 

2,3-DNT 

a The number corresponds to entries in Figure 3-1. b Used to derive an acute-duration oral minimal risk level (MRL) of 0.09 mg/kg/day; the MRL was derived by dividing the LOAEL of 275 mg/kg/day by an uncertaint(10 for use of a LOAEL, 10 for animal-to-human extrapolation, 10 for human variability, and 3 for database limitations). Bd Wt = body weight; Cardio = cardiovascular; d = day(s); (G) = gavage; Hemato = hematological; LD50 = lethal dose, 50% kill; LOAEL = lowest-observed-adversemale; NOAEL = no-observed-adverse-effect level; x = time(s) 
Death 

CardiovascHemHepatiRenal Body NeurologicaReproductiv

atologi

c 

Weigh

mg/kg/day 

10000 

3m 2r

1r 

4r 

1000 

4r 4r 4r 4r 5r 6r 

100 

10 

1 

0.1 

0.01 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

ular 

l 

e 

DINITROTOLUENES 3. HEALTH EFFECTS

cal 

t 

Figure 3-1 Levels of Significant Exposure to 2,3-Dinitrotoluene - Oral
	

Acute (≤14 days)
	

Systemic 

50
2

240

214

284

53

568650

16

270

54

19541340

18

1630

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

ACUTE EXPOSURE Death 

1 Rat 

(SpragueDawley) 

5 d 1 x/d (GO) 

System 

NOAEL 

(mg/kg/day) 

Less Serious 

(mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

240 M (8/15 died) 

Reference Chemical Form 

Lane et al. 1985 2,4-DNT 

2 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

284 M (6/6 died) Lent et al. 2012a 

2,4-DNT 

3 Rat 

(CD) 

once (GO) 568 M (LD50) 

650 F (LD50) 

U.S. Army 1975; U.S. A1978a 2,4-DNT 

4 Rat 

(SpragueDawley) 

once (G) 270 M (LD50) Vernot et al. 1977 2,4-DNT 

5 Mouse 

(Swiss albino) 

once (GO) 1954 M (LD50) 

1340 F (LD50) 

U.S. Army 1975 2,4-DNT 

6 Mouse 

(CF-1) 

once (G) 1630 M (LD50) Vernot et al. 1977 2,4-DNT 
196

50

99

398198

398

3

60

180 240

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to Species 

Figure (Strain) 

Systemic 

7 Rat 

(SpragueDawley) 

8 Rat 

(SpragueDawley) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

once (GO) Hepatic 50 F 99 F Significantly decreased levels of serum albumin 

(13% lower than controls) 

Deng et al. 2011 2,4-DNT 

Renal Bd Wt 

398 F 198 F 398 F Decreased body weight 

gain (1 to 4 g compared to 17 g for controls) 

5 d 1 x/d (GO) 

Hemato 60 M (slight cyanosis) Lane et al. 1985 

2,4-DNT 

Bd Wt 180 M 240 M (weight loss) 
212

14236

71

18

36

14271

142

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

9 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

Cardio 142 M Lent et al. 2012a 

2,4-DNT 

Hemato 36 M 71 M (mild to moderate 

extramedullary hematopoiesis of the spleen) 

Hepatic 18 M 36 M (trace-to-mild single cell 

necrosis and glycogen deposition) 

Renal 142 M Bd Wt 71 M 142 M (terminal Bd Wt 12% 

lower than control) 
189

260.9272.7260.9272.7260.9272.7

78.381.878.381.8

260.9272.7260.9272.7

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System 

10 Rat 

(SpragueDawley) 

14 d ad lib Resp 

Cardio Gastro Hepatic Renal Dermal Ocular 

LOAEL 

NOAEL 

(mg/kg/day) 

Less Serious 

(mg/kg/day) 

Serious 

(mg/kg/day) 

Reference Chemical Form 

260.9 M McGown et al. 1983 

2,4-DNT 272.7 F 

260.9 M 

272.7 F 260.9 M 

272.7 F 

78.3 M (increased alanine 

aminotransferase and cholesterol) 

81.8 F (increased cholesterol) 78.3 M (hyaline droplet 

formation) 

81.8 F (hyaline droplet 

formation) 

260.9 M 

272.7 F 

260.9 M 272.7 F 
237

134

134134

192

260.9272.7

215

142

191

5

25

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

System 

Hemato 

Musc/skel Bd Wt 

NOAEL 

(mg/kg/day) 

134 F 

260.9 M 

272.7 F 

142 M 

b

5 

LOAEL 

a 

Key to Species 

Figure (Strain) 

11 Mouse 

(C57BL/6N) 

Immuno/ Lymphoret 

12 Rat 

(SpragueDawley) 

Neurological 

13 Rat 

(SpragueDawley) 

14 Dog 

(Beagle) 

Exposure/ 

Duration/ 

Frequency 

(Route) Less Serious Serious (mg/kg/day) (mg/kg/day) 

134 F (swim-to-exhaustion time 

decreased by 82% relative to controls) 

134 F (2.7-fold greater body 

weight loss than control) 

25 (incoordination, stiffness, 

abnormal gait) 

Reference Chemical Form 

Wilbanks et al. 2014 2,4-DNT McGown et al. 1983 2,4-DNT Lent et al. 2012a 2,4-DNT Ellis et al. 1985; U.S. Ar1978b 2,4-DNT 

14 days daily (G) 14 d ad lib 1 x/d 14 d (G) 12 d 1 x/d (C) 
4

60 180

213

71

142

190

78.3

66

250

21

371

6

93145

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

Reproductive 

15 Rat 

(SpragueDawley) 

5 d 1 x/d (GO) 

60 M 180 M (decreased fertility) Lane et al. 1985 

2,4-DNT 

16 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

71 M 142 M (decreased absolute and 

relative testes weight and decreased absolute epididymis weight; tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes) 

Lent et al. 2012a 2,4-DNT 

17 Rat 

(SpragueDawley) 

14 d ad lib 78.3 M (decreased thickness of spermatogenic sperm 

layers) 

McGown et al. 1983 2,4-DNT 

18 Mouse 

DBA/2J 

2 d 1 x/d (G) 

INTERMEDIATE EXPOSURE Death 

19 Rat 

(Wistar) 

6 mo ad lib (F) 

250 M (decreased fertility) 371 M (71% died) 

Soares and Lock 1980 2,4-DNT Kozuka et al. 1979 2,4-DNT 

20 Rat 

(CD) 

4 or 13 wk ad lib (F) 

93 M (1/8 died) 

145 F (8/8 died) 

Lee et al. 1985 2,4-DNT 
140

45.3

135

413

151

25

160

10

24

76.7

156.4

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to Species 

Figure (Strain) 

21 Rat 

(CD) 

22 Mouse 

(CD-1) 

23 Dog 

(Beagle) 

24 Dog 

(Beagle) 

Systemic 

25 Rat 

(SpragueDawley) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

3 or 6 mo ad lib (F) 

45.3 F (increased incidence of 

death during parturition) 

U.S. Army 1979 2,4-DNT 

4 or 13 wk ad lib (F) 

413 (2/16M, 2/16F died) Hong et al. 1985; U.S. A

1978b 2,4-DNT 

4 or 13 wk 1 x/d (C) 

25 (5/8 died) Ellis et al. 1985; U.S. Ar

1978b 2,4-DNT 

6 mo 1 x/d (C) 

10 M (4/6 died) U.S. Army 1979, Ellis et 

1985 2,4-DNT 

3 wk ad lib (F) 

Bd Wt 76.7 M 156.4 M (10% decrease body 

weight) 

Bloch et al. 1988 2,4-DNT 
20

371371

371

371

50

27

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

26 Rat 

(Wistar) 

6 mo ad lib (F) 

Hemato 371 M (increased 

methemoglobin) 

Kozuka et al. 1979 2,4-DNT 

Hepatic 371 M (increased relative liver 

weight; increased SGOT, LDH, alkaline phosphatase, acid phosphatase, triglycerides, glucose; formation of puruloid matter) 

Renal 371 M Bd Wt 371 M (41% decrease body 

weight) 

27 Rat 

(Fischer- 344) 

6 or 26 wk ad lib (F) 

Bd Wt 27 M (11% decrease body 

weight) 

Leonard et al. 1987 2,4-DNT 
5

3438

93108

266145

266145266145

3438

141

34.545.3

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

28 Rat 

(CD) 

4 or 13 wk ad lib (F) 

Hemato 34 M 

38 F 

93 M (reticulocytosis; 

hemosiderosis) 

108 F (reticulocytosis; 

hemosiderosis) 

266 M (anemia) 145 F (anemia) 

U.S. Army 1978b, Lee e1985 2,4-DNT 

Hepatic 266 M 

145 F 

Renal 266 M 

145 F 

Bd Wt 34 M (75% decrease body 

weight gain with decreased food consumption) 

38 F (94% decrease body 

weight gain with decreased food consumption) 

29 Rat 

(CD) 

3 or 6 mo ad lib Bd Wt 34.5 M (23-25% decrease in body weight) U.S. Army 1979 2,4-DNT 

45.3 F (10-23% decrease in 

body weight) 
138

137147

413468

47147

137468

413468

413468

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

30 Mouse 

(CD-1) 

4 or 13 wk ad lib (F) 

Hemato 137 M 

147 F 

413 M 468 F (mild anemia, 

reticulocytosis) 

Hong et al. 1985; U.S. A1978b 2,4-DNT 

Hepatic 47 M 

147 F 

137 M 468 F (mild hepatocellular 

dysplasia) 

Renal 413 M 

468 F 

Bd Wt 413 M 

468 F (body weight loss with 

decreased food consumption) 
149

5 252525

200

1.51.50.2

10101.5

163

371

150

25

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

31 Dog 

(Beagle) 

4 or 13 wk 1 x/d (C) 

Hemato 5 25 (anemia, Heinz bodies) Ellis et al. 1985; U.S. Ar

1978b 2,4-DNT 

Hepatic 25 

Renal 25 

32 Dog 

(Beagle) 

9 mo 1 x/d (C) 

Hemato 1.5 M 

1.5 F 

c

0.2 F 

10 M (increased Heinz bodies 

and methemoglobin) 

10 F (decreased erythrocyte 

count, hemoglobin, and hematocrit; increased reticulocytes and Heinz bodies) 

U.S. Army 1979, Ellis et 1985 2,4-DNT 

1.5 F (increased 

methemoglobin) 

Immuno/ Lymphoret 

33 Rat 

(Wistar) 

6 mo ad lib (F) 

371 M (increased relative 

spleen weight) 

Kozuka et al. 1979 2,4-DNT 

34 Dog 

(Beagle) 

4 or 13 wk 1 x/d (C) 

25 Ellis et al. 1985; U.S. Ar

1978b 2,4-DNT 
22

347

15

34108

93145

136

413468

147

5

25

25

76.7 153.4

23

371

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a 

Key to Species 

Figure (Strain) 

Neurological 

35 Rat 

(Wistar) 

36 Rat 

(CD) 

37 Mouse 

(CD-1) 

38 Dog 

(Beagle) 

Reproductive 

39 Rat 

(SpragueDawley) 

40 Rat 

(Wistar) 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) Serious (mg/kg/day) 

6 mo ad lib (F) 

347 M (humpback 

incoordination) 

4 or 13 wk ad lib (F) 

34 M 

108 F 

93 M 

145 F (demyelination of 

cerebellum and brain stem; widespread and stiff-legged gait) 

4 or 13 wk ad lib (F) 

413 M 468 F 

4 or 13 wk 1 x/d (C) 

5 25 (incoordination, abnormal 

gait, paralysis) 

3 wk ad lib (F) 

76.7 M (multinucleated 

spermatids, mild irregularity of basal lamina, vacuolation and lipid accumulation in Sertoli cells) 

153.4 M (extensive degeneration 

of spermatids and spermatocytes; ultrastructural changes in Sertoli cells; 63% decrease sperm count) 

6 mo ad lib (F) 

371 M (testicular atrophy) 

Reference Chemical Form 

Kozuka et al. 1979 2,4-DNT U.S. Army 1978b, Lee e1985 2,4-DNT Hong et al. 1985; U.S. A1978b 2,4-DNT Ellis et al. 1985; U.S. Ar1978b 2,4-DNT Bloch et al. 1988 2,4-DNT Kozuka et al. 1979 2,4-DNT 
186

9.3 93

7

34

93

139

34.545.3

142

34.5

45

137

137468

413

152

295 1032

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a 

Key to Species 

Figure (Strain) 

41 Rat 

(CD) 

42 Rat 

(CD) 

43 Rat 

(CD) 

44 Rat 

(CD) 

45 Mouse 

(CD-1) 

46 Mouse 

(albinoSwiss) 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) Serious (mg/kg/day) 

13 wk ad lib (F) 

9.3 M 93 M (decreased fertility) 

4 or 13 wk ad lib (F) 

34 M 93 M (severe decrease in 

spermatogenesis) 

3 or 6 mo ad lib (F) 

34.5 M 45.3 F (decreased fertility; 

difficult parturition) 

3 or 6 mo ad lib (F) 

34.5 M 45 M (decreased fertility; 

severe atrophy/degeneration of seminiferous tubules) 

4 or 13 wk ad lib (F) 

137 M 468 F 

413 M (mild degeneration of 

seminiferous tubules) 

4 wk (C) 295 M 1032 M (decreased fertility index) 

Reference Chemical Form 

U.S. Army 1978b, Lee e1985 2,4-DNT U.S. Army 1978b, Lee e1985 2,4-DNT U.S. Army 1979 2,4-DNT U.S. Army 1979 2,4-DNT Hong et al. 1985; U.S. A1978b 2,4-DNT U.S. Army 1978b 2,4-DNT 
148

525

25

185

5.1 45.3

9

3.95.1

130

898

47

2727

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a Frequency Key to Species NOAEL Less Serious (Route) Figure (Strain) System (mg/kg/day) (mg/kg/day) 

47 Dog 4 or 13 wk 5 M 1 x/d (Beagle) 

(C) 25 F 

Developmental 

48 Rat 3 or 6 mo 5.1 F ad lib (CD) 

(F) 

CHRONIC EXPOSURE Death 

49 Rat 1-2 yr 

ad lib (CD) (F) 

50 Mouse 24 mo 

ad lib (CD-1) (F) 

Systemic 

51 Rat 52 wk Hepatic ad lib (Fischer- 344) 

(F) 

Bd Wt 

Serious 

(mg/kg/day) 

25 M (testicular degeneration, 

decreased spermatogenesis) 

45.3 F (difficult parturition) 

3.9 M 

5.1 F (decreased survival) 

898 (decreased survival) 

27 M (hepatocellular 

degeneration and vacuolation; basophilic and acidophilic foci of cellular alteration) 

27 M (25% body weight

decrease)

Reference Chemical Form 

Ellis et al. 1985; U.S. Ar1978b 2,4-DNT U.S. Army 1979 2,4-DNT U.S. Army 1978b; U.S. 1979; Lee et al. 1985 2,4-DNT U.S. Army 1979; Hong e1985 2,4-DNT Leonard et al. 1987 2,4-DNT 
80

88.8

2022

10

0.65.1

3.9 34.5

45.3

34.545.33.95.1

34.545.3

81

14.4

7215.2

76

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a Frequency Key to Species NOAEL Less Serious Serious (Route) Figure (Strain) System (mg/kg/day) (mg/kg/day) (mg/kg/day) 

52 Rat 78 wk Bd Wt 8 M 20 M ad lib (Fischer- 344) 

(F) 8.8 F 22 F (25% decrease body 

weight) 

53 Rat 1-2 yr Hemato 0.6 M 3.9 M (decreased RBC count) 34.5 M (anemia) ad lib (CD) 

(F) 5.1 F 45.3 F (anemia) 

Renal 34.5 M 

45.3 F 

Bd Wt 3.9 M 34.5 M (30% decrease body 

weight with decreased

5.1 F food consumption) 

45.3 F (27% decrease body 

weight with decreased food consumption) 

54 Mouse 78 wk Bd Wt 14.4 M 72 M (18% decrease in body 76 F (24% decrease in body ad lib (C57BL/6N) weight gain) weight gain) (F) 

15.2 F (11% decrease in body 

weight gain) 

Reference Chemical Form 

NCI 1978 2,4-DNT U.S. Army 1978b, 1979al. 1985 2,4-DNT NCI 1978 2,4-DNT 
132

95

898

95 14

89814

1495

95 898

144

0.2

1.5

1.5 1010

207

0.2

11

34.545.3

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to Species 

Figure (Strain) 

55 Mouse 

(CD-1) 

56 Dog 

(Beagle) 

57 Dog 

(Beagle) 

Neurological 

58 Rat 

(CD) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

24 mo ad lib (F) 24 mo 1 x/d (C) 12 mo 1 x/d (C) 1-2 yr ad lib (F) 

LOAEL 

NOAEL Less Serious Serious 

System (mg/kg/day) (mg/kg/day) (mg/kg/day) 

Hemato 95 898 (anemia; reticulocytosis; 

Heinz bodies) 

Hepatic 95 F 14 M (hepatocellular dysplasia) 

898 F (hepatocellular dysplasia) 

Renal 14 M (cystic dysplasia; toxic 

nephropathy) 

Bd Wt 14 M 95 M (16% decrease in body 898 F (20% decrease in body 

weight) weight)

95 F 

Hemato 0.2 1.5 (methemoglobinemia, 

anemia) 

Hepatic 1.5 ( 10 Biliary hyperplasia 

Renal 10 

d

Hemato 0.2 F (increased reticulocytes) 

34.5 M (wide-spread and 

stiff-legged gait) 

45.3 F (wide-spread and 

stiff-legged gait) 

Reference Chemical Form 

U.S. Army 1979; Hong e1985 2,4-DNT U.S. Army 1979, Ellis et 1985 2,4-DNT U.S. Army 1979, Ellis et 1985 2,4-DNT US Army 1978b, 1979; al. 1985 2,4-DNT 
131

95

898

146

0.2

1.5

202

3.9

34.5

12

34

134

95

14898

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a 

Key to Species 

Figure (Strain) 

59 Mouse 

(CD-1) 

60 Dog 

(Beagle) 

Reproductive 

61 Rat 

(CD) 

62 Rat 

(CD) 

63 Mouse 

(CD-1) 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) Serious (mg/kg/day) 

24 mo ad lib 95 898 (stiff-legged gait, hyperactivity) 

24 mo 1 x/d (C) 

0.2 1.5 (loss of hindquarter 

control, convulsions) 

1-2 yr ad lib (F) 

3.9 M 34.5 M Atophy of seminiferous 

tubules, aspermatogenesis 

1-2 yr ad lib (F) 

34 M 

24 mo ad lib 95 F 14 M (decreased spermatogenesis and 

degenerative change; testicular atrophy) 

898 F (ovarian atrophy; 

nonfunctioning follicles) 

Reference Chemical Form 

U.S. Army 1979; Hong e1985 2,4-DNT U.S. Army 1979, Ellis et 1985 2,4-DNT U.S. Army 1978b, 1979al. 1985 2,4-DNT U.S. Army 1978b; U.S. 1979; Lee et al. 1985 2,4-DNT U.S. Army 1979; Hong e1985 2,4-DNT 
145

10

78

7.522

13

34.545.3

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a Frequency Key to Species NOAEL Less Serious Serious (Route) Figure (Strain) System (mg/kg/day) (mg/kg/day) (mg/kg/day) 

64 Dog 24 mo 10 M 1 x/d (Beagle) 

(C) 

Cancer 

65 Rat 78 wk 7.5 M (CEL: skin and ad lib (Fischer- 344) subcutaneous fibroma) (F) 

22 F (CEL: mammary 

fibroadenoma) 

66 Rat 1-2 yr 34.5 M (CEL: hepatocellular ad lib (CD) carcinoma; mammary (F) and skin tumors) 

45.3 F (CEL: hepatocellular 

carcinoma, mammary and skin tumors) 

Reference Chemical Form 

U.S. Army 1979, Ellis et 1985 2,4-DNT NCI 1978 2,4-DNT U.S. Army 1978b, 1979al. 1985 2,4-DNT 
133

95

Table 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

67 Mouse 

(CD-1) 

24 mo ad lib U.S. Army 1979; Hong e

1985 2,4-DNT 

95 M (CEL: renal solid 

carcinoma, cystic papillary carcinoma and adenoma, cystic adenoma) 

a The number corresponds to entries in Figure 3-2. b Used to derive an acute-duration oral minimal risk level (MRL) of 0.05 mg/kg/day; the MRL was derived by dividing the NOAEL of 5 mg/kg/day by an uncertainty fanimal-to-human extrapolation and 10 for human variability). c Used to derive an intermediate-duration oral minimal risk level (MRL) of 0.007 mg/kg/day; the MRL was derived by dividing the BMDL1SD of 0.67 mg/kg/day by afactor of 100 (10 for animal-to-human extrapolation and 10 for human variability). d Used to derive a chronic-duration oral minimal risk level (MRL) of 0.001 mg/kg/day; the MRL was derived by dividing the BMDL1SD of 0.12 mg/kg/day by an unce100 (10 for animal-to-human extrapolation and 10 for human variability). ad lib = ad libitum; Bd Wt = body weight; (C) = capsule; Cardio = cardiovascular; CEL = cancer effect level; d = day(s); (F) = feed; F = Female; (G) = gavage; Gastrgastrointestinal; (GO) = gavage in oil; Hemato = hematological; LD50 = lethal dose, 50% kill; LDH = ; LOAEL = lowest-observed-adverse-effect level; M = male; mo NOAEL = no-observed-adverse-effect level; RBC = red blood cell; Resp = respiratory; SGOT = ; x = time(s); wk = week(s); yr = year(s) 
Death

RespiCardiGastrHemMuscHepatRenalDerm

a

mg/kg/day 

10000 

5m 6m 5m 

1000 

3r 3r 

7r 

1r 2r 4r 10r 10r 10r 10r 10r 10r 10r 10r

9r 11m 11m 9r

100 7r 10r 10r 10r 10r 9r 8r 7r 

9r 9r 

9r 

10 

1 

0.1 

0.01 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

ratory 

ovascular 

ointestinal 

uloskeletal 

i

DINITROTOLUENES 3. HEALTH EFFECTS

tological 

c 

al 

Figure 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral
	

Acute (≤14 days)
	

Systemic 

70
mg/kg/day Oc

BodImNeRe

10000 

1000 

7r 

10r 10r 8r 12r 12r 18m

7r 8r 15r

11m 9r 13r 16r

100 

17r 9r 16r 15r 

14d 

10 

14d 

1 

0.1 

0.01 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

ular 

y 

muno/Lymphor 

urological 

productive 

DINITROTOLUENES 3. HEALTH EFFECTS

Weight 

Figure 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (Continued)
 

Acute (≤14 days)
	

Systemic
	

71
Death 

HemHepatiRenal Body ImNeurologic

atolog

c 

Weig

muno/L

mg/kg/day 

10000 

1000 

22m 19r 30m 30m 26r 30m 26r 30m 30m 26r 30m 30m 26r 33r 37m 37m 35r 28r 28r 28r 

20r 30m 30m 28r 30m 30m 28r 28r 25r 36r

100 28r 36r 20r 28r 36r 25r 

21r 30m 29r 28r 28r 28r 28r 29r 36r

23d 31d 31d 31d 27r 34d 38d
	

10
	 24d 32d 32d 

31d 38d 

32d 32d 32d 

1 

32d 

0.1 

0.01 

0.001 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

al 

DINITROTOLUENES 3. HEALTH EFFECTS

ical 

ht 

ymphor 

Figure 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (Continued)

Intermediate (15-364 days) 

Systemic 

72
Reproductive

Development

mg/kg/day 

10000 

46m 

45m 

1000 

45m 40r 46m 

39r 45m 

100 41r 42r 39r 

42r 

47d 47d
	

10
	 41r 

47d 

1 

0.1 

0.01 

0.001 

43r 43r 44r 44r 48r 

48r 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

DINITROTOLUENES 3. HEALTH EFFECTS

al 

Figure 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (Continued)
 

Intermediate (15-364 days)
	

73
Deat

HepaRenaBodyNeurReprCanc

 

 

mg/kg/day Hem

1000 50m 55m 55m 55m 59m 63m 

100 55m 55m 55m 55m 59m 63m 67m 54m 54m 

53r 53r 53r 58r 66r 

53r 53r 53r 58r 61r 62r 66r

51r 51r 52r 65r 52r 

55m 55m 54m 54m 55m 63m 

10 56d 56d 64d 52r 

49r 53r 

49r 53r 

56d 56d 

1 

53r 

56d 57d 

0.1 

0.01 

0.001 

52r 65r 

53r
	 53r
	 61r 

60d 

60d 

*Doses represent the lowest dose tested per study that produced a tumorigenic 

response and do not imply the existence of a threshold for the cancer endpoint. 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

h 

ti

l 

ological 

oductive 

er * 

DINITROTOLUENES 3. HEALTH EFFECTS

atological 

c 

Weight 

Figure 3-2 Levels of Significant Exposure to 2,4-Dinitrotoluene - Oral (Continued)
 

Chronic (≥365 days)
	

Systemic 

74
228

308

206

710

204

1230

231

154

30839

308308308

229

308

Table 3-3 Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

ACUTE EXPOSURE Death 

1 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

System 

NOAEL 

(mg/kg/day) 

Less Serious 

(mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

308 M (1/6 died) 

Reference Chemical Form 

Lent et al. 2012a 2,5-DNT 

2 Rat 

(SpragueDawley) 

once (G) 710 M (LD50) Vernot et al. 1977 2,5-DNT 

3 Mouse 

(CF-1) 

Systemic 

4 Rat 

(SpragueDawley) 

once (G) 1 x/d 14 d (G) 

Cardio 154 M 308 M (increased absolute and 

relative heart weight; mild-to-moderate fibrosis and trace-to-moderate inflammation) 

1230 M (LD50) Vernot et al. 1977 

2,5-DNT Lent et al. 2012a 2,5-DNT 

Hemato 

b

39 M (dark spleen; mild to 

moderate extramedullary hematopoiesis) 

Hepatic 308 M Renal 308 M Bd Wt 308 M 

Neurological 

5 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

308 M Lent et al. 2012a 

2,5-DNT 
230

308

Table 3-3 Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

Reproductive 

6 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

308 M Lent et al. 2012a 

2,5-DNT 

a The number corresponds to entries in Figure 3-3. b Used to derive an acute-duration oral minimal risk level (MRL) for 2,5-DNT of 0.007 mg/kg/day; the MRL was derived by dividing the BMDL10 of 2.05 mg/kg/day bfactor of 300 (10 for animal-to-human extrapolation, 10 for human variability, and 3 for database limitations). Bd Wt = body weight; Cardio = cardiovascular; d = day(s); (G) = gavage; Hemato = hematological; LD50 = lethal dose, 50% kill; LOAEL = lowest-observed-adversemale; NOAEL = no-observed-adverse-effect level; x = time(s) 
Death 

CardioHematHepatiRenal Body NeurolReprod

c

W

mg/kg/day 

10000 

3m 1000 

2r 

1r 4r 4r 4r 4r 5r 6r 

4r 

100 

4r 

10 

1 

0.1 

0.01 

0.001 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

vascular ogical 

uctive 

DINITROTOLUENES 3. HEALTH EFFECTS

ological 

eight 

Figure 3-3 Levels of Significant Exposure to 2,5-Dinitrotoluene - Oral 

Acute (≤14 days) 

Systemic 

77
55

535795

17

180

56

621807

19

1000

197

19925

50

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

ACUTE EXPOSURE Death 

1 Rat 

(CD) 

once (GO) 

System 

NOAEL 

(mg/kg/day) 

Less Serious 

(mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

535 M (LD50) 795 F (LD50) 

Reference Chemical Form 

U.S. Army 1975; U.S. A1978a 2,6-DNT 

2 Rat 

(SpragueDawley) 

once (G) 180 M (LD50) Vernot et al. 1977 2,6-DNT 

3 Mouse 

(CD) 

once (GO) 621 M (LD50) 

807 F (LD50) 

U.S. Army 1975 2,6-DNT 

4 Mouse 

(CF-1) 

Systemic 

5 Rat 

(SpragueDawley) 

once (G) once (GO) Renal 199 F 

1000 M (LD50) Vernot et al. 1977 

2,6-DNT Deng et al. 2011 2,6-DNT 

Bd Wt 25 F 50 F Decreased body weight 

gain (1 to 4 g compared to 17 g for controls) 
235

134

68

14

35134

14

35

218

100

100

201

4

20

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

6 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

Cardio 134 M Lent et al. 2012a 

2,6-DNT 

Hemato 68 M (extramedullary 

hematopoiesis and lymphoid depletion of the spleen) 

Hepatic 14 M 35 M (hepatocellular 

hyperplasia, oval cell hyperplasia, and hepatocellular hypertrophy) 

Renal 134 M (proximal tubule 

degeneration and renal tubular basophilia) 

Bd Wt 14 M 35 M (terminal body weight 

10% lower than controls) 

7 Rat 

(SpragueDawley) 

1 x/d 3 d (G) 

Hepatic 100 M (diffuse hepatocellular 

hypertrophy) 

Rothfuss et al. 2010 2,6-DNT 

Bd Wt 100 M 

8 Dog 

(Beagle) 

2 wk ad lib (C) 

Hemato 

b

4 20 (decreased erythrocytes 

and increased mean cell hemoglobin) 

US Army 1976 2,6-DNT 
233

134

234

35

68

216

33

179

5155

174

20

51

7

14

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) System 

Exposure/ 

Duration/ 

Frequency 

(Route) NOAEL (mg/kg/day) 

LOAEL 

Less Serious 

(mg/kg/day) 

Serious 

(mg/kg/day) 

Reference Chemical Form 

Neurological 

9 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

Reproductive 

10 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

134 M 

35 M 68 M (tubular degeneration, 

multinucleated giant cell formation, and interstitial atrophy of the testes) 

Lent et al. 2012a 2,6-DNT Lent et al. 2012a 2,6-DNT 

INTERMEDIATE EXPOSURE Death 

11 Rat 

(SpragueDawley) 

1 x/d 29 d (G) 

33 M Rothfuss et al. 2010 

2,6-DNT 

12 Mouse 

(Swissalbino) 

4 or 13 wk ad lib (F) 

51 M (8/16 died) 55 F (6/16 died) 

US Army 1976 2,6-DNT 

13 Dog 

(Beagle) 

4 or 13 wk ad lib (C) 

Systemic 

14 Bd Wt Rat (Fischer- 344) 6 or 26 wk ad lib 

(F) 

7 M 

20 F (2/8 died) 14 M (20% decrease body 

weight) 

US Army 1976 2,6-DNT Leonard et al. 1987 2,6-DNT 
217

3333

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

15 Rat 

(SpragueDawley) 

1 x/d 29 d (G) 

Hepatic 33 M (hepatocellular 

hypertrophy, single cell necrosis, bile duct hyperplasia, focal hepatocellular vacuolation) 

Rothfuss et al. 2010 2,6-DNT 

Bd Wt 33 M (decreased body weight) 
171

7

3773537

1451557 35

37

145155

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

16 Rat 

(CD) 

4 or 13 wk ad lib (F) 

Hemato 7 F 37 F (splenic hemosiderosis; 

extramedullary hematopoiesis) 

U.S. Army 1976 2,6-DNT 

7 F Increased incidence of 

extramedullary hematopoiesis of the spleen at 4 weeks 

Hepatic 35 M (bile duct 

hyperplasia;hemosiderosi 

37 F (bile duct hyperplasia; 

hemosiderosis) 

Renal 145 M 

155 F 

Bd Wt 7 35 M 145 M 

37 F (decreased body weight 

gain) 

155 F (body weight loss) 
183

1111

5155

11 51

55

28929911 51

55

178

4

4

20

4

20

4

20

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

17 Mouse 

(Swissalbino) 

4 or 13 wk ad lib (F) 

Hemato 11 M 

11 F 

51 M (extramedullary 

hematopoiesis) 

US Army 1976 2,6-DNT 

55 F (extramedullary 

hematopoiesis) 

Hepatic Renal 

11 

289 M 

51 M (bile duct hyperplasia) 55 F (bile duct hyperplasia) 

299 F 

Bd Wt 11 51 M (weight loss) 

55 F (weight loss) 

18 Dog 

(Beagle) 

4 or 13 wk ad lib (C) 

Hemato 

c

4 (mild extramedullary 

erythropoiesis) 

US Army 1976 2,6-DNT 

Hepatic 4 20 (bile duct hyperplasia; 

degenerative and inflammatory liver changes) 

Renal 4 20 (dilated tubules, 

degenerative foci) 

Bd Wt 4 20 (body weight loss with 

decreased food consumption) 
169

145155

176

20 100

168

145155

180

289299

175

4

20

170

7155

35

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) System NOAEL (mg/kg/day) Less Serious (mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

Immuno/ Lymphoret 

19 Rat 

(CD) 

4 or 13 wk ad lib (F) 

145 M 155 F 

U.S. Army 1976 2,6-DNT 

20 Dog 

(Beagle) 

Neurological 

21 Rat 

(CD) 

4 or 13 wk ad lib (C) 4 or 13 wk ad lib (F) 

20 

145 M 155 F 

100 (thymic involution) US Army 1976 

2,6-DNT U.S. Army 1976 2,6-DNT 

22 Mouse 

(Swissalbino) 

4 or 13 wk ad lib (F) 

289 M 299 F 

US Army 1976 2,6-DNT 

23 Dog 

(Beagle) 

Reproductive 

24 Rat 

(CD) 

4 or 13 wk ad lib (C) 4 or 13 wk ad lib (F) 

4 7 M 

155 F 

20 (incoordination, lack of 

balance) 

35 M (decreased 

spermatogenesis; degeneration of testes) 

US Army 1976 2,6-DNT U.S. Army 1976 2,6-DNT 
182

11299

51

177

4100

20

48

7

7

45

7

Table 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (continued) Exposure/ LOAEL 

Duration/

a Frequency Key to Species NOAEL Less Serious Serious (Route) Figure (Strain) System (mg/kg/day) (mg/kg/day) (mg/kg/day) 

25 Mouse 4 or 13 wk 11 M 51 M (decreased ad lib (Swiss- spermatogenesis) albino) (F) 299 F 

26 Dog 4 or 13 wk 4 M 20 M (testicular degeneration) ad lib (Beagle) 

(C) 100 F 

CHRONIC EXPOSURE Systemic 

27 Rat 52 wk Hepatic 7 M (hepatocellular ad lib (Fischer- 344) degeneration, (F) vacuolation; acidophilic 

and basophilic foci of cellular alteration) 

Bd Wt 7 M (18% decrease body 

weight) 

Cancer 

28 Rat 52 wk 7 M (CEL: ad lib (Fischer- 344) cholangiocarcinoma, (F) hepatocellular 

carcinoma) 

Reference Chemical Form 

US Army 1976 2,6-DNT US Army 1976 2,6-DNT Leonard et al. 1987 2,6-DNT Leonard et al. 1987 2,6-DNT 

a The number corresponds to entries in Figure 3-4. b Used to derive an acute-duration oral minimal risk level (MRL) of 0.09 mg/kg/day; the MRL was derived by dividing the BMDL1SD of 9.31 mg/kg/day by an uncert(10 for animal-to-human extrapolation and 10 for human variability). c Used to derive an intermediate-duration oral minimal risk level (MRL) of 0.004 mg/kg/day; the MRL was derived by dividing the LOAEL of 4 mg/kg/day by an unce1000 (10 for use of a LOAEL, 10 for animal-to-human extrapolation and 10 for human variability). ad lib = ad libitum; Bd Wt = body weight; (C) = capsule; Cardio = cardiovascular; CEL = cancer effect level; d = day(s); (F) = feed; F = Female; (G) = gavage; (GO) Hemato = hematological; LD50 = lethal dose, 50% kill; LOAEL = lowest-observed-adverse-effect level; M = male; NOAEL = no-observed-adverse-effect level; x = tiweek(s) 
Death 

CardiovasculHemHepatiRenal Body Neurological Reproductive 

atological

c 

Weight 

mg/kg/day 

10000 

1000 4m3m 1r 

3m 1r 

5r 2r 

6r 6r 9r 

100 7r 7r 

6r 10r 

5r 

6r 6r 10r 

5r 8d 

6r 6r 

10 

8d 

1 

0.1 

0.01 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

ar 

DINITROTOLUENES 3. HEALTH EFFECTS

Figure 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral
	

Acute (≤14 days)
	

Systemic 

86
Death 

HemHepatiRenal Body ImNeurologReprodu

1000 

atolo

c 

Wei

muno/

mg/kg/day 

25m 

24r 

17m 17m 22m 22m

16r 16r 16r 16r 19r 19r 21r 21r

26d 

12m 

100 20d 

12m 17m 17m 17m 17m 17m 17m 25m

16r 16r 16r 16r 16r 24r 11r 15r 15r 

13d 18d 18d 18d 20d 23d 26d

14r 

17m 17m 17m 17m 25m 10 

24r 

18d 

16r 16r 14r 16r 

18d 18d 18d 23d 26d

1 

0.1 

0.01 

0.001 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

ical 

ctive 

DINITROTOLUENES 3. HEALTH EFFECTS

gical 

ght 

Lymphor 

Figure 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (Continued)
 

Intermediate (15-364 days)
	

Systemic
	

87
Hepati

Body Cancer * 

 

 

10 

c 

Weight 

Systemic
	

mg/kg/day 

27r 27r 28r 

*Doses represent the lowest dose tested per study that produced a tumorigenic 

response and do not imply the existence of a threshold for the cancer endpoint. 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

1 

DINITROTOLUENES 3. HEALTH EFFECTS

Figure 3-4 Levels of Significant Exposure to 2,6-Dinitrotoluene - Oral (Continued)
 

Chronic (≥365 days)
	

88
227

113

22757

227

227

113

227

225

113

227

226

227

Table 3-5 Levels of Significant Exposure to 3,4-Dinitrotoluene - Oral 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

ACUTE EXPOSURE Systemic 

1 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

System 

Cardio 

NOAEL 

(mg/kg/day) 

113 M 

Less Serious 

(mg/kg/day) 

227 M (myocardial fibrosis, 

inflammation, and necrosis) 

LOAEL 

Serious 

(mg/kg/day) 

Reference Chemical Form 

Lent et al. 2012a 3,4-DNT 

Hemato 

b

57 M (extramedullary 

hematopoiesis and lymphoid hyperplasia) 

Hepatic 227 M Renal 227 M (proximal tubule 

degeneration, renal tubule basophilia, and lymphocytic infiltration) 

Bd Wt 113 M 227 M (terminal body weight 

10% less than controls) 

Neurological 

2 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

113 M 227 M (facial twitching, 

hypoactivity, staring) 

Lent et al. 2012a 3,4-DNT 

Reproductive 

3 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

227 M Lent et al. 2012a 

3,4-DNT 

a The number corresponds to entries in Figure 3-5. b Used to derive an acute-duration oral minimal risk level (MRL) of 0.03 mg/kg/day; the MRL was derived by dividing the BMDL10 of 8.05 mg/kg/day by an uncertai(10 for animal-to-human extrapolation, 10 for human variability, and 3 for database limitations). Bd Wt = body weight; Cardio = cardiovascular; d = day(s); (G) = gavage; Hemato = hematological; LOAEL = lowest-observed-adverse-effect level; M = male; NOAEno-observed-adverse-effect level; x = time(s) 
Cardiovascular 

Hematological HepatiRenal Body Neurological Reproductive 

c 

Weight 

Systemic 

mg/kg/day 

1000 

1r 1r 1r 1r 2r 3r 

1r 1r 2r 100 

1r 

10 

1 

0.1 

0.01 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

DINITROTOLUENES 3. HEALTH EFFECTS

Figure 3-5 Levels of Significant Exposure to 3,4-Dinitrotoluene - Oral 

Acute (≤14 days) 

90
220

39

223

3939393939

221

19

39

222

10

19

Table 3-6 Levels of Significant Exposure to 3,5-Dinitrotoluene - Oral 

a 

Key to 

Figure 

Species 

(Strain) 

Exposure/ 

Duration/ 

Frequency 

(Route) 

ACUTE EXPOSURE Death 

1 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

System 

NOAEL 

(mg/kg/day) 

Less Serious 

(mg/kg/day) 

LOAEL 

Serious 

(mg/kg/day) 

39 M (1/6 died) 

Reference Chemical Form 

Lent et al. 2012a 3,5-DNT 

Systemic 

2 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

Cardio 39 M Lent et al. 2012a 

3,5-DNT 

Hemato 39 M Hepatic 39 M Renal 39 M Bd Wt 39 M 

Neurological 

3 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

19 M 39 M (facial twitching and 

paralysis; inflammatory infiltrates in the brain) 

Lent et al. 2012a 3,5-DNT 

Reproductive 

4 Rat 

(SpragueDawley) 

1 x/d 14 d (G) 

b

10 M 19 M (small testes; significantly 

reduced testes weight; tubular degeneration and multinucleated giant cell formation in the testes) 

Lent et al. 2012a 3,5-DNT 

a The number corresponds to entries in Figure 3-6. b Used to derive an acute-duration oral minimal risk level (MRL) 3,5-DNT of 0.03 mg/kg/day; the MRL was derived by dividing the NOAEL of 10 mg/kg/day by an un300 (10 for animal-to-human extrapolation, 10 for human variability, and 3 for database limitations). Bd Wt = body weight; Cardio = cardiovascular; d = day(s); (G) = gavage; Hemato = hematological; LOAEL = lowest-observed-adverse-effect level; M = male; NOAEno-observed-adverse-effect level; x = time(s) 
Death 

Cardiovascular Hematological HepatiRenal Body Neurological Reproductive 

c 

Weight 

Systemic 

mg/kg/day 

100 

1r 2r 2r 2r 2r 2r 3r 

3r 4r 

10 4r 

1 

0.1 

0.01 

c-Cat 

d-Dog

r-Rat 

p-Pig

q-Cow 

k-Monkey

m-Mouse 

h-Rabbit 

a-Sheep 

f-Ferret 

j-Pigeon

e-Gerbil 

s-Hamster 

g-Guinea Pig 

n-Mink 

o-Other

 Cancer Effect Level-Animals

 LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

 Cancer Effect Level-Humans

 LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

 LD50/LC50

Minimal Risk Level

 for effects

 other than

 Cancer 

DINITROTOLUENES 3. HEALTH EFFECTS

Figure 3-6 Levels of Significant Exposure to 3,5-Dinitrotoluene - Oral 

Acute (≤14 days) 

92
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 93 

3. HEALTH EFFECTS 

3.2.2.2 Systemic Effects 

The systemic effects observed after oral exposure of humans and animals to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT are discussed below. No studies were located regarding musculoskeletal effects in humans or 

animals after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

All LOAEL values from each reliable study for systemic effects in each species and duration category for 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, 

respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively. 

Respiratory Effects. No studies were located regarding respiratory effects in humans after oral 

exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

No histopathological effects on the lungs were found when Sprague-Dawley rats were fed 261 mg/kg/day 

(males) or 273 mg/kg/day (females) 2,4-DNT for 14 days (McGown et al. 1983). 

No respiratory system effects were observed when CDF rats were fed 14 mg/kg/day Tg-DNT for 2 years 

(Hazleton Laboratories 1982). Histopathological examination of the lungs and respiratory tract tissues of 

rats exposed to 14 mg/kg/day Tg-DNT for 2 years or 35 mg/kg/day for 1 year did not reveal any 

abnormalities (Hazleton Laboratories 1982). 

No studies were located regarding respiratory effects in animals after oral exposures to 2,3-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 

Cardiovascular Effects. No studies were located regarding cardiovascular effects in humans after 

oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Neither changes in heart weight nor microscopic lesions in the heart were observed in male SpragueDawley rats receiving gavage doses up to 275 mg/kg/day 2,3-DNT for 14 days (Lent et al. 2012a; 

USAPHC 2011a). 

No histopathological effects on the cardiovascular system were found after Sprague-Dawley rats received 

261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT in the diet for 14 days (McGown et al. 

1983). Neither changes in heart weight nor microscopic lesions in the heart were observed in male 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 94 

3. HEALTH EFFECTS 

Sprague-Dawley rats receiving gavage doses up to 142 mg/kg/day 2,4-DNT for 14 days (Lent et al. 

2012a; USAPHC 2011b). 

Increased absolute and relative heart weight, along with mild-to-moderate fibrosis in 4/5 animals and 

trace-to-moderate inflammation in 2/5 animals, were noted in male rats receiving 308 mg/kg/day 

2,5-DNT via gavage for 14 days (Lent et al. 2012a; USAPHC 2011c). 

One of six male Sprague-Dawley rats exposed to 227 mg/kg/day 3,4-DNT (the highest dose tested) 

exhibited cardiac lesions consisting of mild myocardial fibrosis, inflammation, and necrosis; heart weight 

was not affected at this dose or at the lower doses tested (Lent et al. 2012a; USAPHC 2011e). Exposure 

to 14 days of gavage doses up to 39 mg/kg/day 3,5-DNT did not result in changes in heart weight or 

microscopic lesions in the hearts of male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011f). 

At the 26-week interim sacrifice in a 104-week study in which CDF rats were fed 0, 3.5, 14, or 

35 mg/kg/day Tg-DNT in the diet, an increased incidence and severity of myocarditis was noted in males 

at 35 mg/kg/day (Hazleton Laboratories 1982). It was believed that this spontaneous inflammatory 

condition was exacerbated by ingestion of Tg-DNT in the high-dose animals. Although this condition 

was also observed at the 55-week sacrifice, it was not observed at 52 or 104 weeks. 

Gastrointestinal Effects. No studies were located regarding gastrointestinal effects in humans after 

oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

There were no histopathological effects on the gastrointestinal tract of Sprague-Dawley rats fed 

261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT in the diet for 14 days (McGown et al. 

1983). 

Treatment of rats with up to 35 mg/kg/day Tg-DNT for up to 1 year or 14 mg/kg/day for up to 2 years did 

not cause any histopathological changes in the gastrointestinal tract (Hazleton Laboratories 1982). 

No studies were located regarding gastrointestinal effects in animals after oral exposures to 2,3-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. 

Hematological Effects. No studies were located regarding hematological effects in humans after 

oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 95 

3. HEALTH EFFECTS 

Hematological effects were noted in virtually all animal studies of oral exposure to DNTs in which 

circulating blood was examined. The most common findings were methemoglobinemia, anemia, 

reticulocytosis, and an increase in Heinz bodies. The hematological effects are caused by oxidation of the 

iron in hemoglobin, producing methemoglobin. Heinz bodies are granules in erythrocytes that are 

believed to result from denatured hemoglobin. Reticulocytosis, a finding in many animals in these 

studies, is caused by the increased production of immature erythrocytes (red blood cells) and is seen as a 

compensatory mechanism in anemia resulting from exposure to 2,4- and 2,6-DNT. This hematotoxic 

syndrome is a common effect of exposure to aromatic amines and most organic and inorganic nitrates, 

and it has been implicated for many oxidizing agents (Smith 1996; U.S. Army 1979). 

When male Sprague-Dawley rats were treated for 14 days with gavage doses of 275 mg/kg/day 2,3-DNT, 

hematological effects included extramedullary hematopoiesis and lymphoid hyperplasia of the spleen and 

lymphoid depletion (Lent et al. 2012a; USAPHC 2011a). 

Female Sprague-Dawley rats (5/group) administered 2,4-DNT via gavage (in 5% v/v DMSO in corn oil) 

and observed for 24 or 48 hours after dosing showed evidence of erythrocytosis, as indicated by 

significant increases in hemoglobin, hematocrit, and/or erythrocyte and granulocyte counts, at doses 

≥99 mg/kg (Deng et al. 2011). Relative to controls, erythrocyte and granulocyte counts were increased by 

11 and 552% at 99 mg/kg after 24 hours; elevations in hemoglobin (27–31%) and hematocrit (29–33%) 

were only statistically significant in rats treated at 198 or 398 mg/kg and evaluated at 48 hours. No 

significant changes in hematological end points were observed in rats treated with 2,4-DNT at 5 or 

50 mg/kg and evaluated 24 or 48 hours after dosing. Development of erythrocytosis 48 hours following a 

single exposure to 2,4-DNT may be a secondary effect of dehydration, rather than a direct effect on the 

hematological system; however, no information on drinking water consumption was reported in this 

study. Slight cyanosis was observed in rats administered 60 mg/kg 2,4-DNT by gavage for 5 days (Lane 

et al. 1985). No changes in hematological parameters were found in Sprague-Dawley rats fed 

261 mg/kg/day (males) or 273 mg/kg/day (females) 2,4-DNT in the diet for 14 days (McGown et al. 

1983). In male rats receiving 2,4-DNT by gavage for 14 days, red blood cell count was significantly 

decreased at 142 mg/kg/day, while extramedullary hematopoiesis and lymphoid hyperplasia of spleen 

were seen at doses ≥71 mg/kg/day (Lent et al. 2012a; USAPHC 2011b). Hematocrit, percent 

reticulocytes, and relative spleen weight were not different from controls in C57Bl/6N mice exposed to 

134 mg/kg/day 2,4-DNT via gavage for 14 days (Wilbanks et al. 2014). Kozuka et al. (1979) found 

methemoglobin concentrations increased to 7 times those of controls in the blood of rats fed a time­
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 96 

3. HEALTH EFFECTS 

weighted average (TWA) dose of 371 mg/kg/day 2,4-DNT in the diet for 6 months. Anemia was 

observed in a 13-week feeding study in which male and female CD rats were fed 266 and 145 mg/kg/day, 

respectively, in the diet; milder effects, such as reticulocytosis and hemosiderosis or abnormal pigment in 

the spleen, were found at 93 and 108 mg/kg/day in males and females, respectively (Lee et al. 1985; U.S. 

Army 1978b). No hematological effects were observed in males and females administered 34 and 

38 mg/kg/day, respectively. Mild anemia (as indicated by decreases in erythrocyte count, hematocrit, or 

hemoglobin concentration) and concurrent reticulocytosis were also observed in male and female CD-1 

mice administered 413 and 468 mg/kg/day 2,4-DNT, respectively, in the diet for 13 weeks (Hong et al. 

1985; U.S. Army 1978b). Anemia, accompanied by the presence of Heinz bodies, was observed in beagle 

dogs given 25 mg/kg/day 2,4-DNT in capsules (Ellis et al. 1985; U.S. Army 1978b). 

As part of a 2-year study in Beagle dogs (6/sex/group) exposed to 2,4-DNT at doses of 0.2, 1.5, or 

10 mg/kg/day, effects on hematological parameters were evaluated after 3, 6, and 9 months of treatment 

(U.S. Army 1979). At these intermediate-duration timepoints, hematological effects consistent with 

development of methemoglobinemia, anemia, and compensatory hematopoiesis (including decreased 

hemoglobin, hematocrit, and erythrocyte counts, and increased serum methemoglobin and reticulocyte 

counts) were observed in beagle dogs administered oral 2,4-DNT at 1.5 or 10 mg/kg/day. In female dogs 

administered 10 mg/kg/day, statistically significant decreases in erythrocyte count, hematocrit, and 

hemoglobin, a statistically significant increase in reticulocyte count, and the presence of Heinz bodies in 

serum were observed. Similar hematological effects were observed in female dogs administered 0.2 and 

1.5 mg/kg/day, although effects did not reach statistical significance, most likely because the power of the 

study to detect statistically significant changes was compromised by the small number of dogs per 

treatment group. However, a clinically significant increase in methemoglobin levels of 225% was 

observed in female dogs administered 1.5 mg/kg/day; no significant hematological effects were observed 

at 0.2 mg/kg/day. Although effects at all time points were qualitatively similar, hematological changes 

observed after 9 months of exposure were more consistent and pronounced than those observed at the 

3- and 6-month time periods. 

Chronic studies of animals administered 2,4-DNT provide data that strengthen the weight-of-evidence 

supporting hematological effects. In a 24-month study, hematological effects consistent with 

development of methemoglobinemia, anemia, and compensatory hematopoiesis (including decreased 

hemoglobin, hematocrit, and erythrocyte counts, and increased serum methemoglobin and reticulocyte 

counts) were observed in beagle dogs (6/sex/group) administered oral 2,4-DNT at 1.5 or 10 mg/kg/day for 

12 months of continuous dosing (U.S. Army 1979). No significant hematological effects were observed 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 97 

3. HEALTH EFFECTS 

in dogs administered 2,4-DNT at 0.2 mg/kg/day. After treatment for 18 or 24 months, only slight or no 

anemia, near normal reticulocyte levels, no Heinz bodies, and minimal amounts of methemoglobin were 

detected, likely reflective of an adaptive response (Ellis et al. 1985; U.S. Army 1978b). 

In a 2-year study (with a 1-year interim sacrifice) in which CD rats were fed 0.6, 3.9, or 34.5 mg/kg/day 

(males) or 0.7, 5.1, or 45.3 mg/kg/day (females) 2,4-DNT, significant decreases in red blood cell count 

were found in mid-dose males compared to controls, and anemia (as indicated by further reductions in red 

blood cell count, decreased hematocrit, decreased hemoglobin, and a compensatory increase in 

reticulocytes) was found in high-dose animals after 1 year (Lee et al. 1985; U.S. Army 1978b, 1979). No 

changes in methemoglobin or Heinz bodies were found. CD-1 mice that were administered 14, 95, or 

898 mg/kg/day 2,4-DNT in the diet for 24 months were found to be anemic (as shown by significant 

reductions in erythrocytes and hemoglobin) at the high concentration, with compensatory increases in 

reticulocytes (Hong et al. 1985; U.S. Army 1979). 

Dark spleen and mild-to-moderate extramedullary hematopoiesis were observed in male Sprague-Dawley 

rats treated for 14 days with gavage doses ≥39 mg/kg/day 2,5-DNT (Lent et al. 2012a; USAPHC 2011c). 

Dose-related hematology changes were noted, including decreased red blood cells at ≥77 mg/kg/day; 

increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) at 

≥154 mg/kg/day; and increased red blood cell distribution width (at 77 and 154 mg/kg/day, but not at 

308 mg/kg/day). Total white blood cell count was increased at 308 mg/kg/day, resulting from a 

nonsignificant increase in lymphocytes; in addition, the percent of eosinophils was decreased at this dose 

(Lent et al. 2012a; USAPHC 2011c). 

Female Sprague-Dawley rats (5/group) administered 199 mg/kg 2,6-DNT as a single dose via gavage (in 

5% v/v DMSO in corn oil) showed evidence of erythrocytosis, as indicated by statistically significant 

increases in serum hemoglobin (45%), hematocrit (41%), and erythrocyte (61%) and granulocyte 

(11-fold) counts 48 hours after dosing (Deng et al. 2011). Increased numbers of reticulocytes (44% 

higher than controls), which were associated with mature erythrocytes containing Heinz bodies, were also 

observed in rats administered 199 mg/kg and evaluated at 24 hours. No significant changes in 

hematological end points were observed in rats administered 2,6-DNT at 5–99 mg/kg and evaluated 24 or 

48 hours after dosing. Development of erythrocytosis 48 hours following a single exposure to 2,6-DNT 

may be a secondary effect of dehydration, rather than a direct effect on the hematological system; 

however, no information on drinking water consumption was reported in this study. Lent et al. (2012a; 

USAPHC 2011d) reported hematology changes consisting of significantly reduced hemoglobin and 
 

 

 

 

 

 

 

 

DINITROTOLUENES 98 

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hematocrit, as well as increased neutrophils, increased monocytes, and increased percentages of 

neutrophils and lymphocytes, in male rats exposed by gavage to 134 mg/kg/day of 2,6-DNT. 

Nonsignificant decreases in hemoglobin and hematocrit were seen at all doses in this study (4– 

134 mg/kg/day). As part of a 13-week study in Beagle dogs (4/sex/group) exposed to 2,6-DNT at doses 

of 4, 20, or 100 mg/kg/day, effects on hematological parameters were evaluated after 2 weeks of 

treatment (U.S. Army 1976). Dogs treated at 20 mg/kg/day showed a statistically significant decrease in 

erythrocyte count (16%) and a significant increase in mean cell hemoglobin (5%) after dosing for 

2 weeks. At 100 mg/kg/day, more pronounced changes consistent with development of 

methemoglobinemia, anemia, and compensatory hematopoiesis were observed; dogs showed statistically 

significant reductions in erythrocyte count, hematocrit, and hemoglobin and an increase in reticulocyte 

count after 2 weeks of continuous dosing (U.S. Army 1976). 

Subchronic administration (13 weeks) of 2,6-DNT in dogs and rats provide data that strengthen the 

weight-of-evidence supporting hematological effects. Significant hematological effects were observed in 

beagle dogs (4/sex/group) after administration of 2,6-DNT at 20 and 100 mg/kg/day, but not 4 mg/kg/day 

(U.S. Army 1976). Dogs treated at 20 mg/kg/day showed a statistically significant decrease in 

erythrocyte count (12%) after dosing for 4 weeks. At 100 mg/kg/day, more pronounced changes 

consistent with development of methemoglobinemia, anemia, and compensatory hematopoiesis were 

observed; dogs showed statistically significant reductions in erythrocyte count, hematocrit, and 

hemoglobin and an increase in reticulocyte count after 4 weeks of continuous dosing. Histopathological 

evaluation of the spleen showed an increased incidence of extramedullary erythropoiesis in dogs treated 

with 2,6-DNT at 4, 20, or 100 mg/kg/day for 4 or 13 weeks; this effect is an adaptive response to 

2,6-DNT-induced methemoglobinemia and anemia. 

Subchronic (13-week) administration of 2,6-DNT in CD rats induced changes in hematological 

parameters (measured at 4, 8, and 13 weeks) indicative of anemia and compensatory hematopoiesis 

(including significant decreases in erythrocytes, hematocrit, and hemoglobin and increased reticulocytes) 

at the highest tested dose (145 and 155 mg/kg/day for male and female rats, respectively) only; these 

effects were most pronounced after treatment for 4 weeks (U.S. Army 1976). No significant 

hematological changes were observed at 7 and 35 mg/kg/day (males) or 7 and 37 mg/kg/day (females). 

However, histopathological effects (extramedullary hematopoiesis and/or splenic hemosiderosis), 

indicative of an adaptive response to anemia and compensatory erythropoiesis, were observed in male and 

female rats administered 2,6-DNT at doses ≥7 mg/kg/day. Although histopathological effects 

(extramedullary hematopoiesis) were observed in CD-1 mice administered 2,6-DNT at ≥51 mg/kg/day 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 99 

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(but not 11 mg/kg/day) for 4 or 13 weeks, no statistically significant changes in hematological parameters 

were seen at levels up to 289 mg/kg/day (males) or 299 mg/kg/day (females). The study authors indicated 

that some blood samples clotted, making hematological analyses impossible to perform. The small 

number of animals evaluated likely contributed to the identification of histopathological findings of the 

spleen in the apparent absence of 2,6-DNT-induced hematological effects. The 2,6-DNT isomer was not 

tested for hematological end points in studies of chronic duration. 

Fourteen-day gavage exposure of male Sprague-Dawley rats to 3,4-DNT resulted in reduced hemoglobin 

and hematocrit at all doses, with statistically significant reductions at doses of 14, 28, and 113 mg/kg/day; 

however, the authors indicated that both parameters remained within reference ranges at all doses (Lent et 

al. 2012a; USAPHC 2011e). Splenic lesions, of trace-to-mild severity, were observed at doses 

≥57 mg/kg/day, including extramedullary hematopoiesis and lymphoid hyperplasia. 

In the corresponding 14-day rat study of 3,5-DNT, Lent et al. (2012a; USAPHC 2011f) observed no 

changes in hematology parameters and no splenic lesions at doses up to 39 mg/kg/day. 

Hematological changes consistent with those observed in anemia were found in pregnant F344 rats 

administered 100 mg/kg Tg-DNT by gavage during gestation days 7–20 (Jones-Price et al. 1982). 

Administration of Tg-DNT to rats in the diet for 4 weeks (Hazleton Laboratories 1977) or 26 weeks 

(Hazleton Laboratories 1982) resulted in dose- and duration-related adverse effects on hematological 

parameters. In the 4-week study at 37.5 mg/kg/day, significant increases in reticulocytes and percentage 

of Heinz bodies were noted in both sexes and significant increases in methemoglobin levels were found in 

females; anemia was observed at 100 mg/kg/day in both sexes (Hazleton Laboratories 1977). Spleens of 

rats fed 150 mg/kg Tg-DNT for 30 days in the diet were altered in appearance; these alterations included 

discoloration, enlargement, and surface irregularity (Hazleton Laboratories 1977). An increased 

incidence of extramedullary hematopoiesis was noted in the splenic red pulp of male, but not female, rats 

fed 35 mg/kg/day Tg-DNT in the diet for 52 weeks (Hazleton Laboratories 1982). In rats sacrificed after 

26 weeks in a 24-month study, no effects on hematological parameters were observed at 14 mg/kg/day 

Tg-DNT. However, at 35 mg/kg/day, there were increases in reticulocytes and methemoglobin and 

decreases in red blood cells along with hemosiderosis and extramedullary hematopoiesis in males, and 

increases in MCV in females (Hazleton Laboratories 1982). After 1 year, slight-to-moderate myeloid and 

erythroid hyperplasia was noted in the bone marrow of most male rats treated with 35 mg/kg/day Tg-DNT 

(Hazleton Laboratories 1982). In a 24-month study in which Tg-DNT was administered to rats in the 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 100 

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diet, anemia was observed at 14 mg/kg/day in males but not in females; the NOAEL for this effect in 

males was 3.5 mg/kg/day Tg-DNT (Hazleton Laboratories 1982). 

The consistent observation of adverse hematological effects following exposure of laboratory animals to 

DNTs indicates that the blood is a primary target of DNT toxicity. As noted previously and shown in 

Table 3-7, the various DNT isomers elicited isomer-specific differences in selected hematological 

parameters following 14 days of gavage treatment of male Sprague-Dawley rats (Lent et al. 2012a; 

USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f). The 2,4- and 2,5-DNT isomers were the only 

ones causing decreases in red blood cell counts, with 2,5-DNT being more potent than 2,4-DNT. 

Increased spleen weight was observed at similar concentrations of 2,4-, 2,5-, and 2,6-DNT. Splenic 

lesions were observed after acute exposure to all DNT isomers except 3,5-DNT; the available data 

suggest that the 2,5-DNT isomer is more potent than the other isomers. 

Musculoskeletal Effects. No studies were located regarding musculoskeletal effects in humans after 

oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, or Tg-DNT or in animals after oral exposure to 2,3-, 2,5-, 

2,6-, 3,4-, 3,5-, or Tg-DNT. Swim-to-exhaustion time was decreased by 82% (relative to controls) in 

C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days (Wilbanks et al. 2014). 

Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

The hepatotoxic effects of DNTs have been consistently observed in animals. The liver appears to be a 

target organ of DNT toxicity, particularly when administered to rats, but hepatotoxic effects have also 

been observed in mice and dogs. Hepatic effects of DNTs include liver discoloration and inflammation, 

alteration of hepatocytes, proliferation of bile duct epithelium, and hyperplastic foci. However, as 

discussed in Section 3.2.2.7 (Cancer), 2,4-, 2,6-, and Tg-DNT have been shown to induce hepatocellular 

carcinoma following chronic-duration oral exposure. Thus, hepatic effects observed at less-than-chronic 

exposure durations or at lower doses may represent early stages of progressive development to hepatic 

cancer. 

In male Sprague-Dawley rats receiving gavage doses of 2,3-DNT for 14 days, mean relative (but not 

absolute) liver weight was increased at 275 mg/kg/day, but this increase was likely attributable to lower 

body weight at this dose (Lent et al. 2012a; USAPHC 2011a). Dose-related clinical chemistry changes 

were not observed, and no histopathology lesions were seen in the liver. 
 

 

DINITROTOLUENES 101 

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Table 3-7. NOAELs and LOAELs for Hematological Effects Following 14-Day 

Gavage Dosing of Male Sprague-Dawley Rats with Individual DNT Isomers
 

Decreased red blood Increased spleen Increased incidence 

Effect cell count weight splenic lesions 

Effect level (mg/kg/day) NOAEL LOAEL NOAEL LOAEL NOAEL LOAEL 

Isomer 

2,3-DNT 275 – 275 – 138 275 

2,4-DNT 71 142 36 71 36 71 

2,5-DNT 39 77 39 77 19 39 

2,6-DNT 134 – 35 68 35 68 

3,4-DNT 227 – 227 – 28 57 

3,5-DNT 39 – 39 – 39 – 

DNT = dinitrotoluene; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level 

Sources: Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 102 

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In female Sprague-Dawley rats (5/group) administered 2,4-DNT via gavage (in 5% v/v DMSO in corn 

oil) and observed for 48 hours, significantly decreased levels of serum albumin (13–51% lower than 

controls) were observed at doses ≥99 mg/kg (Deng et al. 2011). Although relative liver weight was also 

significantly increased at 99 mg/kg, this effect was not observed at 198 or 398 mg/kg. Hepatic sinusoid 

congestion was observed in rats administered 398 mg/kg 2,4-DNT in the absence of other 

histopathological effects. Significantly increased (14% higher than controls) relative liver weight was 

observed in wild type C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days; serum 

triglycerides and glucose levels were not affected (Wilbanks et al. 2014). Increased blood cholesterol was 

found in male and female Sprague-Dawley rats fed 78 or 82 mg/kg/day 2,4-DNT, respectively, in the diet 

for 14 days, and increased alanine aminotransferase levels were found in males (McGown et al. 1983). 

Blood glucose levels trended upward in all male and female groups in this study, but were increased 

significantly only in females fed 273 mg/kg/day. Trace-to-mild single cell necrosis occurred in the livers 

of male Sprague-Dawley rats receiving gavage doses of 18, 36, 71, and 142 mg/kg/day 2,4-DNT; vehicle 

controls and rats exposed to 9 mg/kg/day did not exhibit this effect (Lent et al. 2012a; USAPHC 2011b). 

In addition, the authors observed trace apoptosis in the livers of 1/6 rats in each of the 36 and 

71 mg/kg/day groups, and increases in glycogen deposition at ≥36 mg/kg/day. No clinical chemistry 

changes were seen at any dose. 

Oral administration of 2,4-DNT for 13 weeks to rats (266 or 145 mg/kg/day in males and females, 

respectively) and dogs (25 mg/kg/day) did not result in liver toxicity (Ellis et al. 1985; U.S. Army 1978b). 

After 26 weeks of treatment, rats fed 27 mg/kg/day in the diet had significant increases in epoxide 

hydrolase (EH) activity, which is sometimes considered to be a phenotypic marker of neoplastic nodules; 

however, hepatocellular lesions did not develop in these animals when treatment was carried through 

52 weeks (Leonard et al. 1987). Mild hepatocellular dysplasia was observed in mice fed 137 mg/kg/day 

(males) or 468 mg/kg/day (females) of 2,4-DNT for 13 weeks (Hong et al. 1985; U.S. Army 1978b). 

Hepatic effects have also been observed in laboratory animals following chronic-duration oral exposure to 

DNTs. However, these effects are often observed in conjunction with the development of hepatocellular 

carcinoma and may represent precancerous changes. For example, dietary exposure of male rats to 

0.6 mg/kg/day 2,4-DNT induced “hepatocellular” alterations; however, neoplastic nodules were noted at 

this dose level and higher as well (Lee et al. 1985). Hepatocellular degeneration and vacuolation 

accompanied by acidophilic foci and occasional basophilic foci of cellular alteration were found in F344 

rats fed 27 mg/kg/day 2,4-DNT for 52 weeks (Leonard et al. 1987). The incidences of focal areas of 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 103 

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alteration were less in the 2,4-DNT-treated rats than they were in rats similarly treated with 2,6-DNT or 

Tg-DNT. Wistar rats fed a TWA dose of 371 mg/kg/day 2,4-DNT in the diet for 6 months had increased 

relative liver weights, formation of puruloid matter, and increased levels of serum glutamic-oxaloacetic 

transaminase (SGOT), lactate dehydrogenase (LDH), alkaline and acid phosphatase, triglycerides, and 

blood glucose levels compared to controls (Kozuka et al. 1979). In this study, the levels of serum 

albumin and the albumin/globulin ratios were decreased. Hepatocellular dysplasia was found in male and 

female CD-1 mice fed 14 or 898 mg/kg/day 2,4-DNT, respectively, for 24 months (Hong et al. 1985; U.S. 

Army 1979). Administration of 10 mg/kg/day 2,4-DNT for 24 months resulted in biliary hyperplasia in 

dogs; this effect was not seen in dogs administered 1.5 mg/kg/day (Ellis et al. 1985; U.S. Army 1979). 

When 2,5-DNT was administered to male Sprague-Dawley rats via gavage for 2 weeks at doses up to 

308 mg/kg/day, there were no treatment-related adverse effects on serum liver enzyme activity, liver 

weight, or liver histology (Lent et al. 2012a; USAPHC 2011c). 

Increased ALT activity and histopathological changes to the liver were observed in individual female 

Sprague-Dawley rats administered 50 and 99 mg/kg 2,6-DNT (in 5% DMSO in corn oil) by gavage and 

observed for 48 hours (Deng et al. 2011). Serum ALT activity was significantly increased 7-fold 

compared to controls in rats administered 50 and 99 mg/kg 2,6-DNT, but no increase in serum ALT level 

was observed at lower dose levels (5–25 mg/kg). Histopathological changes to the liver observed in rats 

administered 2,6-DNT at 50 or 99 mg/kg included congested sinusoids with sloughed hepatocytes and 

segmented neutrophils, disorganized midzonal regions characterized by infiltration of erythrocytes and 

hepatocytes with pyknotic nuclei and microvesiculated cytoplasm, and apoptotic hepatocytes (severity of 

effects not specified). ALT levels were similar to controls and hepatocytes were mostly undamaged in 

rats administered 2,6-DNT at 199 mg/kg, although evidence of sinusoid congestion, occasional 

erythrocyte infiltration (with no signs of necrosis), and enlarged nuclei in several hepatocytes were noted. 

Three consecutive daily oral doses of 100 mg/kg/day 2,6-DNT resulted in mild diffuse hepatocellular 

hypertrophy in male Sprague-Dawley rats (Rothfuss et al. 2010). After 2 weeks of gavage doses of 68 or 

134 mg/kg/day 2,6-DNT, male rats exhibited significant increases in serum ALT and aspartate 

aminotransferase (AST), as well as a nonsignificant but dose-related increase in alkaline phosphatase 

(ALP) (Lent et al. 2012a; USAPHC 2011d). Liver lesions consisting of hepatocellular and oval cell 

hyperplasia and hepatocellular hypertrophy occurred at doses ≥35 mg/kg/day. Additional lesions 

(increases in mitotic activity, single cell necrosis, and karyocytomegaly) occurred at higher doses of 68 

and 134 mg/kg/day (Lent et al. 2012a; USAPHC 2011d). 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 104 

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Intermediate duration exposure to 2,6-DNT also results in liver toxicity. After oral exposure to 

33 mg/kg/day 2,6-DNT for 29 consecutive days, male rat livers exhibited hepatocellular hypertrophy, 

single cell necrosis, bile duct hyperplasia, and focal hepatocellular vacuolation, in the absence of changes 

in serum liver enzyme levels (Rothfuss et al. 2010). Six weeks of dietary consumption of 7 mg/kg/day 

2,6-DNT caused a 380% increase in EH levels in rats but did not increase the level of DT-diaphorase 

(DTD) (Leonard et al. 1987). In the same study, both of these enzymes were elevated after 6 weeks of 

treatment with 14 mg/kg/day of 2,6-DNT. Dosing of rats, mice, and dogs with 2,6-DNT for 13 weeks 

resulted in liver toxicity (U.S. Army 1976). Bile duct hyperplasia was observed in rats fed 35 mg/kg/day 

and mice fed 51 mg/kg/day 2,6-DNT for 13 weeks (U.S. Army 1976). Liver degeneration and bile duct 

hyperplasia were observed in dogs dosed with 20 mg/kg/day 2,6-DNT but were not seen in dogs dosed 

with 4 mg/kg/day (U.S. Army 1976). After 52 weeks of treatment with 7 mg/kg/day 2,6-DNT, 

hepatocellular degeneration and vacuolation accompanied by acidophilic and basophilic foci of cellular 

alteration were found in F344 rats (Leonard et al. 1987). 

Gavage doses up to 227 mg/kg/day 3,4-DNT did not alter clinical chemistry or liver histology in male 

Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011e). Significant increases in relative, but not 

absolute liver weight were noted at 113 and 227 mg/kg/day, but were likely attributable to reduced body 

weights at these doses. 

In the 14-day gavage study of rats by Lent et al. (2012a; USAPHC 2011f), no exposure-related changes in 

serum liver enzymes, liver weight, or histopathology were seen at doses up to 39 mg/kg/day of 3,5-DNT. 

Irregular liver surfaces were found in male F344 rats fed 37.5 mg/kg/day Tg-DNT for 30 days (Hazleton 

Laboratories 1977). Hepatocytic necrosis, nonsupportive pericholangitis, and periportal megalocytosis 

were found in CDF rats fed 14 mg/kg/day for 26 weeks, and when treatment of these animals was 

extended to 2 years, slight-to-severe biliary cirrhosis was found in males (Hazleton Laboratories 1982). It 

has been suggested that this latter lesion may be a precursor to cholangiocarcinoma (Hazleton 

Laboratories 1982). Hepatocytic degeneration and acidophilic and basophilic foci of cellular alteration 

were observed in F344 rats fed 35 mg/kg/day Tg-DNT in the diet for 52 weeks (Leonard et al. 1987). 

When administration of Tg-DNT was continued for 24 months, liver discoloration resulted at 

3.5 mg/kg/day and liver nodules and malignancies at 14 mg/kg/day (Hazleton Laboratories 1982). 

As shown in Table 3-8, 2,4- and 2,6-DNT were the only isomers that elicited hepatic effects in SpragueDawley rats following 14 days of gavage treatment (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 
 

 

DINITROTOLUENES 105 

3. HEALTH EFFECTS 

Table 3-8. NOAELs and LOAELs for Hepatic Effects Following 14-Day Gavage 

Dosing of Male Sprague-Dawley Rats with Individual DNT Isomers
 

Increased serum liver Increased incidence 

Effect Increased liver weight enzyme activity hepatic lesions 

Effect level (mg/kg/day) NOAEL LOAEL NOAEL LOAEL NOAEL LOAEL 

Isomer 

2,3-DNT 275 – 275 – 275 – 

2,4-DNT 71 142 142 – 18 36 

2,5-DNT 308 – 308 – 308 – 

2,6-DNT 134 – 35 68 14 35 

3,4-DNT 227 – 227 – 227 – 

3,5-DNT 39 – 39 – 39 – 

DNT = dinitrotoluene; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level 

Sources: Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 106 

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2011d, 2011e, 2011f). The 2,4-DNT isomer caused increased liver weight (LOAEL=142 mg/kg/day) and 

increased incidences of single cell necrosis and glycogen deposition (LOAEL=36 mg/kg/day). The 

2,6-DNT isomer caused increased serum liver enzyme activity (LOAEL=68 mg/kg/day) and increased 

incidences of hepatocellular hypertrophy and hyperplasia and oval cell hyperplasia 

(LOAEL=35 mg/kg/day). 

Renal Effects. No studies were located regarding renal effects in humans after oral exposure to 2,3-, 

2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Exposure to 2,3-DNT by daily gavage for 14 days did not result in serum chemistry changes indicative of 

renal toxicity, or changes in kidney weight in male rats; however, increased incidences of renal tubular 

dilatation (2/6 rats) and renal lymphocytic infiltration (4/6) were observed (Lent et al. 2012a; USAPHC 

2011a). 

Sprague-Dawley rats (5/group) administered 2,4-DNT (in 5% DMSO in corn oil) via gavage at doses up 

to 398 mg/kg and evaluated at 24 or 48 hours showed no effects on levels of serum creatinine or urea 

(Deng et al. 2011). Hyaline droplet accumulation in the epithelium of the proximal convoluted tubule 

was found in both sexes of Sprague-Dawley rats after they were administered 78, 104, 165, or 

261 mg/kg/day 2,4-DNT (males) or 82, 109, 173, or 273 mg/kg/day 2,4-DNT (females) in the diet 

(McGown et al. 1983). Although this effect was observed at all concentrations, there was no dose 

response evident. The kidney was not a target organ of 2,4-DNT in male rats exposed via gavage for 

14 days at doses up to 142 mg/kg/day; serum chemistry and kidney weight were not affected by exposure, 

nor were there histopathology findings in the kidney (Lent et al. 2012a; USAPHC 2011b). Oral 

administration of 2,4-DNT to mice (413 mg/kg/day), rats (145 mg/kg/day), and dogs (25 mg/kg/day) for 

13 weeks did not result in significant adverse effects in the kidney (Hong et al. 1985; U.S. Army 1978b). 

Treatment of the same species for 24 months resulted in renal dysplasia in male mice at a dose of 

14 mg/kg/day of 2,4-DNT, but no renal effects were observed in rats or dogs dosed with 34.5 mg/kg/day 

or 10 mg/kg/day, respectively (U.S. Army 1979). Adverse effects in the kidneys of mice included cystic 

dysplasia in the tubular epithelium, atypical epithelium lining the cysts, and a variety of tumors (Hong et 

al. 1985). These effects were more pronounced in male mice than in female mice. 

Exposure to 2,5-DNT for 14 days at gavage doses up to 308 mg/kg/day did not result in kidney effects in 

male rats, as assessed by serum chemistry, kidney weight, and histopathology (Lent et al. 2012a; 

USAPHC 2011c). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 107 

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Renal tubular degeneration and proximal tubule degeneration were observed in male rats exposed by 

gavage to 2,6-DNT at 134 mg/kg/day (the highest dose tested) for 14 days (Lent et al. 2012a; USAPHC 

2011d). 

Female Sprague-Dawley rats (5/group) administered 2,6-DNT (in 5% DMSO in corn oil) via gavage at 

doses up to 199 mg/kg showed no effects on levels of serum creatinine or urea at 24 or 48 hours (Deng et 

al. 2011). Dosing of dogs with 20 mg/kg/day 2,6-DNT for 13 weeks resulted in dilated tubules, foci of 

inflammation, and degeneration of the kidney (U.S. Army 1976). No treatment-related effects on the 

kidney were found when rats were fed 2,6-DNT for 13 weeks (U.S. Army 1976). The severe renal effects 

observed after 2,4-DNT administration in mice were not observed when mice were fed 289 mg/kg/day 

2,6-DNT for 13 weeks (U.S. Army 1976). However, the renal toxicity of 2,4-DNT in mice was observed 

only after chronic administration. Chronic studies of 2,6-DNT have not been performed in mice. 

After 26 or 52 weeks of dietary consumption of 35 mg/kg/day Tg-DNT, BUN levels were significantly 

increased in CDF rats (Hazleton Laboratories 1982). Exacerbation of chronic interstitial nephritis that 

was also observed in controls was observed at 14 mg/kg/day Tg-DNT in a chronic study in rats (Hazleton 

Laboratories 1982). 

Renal effects were observed in male rats exposed to 3,4-DNT by gavage for 14 consecutive days (Lent et 

al. 2012a; USAPHC 2011e). These effects consisted of proximal tubule degeneration, renal tubule 

basophilia, and lymphocytic infiltration at the highest dose tested (227 mg/kg/day); however, renal 

histopathology was not examined at lower doses. 

In the corresponding study of 3,5-DNT (Lent et al. 2012a; USAPHC 2011f), no effects on kidney weight, 

kidney histopathology, or serum parameters indicative of renal toxicity were seen in male rats exposed by 

gavage to doses up to 39 mg/kg/day for 14 days. 

The kidney does not appear to be a sensitive target of DNT toxicity for all species tested. Severe renal 

effects were observed only in CD-1 mice fed 14 mg/kg/day 2,4-DNT for 24 months, and less severe renal 

effects were observed in dogs administered 20 mg/kg/day 2,6-DNT for 13 weeks. 

In the 14-day gavage studies that compared the effects of the various DNT isomers on the kidneys of male 

Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f), only the 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 108 

3. HEALTH EFFECTS 

2,3-, 2,6-, and 3,4-DNT elicited adverse effects. Increased kidney weight accompanied by increased 

incidences of histopathologic lesions (trace tubular dilatation and lymphocytic infiltration) were observed 

for 2,3-DNT (LOAEL=275 mg/kg/day). Increased kidney weight (LOAEL=68 mg/kg/day) and increased 

incidences of proximal tubule degeneration and renal tubular basophilia were observed for 2,6-DNT 

(LOAEL=134 mg/kg/day). Increased incidences of proximal tubule degeneration, renal tubule 

basophilia, and lymphocytic infiltration were noted for 3,4-DNT (LOAEL=227 mg/kg/day). 

Endocrine Effects. No studies were located regarding endocrine effects in humans after oral 

exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Administration of 2,4-DNT in the diet for 14 days, at 78 mg/kg/day for males or 82 mg/kg/day for 

females did not cause any histopathological changes in adrenal, pituitary, or thyroid glands of SpragueDawley rats (McGown et al. 1983). 

No histopathological effects on adrenal, pituitary, or thyroid glands were found in rats treated with 

14 mg/kg/day Tg-DNT for up to 2 years or 35 mg/kg/day for 1 year (Hazleton Laboratories 1982). 

Increases in the incidence and severity of parathyroid hyperplasia (males) and increases in the incidence 

and severity of fatty metamorphosis and vascular ectasia (males and females) were found in rats fed 

14 mg/kg/day Tg-DNT in the diet in a chronic study (Hazleton Laboratories 1982). 

No studies were located regarding endocrine effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, 

or 3,5-DNT. 

Dermal Effects. No studies were located regarding dermal effects in humans after oral exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Concentrations of up to 261 mg/kg/day 2,4-DNT for males or 273 mg/kg/day 2,4-DNT for females 

administered in the diet for 14 days to Sprague-Dawley rats caused no histopathological changes in their 

skin (McGown et al. 1983). 

No effects were found on the skin of rats treated for up to 2 years with 14 mg/kg/day Tg-DNT or up to 

1 year with 35 mg/kg/day Tg-DNT (Hazleton Laboratories 1982). 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 109 

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No studies were located regarding dermal effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT. 

Ocular Effects. No studies were located regarding ocular effects in humans after oral exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

The eyes of male and female Sprague-Dawley rats administered up to 261 or 273 mg/kg/day 2,4-DNT, 

respectively, in the diet for 14 days did not exhibit any alterations upon histopathological examination 

(McGown et al. 1983). 

No effects were found on the eyes of rats treated for up to 2 years with 14 mg/kg/day Tg-DNT in feed or 

up to 1 year with 35 mg/kg/day Tg-DNT in feed (Hazleton Laboratories 1982). 

No studies were located regarding ocular effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT. 

Body Weight Effects. No studies were located regarding body weight effects in humans after oral 

exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Adverse effects on body weight and body weight gain in rats, mice, and dogs were observed after oral 

administration of 2,4-DNT, 2,6-DNT, and Tg-DNT, and in rats exposed to 3,4-DNT. In most of these 

studies, a concurrent decrease in food consumption was also observed. Because exposure resulted from 

intake of the test article in feed in most of these studies, it is possible that some of the body weight 

changes resulted from unpalatability. 

Body weight was not altered in male rats receiving doses up to 275 mg/kg/day 2,3-DNT by gavage for 

14 days (Lent et al. 2012a; USAPHC 2011a). 

Adverse effects on body weight, including body weight loss, have been reported after almost all acute-, 

intermediate-, and chronic-duration oral administration of 2,4-DNT (Bloch et al. 1988; Deng et al. 2011; 

Ellis et al. 1985; Hazleton Laboratories 1982; Hong et al. 1985; Kozuka et al. 1979; Lane et al. 1985; Lee 

et al. 1985; Lent et al. 2012a; USAPHC 2011b; Leonard et al. 1987; McGown et al. 1983; NCI 1978; U.S. 

Army 1978b, 1979). Female Sprague-Dawley rats (5/group) administered 2,4-DNT (in 5% DMSO in 

corn oil) via gavage at 398 mg/kg showed decreased body weight gain (1–4 g compared to 17 g for 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 110 

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controls) 48 hours after dosing (Deng et al. 2011). In another acute study, rats dosed by gavage with 

240 mg/kg 2,4-DNT for 5 days lost weight (Lane et al. 1985). Lent et al. (2012a; USAPHC 2011b) 

reported a 12% decrease in body weight (relative to controls) in male rats given 2,4-DNT by gavage for 

14 days at a dose of 142 mg/kg/day. Wilbanks et al. (2014) observed greater body weight loss 

(0.96 versus 0.35 g), compared with untreated controls, in wild type C57Bl/6N mice exposed to 

134 mg/kg/day 2,4-DNT via gavage for 14 days. In general, there were losses of 10–40% in body weight 

in acute-, intermediate-, and chronic-duration studies in rats. After 6 months, decreases in body weight 

gain were noted in rats fed 27 mg/kg/day 2,4-DNT (Leonard et al. 1987), and a 25% decrease in body 

weight was seen in rats fed 34.5 mg/kg/day (Lee et al. 1985; U.S. Army 1978b, 1979). This reduction in 

body weight gain tended to become more pronounced when 2,4-DNT was continued for periods of 1– 

2 years (Leonard et al. 1987). Body weight was decreased 25% in rats that received 20 mg/kg/day 

2,4-DNT in the diet for 78 weeks; the NOAEL in this study was 8 mg/kg/day (NCI 1978). Mice showed 

similar decreases in body weight after intermediate- (13 weeks) and chronic-duration (78–104 weeks) 

exposure, but the doses of the test article needed to evoke this effect were considerably higher than in rats 

(Hong et al. 1985; NCI 1978; U.S. Army 1978b). An 18–24% decrease in body weight was seen in rats 

receiving 72–76 mg/kg/day 2,4-DNT in the diet for 78 weeks (NCI 1978). 

In a 14-day study of 2,5-DNT, body weight was not altered in male rats receiving doses up to 

308 mg/kg/day (Lent et al. 2012a; USAPHC 2011c). 

Female Sprague-Dawley rats (5/group) administered 2,6-DNT (in 5% DMSO in corn oil) via gavage at 

50 or 99 mg/kg gained less weight than control animals over the 48-hour observation period (1–4 g 

compared to 17 g for controls); rats from the same study administered 2,6-DNT at 199 mg/kg lost weight 

over the course of 48 hours (Deng et al. 2011). Body weight reductions of at least 10% were observed in 

male rats receiving gavage doses ≥35 mg/kg/day for 14 days, although the reductions were statistically 

significant only at the highest dose of 134 mg/kg/day (Lent et al. 2012a; USAPHC 2011d). Reduced 

body weight was reported in male Sprague-Dawley rats exposed to 29 consecutive daily oral doses of 

33 mg/kg/day 2,6-DNT (Rothfuss et al. 2010). Administration of 2,6-DNT also caused decreased body 

weight gain or body weight loss in rats, mice, and dogs at doses ranging from 14 to 145 mg/kg/day in 

intermediate-duration studies (U.S. Army 1976). Treatment with 7 mg/kg/day 2,6-DNT for 52 weeks 

decreased body weight in rats by 18% (Leonard et al. 1987). 
 

 

 

 

 

 

DINITROTOLUENES 111 

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A 10% reduction in terminal body weight was seen in male rats exposed to 227 mg/kg/day 3,4-DNT by 

gavage for 14 days; however, the difference from control weight was not statistically significant (Lent et 

al. 2012a; USAPHC 2011e). 

Body weight was not altered in male rats receiving gavage doses up to 39 mg/kg/day 3,5-DNT for 

14 days (Lent et al. 2012a; USAPHC 2011f). 

A 29% decrease in absolute maternal weight gain was observed in dams fed 14 mg/kg/day Tg-DNT for 

14 days during gestation (Jones-Price et al. 1982). Decreased body weight or decreased body weight gain 

was reported in rats at levels as low as 14 mg/kg/day Tg-DNT in intermediate- or chronic-duration studies 

(Hazleton Laboratories 1982). Other intermediate- and chronic-duration studies also confirmed these 

body weight effects (Hazleton Laboratories 1977; Leonard et al. 1987; NCI 1978). 

Metabolic Effects. No studies were located regarding metabolic effects in humans after oral exposure 

to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Female Sprague-Dawley rats (5/group) administered 2,4-DNT (at 198 or 398 mg/kg) or 2,6-DNT (at 

199 mg/kg) via gavage (in 5% DMSO in corn oil) showed small but statistically significant reductions in 

sodium levels in the serum (4–11% lower than controls) 24 and/or 48 hours after dosing (Deng et al. 

2011). Rats treated with 2,4-DNT at 398 mg/kg also had elevated levels of blood glucose (increased 

2.6-fold) 48 hours after dosing. 

No studies were located regarding metabolic effects in animals after oral exposure to 2,3-, 2,5-, 3,4-, or 

3,5-DNT. 

3.2.2.3 Immunological and Lymphoreticular Effects 

No studies were located regarding immunological and lymphoreticular effects in humans after oral 

exposure to 2,3-, 2,4-, 2,5-, 2,6-, or 3,5-DNT. 

Testing for immunological effects of DNTs is limited. Acute (14-day) exposure of male rats to 2,3-DNT 

resulted in lymphoid hyperplasia of the spleen and lymphoid depletion at gavage doses of 275 mg/kg/day 

(Lent et al. 2012a; USAPHC 2011a). In the corresponding study of 2,4-DNT, lymphoid hyperplasia was 

seen at doses ≥35 mg/kg/day, and lymphoid depletion was seen at 134 mg/kg/day (Lent et al. 2012a; 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 112 

3. HEALTH EFFECTS 

USAPHC 2011b). No changes in serum concentrations of IgE were observed in rats and dogs 

administered 2,4-DNT at levels up to 206 and 25 mg/kg/day, respectively, for 13 weeks (Ellis et al. 1985; 

Lee et al. 1985; U.S. Army 1978b). In these studies, the rats received the test article in feed, while the 

dogs received it in capsules. No histopathological changes were found in the spleen or thymus of 

Sprague-Dawley male rats fed 78 mg/kg/day 2,4-DNT or female rats fed 82 mg/kg/day 2,4-DNT in the 

diet for 14 days (McGown et al. 1983). 

Administration of 2,6-DNT to dogs (up to 100 mg/kg) and rats (up to 145 mg/kg/day) for 13 weeks 

resulted in no observable changes in IgE serum concentrations (U.S. Army 1976). IgE is the antibody 

associated with allergic or hypersensitive reactions, and so it may be expected that the human sensitizing 

potential of 2,4- and 2,6-DNT would be low. Involution of the thymus was noted when dogs were 

administered 100 mg/kg 2,6-DNT, but was not noted when they were administered 20 mg/kg 2,6-DNT by 

capsule for 13 weeks (U.S. Army 1976). 

Exposure to 3,4-DNT by gavage for 14 days induced lymphoid hyperplasia of the spleen in male rats 

exposed to doses ≥57 mg/kg/day (Lent et al. 2012a; USAPHC 2011e). 

All LOAEL values from each reliable study for immunological/lymphoreticular effects in each species 

and duration category for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 

3-4, 3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively. 

No studies were located regarding immunological and lymphoreticular effects in animals after oral 

exposure to 2,5- or 3,5-DNT, although Lent et al. (2012a; USAPHC 2011c, 2011f) did not observe 

splenic lymphoid lesions in rats exposed to these two compounds for 14 days. 

3.2.2.4 Neurological Effects 

No studies were located regarding neurological effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. 

Neurotoxicity appears to be a characteristic syndrome of subchronic and chronic DNT poisoning of 

animals. Neurotoxic symptoms, of decreased severity compared to dogs, were observed in mice and rats 

at doses higher than neurotoxic doses in dogs; however, the test article was administered in feed to 

rodents and in capsules to dogs. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 113 

3. HEALTH EFFECTS 

Acute (14-day) oral exposure of male Sprague-Dawley rats to 2,3-DNT at doses up to 275 mg/kg/day or 

2,4-DNT at doses up to 142 mg/kg/day did not lead to any clinical signs of neurotoxicity, nor was brain 

weight affected (Lent et al. 2012a; USAPHC 2011a). 

In a subchronic study, beagle dogs (4/sex/group) administered 2,4-DNT at 1 or 5 mg/kg/day showed no 

signs of behavioral changes or clinical signs of neurotoxicity. Neurotoxicity was observed at 

25 mg/kg/day, with signs of neurotoxicity (loss of hind leg control) first observed in a female dog on day 

12 of treatment. Three additional male dogs showed similar signs (signs not specified) on day 14 of 

treatment. All dogs administered 25 mg/kg/day showed signs of neurotoxicity after treatment for 12– 

22 days. The onset and severity of toxic signs reportedly varied among dogs within the same treatment 

group; some dogs were moribund at the same time that others began experiencing symptoms. In 

individual dogs, symptom severity varied over time, with no duration-related pattern of severity. The 

specific neurotoxic effects in dogs affected within the first 14 days of treatment were not specified. The 

NOAEL for the neurotoxicity observed after 12 days was 5 mg/kg/day. No histopathological changes 

were found in the brain or spinal cord of male and female Sprague-Dawley rats fed 2,4-DNT for 14 days 

in the diet at doses of 78 and 82 mg/kg/day, respectively (McGown et al. 1983). 

Neurotoxicity has been reported in laboratory animals after intermediate- or chronic-duration exposure to 

2,4-DNT with symptoms ranging from tremors, convulsions, and ataxia to paralysis. These effects were 

observed in 13-week studies of rats and dogs. Administration of 93 mg/kg/day 2,4-DNT in the diet for 

13 weeks caused demyelinization in the cerebellum and brain stem of 1 male rat, while at 266 mg/kg/day, 

some rats exhibited a widespread or stiff-legged gait that did not progress to the rigid paralysis observed 

in dogs (Lee et al. 1985; U.S. Army 1978b). After 3 months of being fed 2,4-DNT at 0.5% in the diet (an 

estimated dose of 350 mg/kg/day based on body weight and feed consumption data provided by the study 

authors), Wistar rats exhibited humpback and jerky incoordination (Kozuka et al. 1979). Dogs that were 

administered 25 mg/kg/day 2,4-DNT in capsules for 13 weeks began to show neurotoxic effects within 

2 months; these effects included incoordination, abnormal gait, rigid paralysis of the hind legs, eventually 

progressing to paralysis up to the neck (Ellis et al. 1985; U.S. Army 1978b). No neurological signs were 

observed in mice fed 413 mg/kg/day in males or 468 mg/kg/day in females 2,4-DNT in the diet for 

13 weeks (Hong et al. 1985; U.S. Army 1978b). 

An abnormal gait was also observed in chronic studies of laboratory animals fed 2,4-DNT. The 

characteristic widespread and stiff-legged gait was observed after feeding 34.5 or 45.3 mg/kg/day 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 114 

3. HEALTH EFFECTS 

2,4-DNT to male and female rats, respectively, for up to 2 years (Lee et al. 1985; U.S. Army 1978b, 

1979). This stiff-legged gait and hyperactive behavior were also noted in mice fed 898 mg/kg/day in the 

diet for 24 months but were not observed in mice at 95 mg/kg/day (Ellis et al. 1985; U.S. Army 1979). In 

dogs dosed at 1.5 mg/kg/day 2,4-DNT in a 2-year study, one of six dogs showed intermittent loss of 

hindquarter control (Ellis et al. 1985; U.S. Army 1979). Central nervous system lesions were identified in 

high-dose (10 mg/kg/day) dogs in this study and included vacuolization, hypertrophy, endothelial mitosis, 

and focal gliosis in the cerebellum, as well as some perivascular hemorrhage in the cerebellum and brain 

stem (Ellis et al. 1985; U.S. Army 1979). 

Male rats exposed for 14 days by gavage to 2,5-DNT at doses up to 308 mg/kg/day or 2,6-DNT at doses 

up to 134 mg/kg/day did not display clinical signs of neurotoxicity or alterations in brain weight (Lent et 

al. 2012a; USAPHC 2011c). 

Dogs dosed with 20 or 100 mg/kg/day of 2,6-DNT for 13 weeks exhibited dose-related neurotoxic 

symptoms that included muscular incoordination, weakness, tremors, and paralysis (U.S. Army 1976). 

Rats and mice dosed at 145 and 289 mg/kg/day of 2,6-DNT, respectively, for 13 weeks did not display 

neurotoxic symptoms (U.S. Army 1976). 

Clinical signs of neurotoxicity consisting of facial twitching, hypoactivity, and staring were noted in male 

rats exposed by gavage to 227 mg/kg/day 3,4-DNT for 14 days (Lent et al. 2012a; USAPHC 2011e). 

Male rats exposed to ≥39 mg/kg/day 3,5-DNT by gavage for 14 days were reported to exhibit 

neurological signs progressing from facial twitching to paralysis of the forelimbs (Lent et al. 2012a; 

USAPHC 2011f). However, all rats exposed to higher doses died or were sacrificed moribund, as did one 

rat exposed to 39 mg/kg/day. It is not clear whether these signs were seen only in the animals that were 

moribund or if some neurological signs were seen in animals that survived treatment. Animals in the 

39 mg/kg/day group also showed a 30% increase (compared with controls) in brain weight relative to 

body weight, as well as mild or moderate inflammatory infiltrates in the brain (Lent et al. 2012a; 

USAPHC 2011f). 

Administration of 150 mg/kg/day Tg-DNT to F344 dams during gestation days 7–20 caused hind limb 

weakness in 7 of 13 animals (Jones-Price et al. 1982). No clinical signs of neurotoxicity or 

histopathological changes were found in rats fed up to 35 mg/kg/day Tg-DNT in the diet for 26 or 

52 weeks (Hazleton Laboratories 1982). 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 115 

3. HEALTH EFFECTS 

In the 14-day gavage studies that compared the effects of the various DNT isomers on neurological end 

points in male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 

2011f), adverse effects were elicited only by 3,4-DNT (facial twitching, hypoactivity, staring; 

LOAEL=227 mg/kg/day) and 3,5-DNT (facial twitching and paralysis, inflammatory infiltrates in the 

brain; LOAEL=39 mg/kg/day). 

All LOAEL values from each reliable study for neurological effects in each species and duration category 

for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, 

respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively. 

3.2.2.5 Reproductive Effects 

No studies were located regarding reproductive effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. 

Studies in laboratory animals have shown that oral exposure to 2,4-DNT can result in adverse effects on 

reproduction, as shown by decreased fertility and the development of lesions of the male and female 

reproductive tracts. The male reproductive system seems to be particularly sensitive; observed effects 

include decreased sperm production, testicular atrophy, changes in Sertoli cell morphology, and 

degenerated seminiferous tubules (Bloch et al. 1988; Kozuka et al. 1979; Lane et al. 1985; McGown et al. 

1983; U.S. Army 1976, 1978b, 1979). In the female reproductive system, ovarian atrophy and 

dysfunction were observed (U.S. Army 1979). 

Fourteen-day exposure to 2,3-DNT at doses up to 275 mg/kg/day did not result in alterations in testes or 

epididymides weights in male Sprague-Dawley rats exposed by gavage (Lent et al. 2012a; USAPHC 

2011a). 

The effects on the male reproductive system have been reported in studies of brief durations. Decreased 

fertility was noted in male rats dosed with 180 mg/kg 2,4-DNT for 5 days; no dominant lethal effect was 

observed at this dose (Lane et al. 1985). Decreased absolute and relative testes weight, seminiferous 

tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes were noted 

in male rats after 14 days of gavage doses of 142 mg/kg/day 2,4-DNT (Lent et al. 2012a; USAPHC 

2011b). Sprague-Dawley rats administered 104, 165, or 261 mg/kg/day 2,4-DNT in the diet for 14 days 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 116 

3. HEALTH EFFECTS 

exhibited oligospermia with degenerative changes, such as syncytial cell formation and focal spermatic 

granuloma, in a dose-related manner (McGown et al. 1983). A dose of 78 mg/kg/day 2,4-DNT caused a 

decrease in the thickness of spermatogenic cell layers. No histopathological changes were found in the 

reproductive organs of females in this study (McGown et al. 1983). Although no changes were found in 

sperm morphology of male mice that were administered 250 mg/kg/day 2,4-DNT for 2 days, significant 

decreases in fertile matings of these animals were observed during weeks 2, 3, and 6 post-treatment 

(Soares and Lock 1980). However, sperm morphology was examined at 8 weeks post-treatment, so it is 

possible that a toxic effect was selective for specific types of sperm cells. 

In intermediate studies of 2,4-DNT, serious effects on the male reproductive system have been observed 

in numerous animal studies. In a series of three dominant lethal studies using male rats for 13 weeks, 

45 mg/kg/day 2,4-DNT in the diet caused severe atrophy and degeneration of the seminiferous tubules, 

resulting in decreased fertility, although no dominant lethal effect was observed (U.S. Army 1979). 

Another study using CD rats found that spermatogenesis was impaired after 4 weeks of feeding 

93 mg/kg/day 2,4-DNT in the diet and had completely ceased after 13 weeks (Lee et al. 1985; U.S. Army 

1978b). This effect was not reversible after a 4-week post-treatment period. Higher concentrations of 

2,4-DNT were needed to cause these effects in mice. Testicular atrophy and aspermatogenesis occurred 

in CD-1 mice fed 413 mg/kg/day 2,4-DNT for 13 weeks (Hong et al. 1985; U.S. Army 1978b) and rats 

fed a TWA dose of 371 mg/kg/day 2,4-DNT for 6 months in feed (Kozuka et al. 1979). Decreased 

fertility was observed after male mice were treated with 1,032 mg/kg/day, but not 295 mg/kg/day, 

2,4-DNT in the feed for 4 weeks in a dominant lethal study (U.S. Army 1978b). The decreased fertility 

was not observed in mice fed 295 mg/kg/day (U.S. Army 1978b). The testicular atrophy was considered 

to be due to a direct toxic effect on spermatogenic cells. Mild-to-severe testicular degeneration with 

decreased spermatogenesis has also been observed in dogs administered 25 mg/kg 2,4-DNT in capsules 

for 13 weeks (Ellis et al. 1985; U.S. Army 1978b). No testicular effects were found at 5 mg/kg in the 

study. 

Histopathological examination of the testes after treatment with 2,4-DNT revealed changes, which 

suggest specific causes for the male infertility observed in animal studies. Dose-related changes in sperm 

cell morphology were found in Sprague-Dawley rats fed 76.7 or 153.4 mg/kg/day 2,4-DNT in the diet for 

3 weeks (Bloch et al. 1988). At the low dose, vacuolation and lipid accumulation were noted in Sertoli 

cells; multinucleated spermatid and irregularities of the basal lamina were also found. These changes 

were limited and variable with most samples, demonstrating patchy damage. More extensive 

degenerative changes in both spermatocytes and spermatids were found at the high dose as well as 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 117 

3. HEALTH EFFECTS 

ultrastructural changes in Sertoli cells; epididymal sperm counts were decreased 63%. The high-dose 

animals also had increased levels of serum luteinizing hormone (LH) and FSH but not testosterone (Bloch 

et al. 1988). 

Chronic-duration studies in laboratory animals have also demonstrated both male and female reproductive 

effects. Male CD-1 mice fed 14 mg/kg/day 2,4-DNT for 12 months showed atrophy of the testes and 

decreased spermatogenesis (Hong et al. 1985). Female mice fed 898 mg/kg/day 2,4-DNT in this study 

had ovarian atrophy with non-functioning follicles, with a NOAEL of 95 mg/kg/day (Hong et al. 1985). 

Male CD rats that received 34 mg/kg/day 2,4-DNT in the diet for 12 months showed an increased 

incidence of seminiferous tubule atrophy compared to controls (100% affected at the high dose versus 0% 

of controls), with a NOAEL of 3.9 mg/kg/day (Lee et al. 1985; U.S. Army 1978b, 1979). No adverse 

reproductive effects were found in dogs fed 10 mg/kg/day 2,4-DNT for 24 months (Ellis et al. 1985; U.S. 

Army 1979). 

A three-generation reproductive toxicity study was performed in rats fed 2,4-DNT for up to 6 months 

before mating of original prenatal animals (U.S. Army 1979). Effects on neonatal viability were observed 

at the highest concentration of 2,4-DNT used, 40 mg/kg/day. Reductions in neonatal viability became 

more severe with successive litters within each generation, such that no second litters were produced by 

the second generation of high-dose animals, which were fed 34.5 mg/kg/day (male) or 45.3 mg/kg/day 

(female) 2,4-DNT. Decreased fetal viability was attributed to maternal neglect and maternal death during 

parturition. Decreases in the number of fetal implants were attributed to the adverse impact of 2,4-DNT 

on sperm production. 

No effects on testes or epididymides weights were observed in male rats receiving 308 mg/kg/day 

2,5-DNT by gavage for 14 days (Lent et al. 2012a; USAPHC 2011c). 

In contrast, seminiferous tubular degeneration, multinucleated giant cell formation, and interstitial atrophy 

of the testes were reported in rats after gavage doses ≥68 mg/kg/day 2,6-DNT for 14 days (Lent et al. 

2012a; USAPHC 2011d). At the highest dose (134 mg/kg/day 2,6-DNT), these effects were accompanied 

by marked decreases (49–57% compared with controls) in absolute and relative testes weight (Lent et al. 

2012a; USAPHC 2011d). 

In studies of rats, mice, and dogs dosed with 2,6-DNT for 13 weeks (U.S. Army 1976), decreased 

spermatogenesis was observed in male mice administered 51 mg/kg/day, but normal spermatogenesis was 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 118 

3. HEALTH EFFECTS 

observed in animals dosed with 11 mg/kg/day. Testicular atrophy was reported in rats administered 

35 mg/kg/day 2,6-DNT, and no effects were observed in rats dosed with 7 mg/kg/day (U.S. Army 1976). 

Dogs dosed with 20 and 100 mg/kg/day had testicular degeneration, but no effects were observed in dogs 

dosed with 4 mg/kg/day (U.S. Army 1976). 

3,4-DNT doses up to 227 mg/kg/day administered by gavage for 14 days to male Sprague-Dawley rats 

did not result in any changes in testicular or epididymides weights (Lent et al. 2012a; USAPHC 2011e). 

Exposure to 3,5-DNT, in contrast, resulted in significant reductions in absolute and relative testes weights 

(≥40% lower than controls), as well as seminiferous tubular degeneration and multinucleated giant cell 

formation in the testes of male rats after exposure to doses of 19 and 39 mg/kg/day for 14 days (Lent et al. 

2012a; USAPHC 2011f). 

Based upon the testicular effects observed after administration of 2,4-, 2,6-, and 3,5-DNT it is not 

surprising that these effects are found after treatment with Tg-DNT. Testicular degeneration was found in 

male rats fed 35 mg/kg/day Tg-DNT for 26 weeks, but since the finding was unilateral, the relationship to 

treatment may be considered equivocal (Hazleton Laboratories 1982). When treatment with this 

concentration was carried through 52 weeks, however, bilateral mild-to-severe testicular degeneration and 

hypospermatogenesis were observed (Hazleton Laboratories 1982). No changes were found in the 

fertility or sperm morphology of male mice that received 250 mg/kg Tg-DNT by gavage for 2 days in a 

dominant lethal study (Soares and Lock 1980). 

In the 14-day gavage studies that compared the effects of the various DNT isomers on reproductive end 

points in male Sprague-Dawley rats (Lent et al. 2012a; USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 

2011f), adverse effects were elicited only by 2,4-DNT (decreased weights of testis and epididymis, 

tubular degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes; 

LOAEL=142 mg/kg/day), 2,6-DNT (decreased testis weight at a LOAEL of 134 mg/kg/day; tubular 

degeneration, multinucleated giant cell formation, and interstitial atrophy of the testes at a LOAEL of 

68 mg/kg/day), and 3,5-DNT (small testes, significantly reduced testes weight, tubular degeneration and 

multinucleated giant cell formation in the testes; LOAEL=19 mg/kg/day). 

The highest NOAEL values and all reliable LOAEL values for reproductive effects in each species and 

duration category for 2,3-, 2,4-, 2,5-, 2,6, 3,4-, and 3,5-DNT are recorded in Tables 3-1, 3-2, 3-3, 3-4, 

3-5, and 3-6, respectively, and plotted in Figures 3-1, 3-2, 3-3, 3-4, 3-5, and 3-6, respectively. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 119 

3. HEALTH EFFECTS 

3.2.2.6 Developmental Effects 

No studies were located regarding developmental effects in humans after oral exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. However, developmental toxicity from DNTs could potentially occur because 

exposure to any substance that depletes the amount of oxygen available to developing fetal tissues may 

have adverse consequences. U.S. Army (1979) conducted a 3-generation reproductive study in which 

2,4-DNT was administered to male and female rats at doses up to 34.5 and 45.3 mg/kg/day, respectively. 

Normal birth weights, liveborn index, and weight at weaning were observed. Decreases in pup viability 

at 45.3 mg/kg/day in this study resulted from maternal neglect and a high incidence of maternal death 

during parturition; these decreases did not appear to result from pup defects since no anomalies were 

detected in offspring from any generation. These effects were not observed in animals fed 5.1 mg/kg/day 

2,4-DNT (U.S. Army 1979). 

Tg-DNT was administered by gavage to pregnant rats for 14 days during gestation, and pups were 

evaluated for developmental toxicity either at gestation day 20 or postpartum day 60 (Jones-Price et al. 

1982). Adverse effects on hematologic parameters and altered organ weights were observed in both dams 

and fetuses when dams were administered 100 or 150 mg/kg/day. However, the fetal toxicity was not 

dose related. A decrease in relative liver weight was observed, however, in the postpartum pups at the 

low dose of 14 mg/kg/day; this dose is considered to be a LOAEL. Dose-related effects on postnatal 

development were not observed in pups when dams were administered 35 or 75 mg/kg/day. Transient 

and statistically significant signs of neurotoxicity, which were not dose-related, included delayed eye 

opening and cliff avoidance when dams were treated with 35 or 75 mg/kg/day. No evidence of toxicity 

was found in pups at postpartum day 60 of the postnatal study. 

No studies were located regarding developmental effects in animals after oral exposure to 2,3-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 

The highest NOAEL values and all reliable LOAEL values for developmental effects in each species and 

duration category for 2,4-DNT are recorded in Table 3-1 and plotted in Figure 3-1. 

3.2.2.7 Cancer 

No studies were located regarding cancer in humans after oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT. 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 120 

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The carcinogenic activity of DNTs has been extensively studied in typical chronic bioassays and in some 

less-than-lifetime studies. 2,4-DNT produced renal tumors in male mice and was hepatocarcinogenic in 

rats. 2,6-DNT and Tg-DNT are potent hepatocarcinogens in rats (Lee et al. 1985; U.S. Army 1978b, 

1979). 

2,4-DNT (98% 2,4-DNT, 2% 2,6-DNT) produced renal tumors (76%) in male CD-1 mice fed 

95 mg/kg/day for 2 years (U.S. Army 1979). A statistically significant increase in renal tumors in female 

mice was not observed. A National Cancer Institute (NCI) bioassay (NCI 1978) of 2,4-DNT (95% 

2,4-DNT, the other components not specified) did not detect a carcinogenic effect in mice dosed with 

72 mg/kg/day for 78 weeks. The NCI bioassay used the C57BL/6N strain of mouse, lower doses, and a 

shorter treatment schedule than did U.S. Army (1979). 

Hepatocellular carcinoma were significantly increased in male CD rats fed 34.5 mg/kg/day 2,4-DNT and 

in females fed 45.3 mg/kg/day 2,4-DNT for 2 years (U.S. Army 1979). The tumor response in females 

was higher than in the males. Two other studies of rats in which malignancies were not observed used the 

F344 strain, lower doses, and shorter exposure durations than did U.S. Army (1979): 10 mg/kg/day for 

78 weeks (NCI 1978) and 27 mg/kg/day for 52 weeks (Leonard et al. 1987). NCI (1978) reported 

significant increases in subcutaneous tissue fibroma in male rats at 7.5–8 mg/kg/day and mammary gland 

fibroadenomas in female rats at 22 mg/kg/day. U.S. Army (1979) found significant increases in 

subcutaneous tissue fibromas in male rats at 34.5 mg/kg/day and mammary gland fibroadenomas in 

female rats at 45.3 mg/kg/day; these were benign tumors. 

2,4-DNT was not found to be carcinogenic in the Strain A/J mouse pulmonary tumor bioassay when 

250 mg/kg was administered by gavage twice a week for 12 weeks (Stoner et al. 1984). 2,4-DNT was a 

hepatic tumor promoter, but not a tumor initiator, using in vivo hepatic initiation-promotion protocols 

(Leonard et al. 1986). 

2,6-DNT administered for 1 year at 7 and 14 mg/kg/day produced hepatocellular carcinomas in 85% and 

100%, respectively, of male F344 rats (Leonard et al. 1987). Pulmonary metastases of hepatocytic origin 

were also observed. Both tumor-initiating and tumor-promoting activities of 2,6-DNT in rat liver were 

reported (Leonard et al. 1983, 1986; Mirsalis and Butterworth 1982). 2,6-DNT was not found to be a 

lung carcinogen in the Strain A/J mouse pulmonary tumor bioassay when 250 mg/kg was administered by 

gavage twice a week for 12 weeks (Schut et al. 1983; Stoner et al. 1984). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 121 

3. HEALTH EFFECTS 

The effect of diet-induced changes in gut microflora on the hepatocarcinogenicity of 2,6-DNT was 

studied in male F344 rats (Goldsworthy et al. 1986). Groups of the rats were placed on one of three diets 

containing 2,6-DNT at doses of 0, 0.6–0.7, or 3–3.5 mg/kg/day. Ten animals from each group were 

sacrificed at 3, 6, and 12 months, and their livers were evaluated histopathologically. The diets used were 

NIH-07, an open formula cereal-based diet high in pectin content; AIN-76A, a purified pectin-free diet; or 

AP, which is AIN-76A supplemented with 5% pectin. The number and size of γ-glutamyl transpeptidasestaining foci in the liver increased in a dose- and time-related manner in animals given 2,6-DNT in the 

NIH-07 diet. Hepatocellular carcinomas and neoplastic nodules were observed only in rats fed NIH-07 

containing 2,6-DNT. No tumor was observed in rats receiving the control diets or 2,6-DNT in the 

AIN-76 diet with or without pectin. This finding suggested that pectin did not influence the tumor 

outcome of the experiment. Unidentified contaminants in cereal-based diets may influence liver foci and 

tumor production in the rat liver during carcinogen treatment. 

Tg-DNT provided positive hepatocarcinogenic results in two bioassays of less-than-lifetime duration. In 

a 52-week study of male rats dosed with 35 mg/kg/day of Tg-DNT, Leonard et al. (1987) observed a 47% 

increase in hepatocellular carcinoma; cholangiocarcinomas were also found in 10% of rats treated with 

35 mg/kg/day Tg-DNT in the Leonard et al. (1987) study. Hazleton Laboratories (1982) reported that 

dietary administration of 35 mg/kg/day Tg-DNT to rats for 55 weeks resulted in an increased incidence 

(100% in males and 55% in females) of hepatocellular carcinoma; this lesion was found in some animals 

treated at this level for 26 weeks. The administration of 3.5 mg/kg/day Tg-DNT for 104 weeks caused 

hepatocellular carcinoma in 9 of 70 males compared to 1 of 61 controls. Mammary fibroadenoma and 

subcutaneous fibroma were also found in both sexes at 3.5 mg/kg/day after 104 weeks (Hazleton 

Laboratories 1982). Administration of 14 mg/kg/day Tg-DNT for 104 weeks caused cholangiocarcinomas and parathyroid adenomas in males and hepatocellular carcinomas and hepatocholangiocarcinomas in females (Hazleton Laboratories 1982). 

Tg-DNT contains about 76% 2,4-DNT and 19% 2,6-DNT, as well as small amounts of other isomers. 

Rats that received 35 mg/kg/day in the Leonard et al. (1987) and Hazleton Laboratories (1982) studies 

were provided approximately 28 and 7 mg/kg/day of 2,4- and 2,6-DNT, respectively. This dose of 

2,6-DNT in Tg-DNT bioassays is equivalent to the low dose of 2,6-DNT administered to rats by Leonard 

et al. (1987) that produced hepatocellular carcinomas. 
 

 

 

 

 

 

 

 

DINITROTOLUENES 122 

3. HEALTH EFFECTS 

In hepatic tumor initiation-promotion protocols, Tg-DNT was reported to have tumor-promoting and 

tumor-initiating activity (Leonard et al. 1983, 1986; Mirsalis and Butterworth 1982). The results of the 

initiation-promotion protocols for 2,4-, 2,6-, and Tg-DNT indicate that 2,6-DNT is a complete 

hepatocarcinogen and is primarily responsible for the carcinogenic activity of Tg-DNT. 

No studies were located regarding cancer in animals after oral exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT. 

3.2.3 Dermal Exposure 

There are no data regarding adverse health effects associated with dermal exposure of humans or animals 

to 2,3-, 2,5-, 3,4-, or 3,5-DNT. There are data on occupational exposure of humans to 2,4-DNT and 

Tg-DNT (see Section 3.2.1) in which dermal exposure probably occurred, but the primary route of 

exposure in these studies is believed to be inhalation. The relative contribution of dermal exposure to 

total occupational exposure cannot be determined from these studies. Levine et al. (1985b) reported that 

small amounts of 2,4-DNT were detected on the hands, face, and forehead when a wipe-sample survey 

was conducted on workers in a DNT manufacturing plant. The highest quantity found on a worker’s skin 

was 180 μg (obtained from wipe sampling of hands and face; total surface area wiped was not reported) 

and may account for the quantity of excreted urinary metabolites that exceeded the amount of inhaled 

DNT in the operators and loaders. Animal data associated with dermal exposure of animals to DNTs are 

limited to studies of dermal irritation, eye irritation, and dermal sensitization of 2,4-, 2,6-, or Tg-DNT in 

rabbits. 

3.2.3.1 Death 

No studies were located regarding mortality in humans associated with dermal exposure to 2,3-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 

One study was located that examined death among humans exposed to DNTs. A retrospective mortality 

study of munitions workers exposed to either 2,4-DNT or Tg-DNT revealed an increased death rate due to 

ischemic heart disease and residual diseases of the circulatory system in the exposed cohort (Levine et al. 

1986a, 1986b). Exposure concentrations to 2,4-DNT or Tg-DNT were not reported. The residual 

diseases included cardiac arrest and arteriosclerosis. Exposure levels were not reported, and the study is 

further limited by the small cohort size and concurrent inhalation exposure of the workers. 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 123 

3. HEALTH EFFECTS 

No studies were located regarding death in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT. 

3.2.3.2 Systemic Effects 

No studies were located regarding respiratory, renal, body weight, or endocrine effects in humans or 

animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Gastrointestinal Effects. No studies were located regarding gastrointestinal effects in humans 

associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Gastrointestinal complaints of munitions workers exposed to either 2,4-DNT or Tg-DNT included nausea 

and vomiting (McGee et al. 1947). These workers also presumably inhaled DNT in the occupational 

setting. Exposure concentrations to 2,4-DNT or Tg-DNT were not reported. 

No studies were located regarding gastrointestinal effects in animals after dermal exposure to 2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Cardiovascular Effects. No studies were identified with respect to cardiovascular effects after 

dermal exposure of humans to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Levine et al. (1986a) reported a significant increase in heart disease mortality in workers involved in the 

manufacture and processing of 2,4-DNT and/or Tg-DNT. 

No studies were located regarding cardiovascular effects in animals after dermal exposure to 2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Hematological Effects. No studies were located regarding hematological effects in humans 

associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Hematological effects, such as anemia and cyanosis (which can be indicative of anemia), have been found 

in men employed at munitions factories (McGee et al. 1947; Perkins 1919). These workers were exposed 

to either 2,4-DNT or Tg-DNT. Because these studies lacked worker histories, exposure data, and 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 124 

3. HEALTH EFFECTS 

reported on small cohorts, the results are equivocal and are best used to qualitatively describe symptoms. 

In addition, the workers probably received their primary exposure via the inhalation pathway. 

No studies were located regarding hematological effects in animals after dermal exposure to 2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Musculoskeletal Effects. No studies were located regarding musculoskeletal effects in humans 

associated with dermal exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Muscle weakness and joint pain have been reported by munitions workers after occupational exposure to 

unspecified concentrations of 2,4-DNT or Tg-DNT (McGee et al. 1947; Perkins 1919). 

In the Perkins (1919) study, joint pain and other incapacitating symptoms were noted following exposure 

to what were presumed to be very high concentrations of Tg-DNT since the processes described required 

direct handling without protective equipment. In both of these studies, however, no exposure data were 

available; exposure to other compounds may have occurred, and concomitant exposure via inhalation was 

also likely. 

No studies were located regarding musculoskeletal effects in animals after dermal exposure to 2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, or 3,5-DNT. 

Hepatic Effects. No studies were located regarding hepatic effects in humans associated with dermal 

exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

In a study of male munitions workers exposed to unspecified concentrations of 2,4-DNT, 29 of 

714 workers displayed tenderness of the liver (McGee et al. 1947). No other clinical evaluation was 

performed that might provide further insight into the significance of this finding. These workers were 

also exposed to DNTs via inhalation. 

No studies were located regarding hepatic effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 

Dermal Effects. No studies were located regarding dermal effects in humans associated with dermal 

exposure to 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 125 

3. HEALTH EFFECTS 

A study of dermal effects of topical exposure to 2,4-DNT in workers employed by a munitions factory 

during World War II reported that 32 of 714 workers complained of dermatitis (McGee et al. 1947). 

Exposure levels were not quantified in this study. 

Both 2,4- and 2,6-DNT were shown to be mild primary dermal irritants in rabbits (U.S. Army 1975, 

1978a). 

No studies were located regarding dermal effects in animals after dermal exposure to 2,3-, 2,5-, 3,4-, or 

3,5-DNT. 

Ocular Effects. No studies were located regarding ocular effects in humans after dermal exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

No ocular irritation was found in rabbits in a primary eye irritation test using unspecified concentrations 

of 2,4- or 2,6-DNT (U.S. Army 1975, 1978a). However, mild eye irritations were reported in rabbits 

treated with 2,4-DNT (Ford 1981). 

No studies were located regarding ocular effects in animals after dermal exposure to 2,3-, 2,5-, 3,4-, or 

3,5-DNT. 

3.2.3.3 Immunological and Lymphoreticular Effects 

No studies were located regarding immunological and lymphoreticular effects in humans after dermal 

exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

In dermal sensitization tests, 2 of 10 guinea pigs exhibited mild sensitization to 2,6-DNT, but no 

sensitization was evident when 2,4-DNT was tested (U.S. Army 1975, 1978a). 

No studies were located regarding immunological and lymphoreticular effects in animals after dermal 

exposure to 2,3-, 2,5-, 3,4-, or 3,5-DNT. 
 

 

 

 

 

 

DINITROTOLUENES 126 

3. HEALTH EFFECTS 

3.2.3.4 Neurological Effects 

No studies were located regarding neurological effects in humans after dermal exposure to 2,3-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 

Various neurological symptoms, including headache, vertigo, and pain or numbness in the extremities, 

have been reported in surveys of munitions workers exposed to unspecified concentrations of 2,4-DNT 

(McGee et al. 1947). Although it is assumed that some dermal exposure to 2,4-DNT occurred in these 

workers, inhalation was the probable primary route of exposure. 

No studies were located regarding neurological effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. 

3.2.3.5 Reproductive Effects 

No significant effects on fertility were observed in workers occupationally exposed to Tg-DNT (Levine et 

al. 1985a). However, Levine et al. estimated that only a 50–70% reduction in fertility could have been 

detected in the worker population that they studied. 

No studies were located regarding reproductive effects in animals after dermal exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. 

3.2.3.6 Developmental Effects 

No studies were located regarding developmental effects in humans or animals after dermal exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.2.3.7 Cancer 

In a case-cohort study of German copper miners with exposure to Tg-DNT (see Section 3.2.1 for 

additional detail), Seidler et al. (2014b) observed elevated Cox proportional HRs for renal cancer among 

workers with medium or high dermal exposure to Tg-DNT (HR 2.73, 95% CI 1.0–7.42 for medium 

exposure and HR 1.81, 95% CI 0.75–4.33 for high exposure). When combined across medium and high 

exposure categories for both dermal and inhalation exposure, the HR for renal cancer was significantly 

increased (HR 2.12, 95% CI 1.03–4.37). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 127 

3. HEALTH EFFECTS 

No studies were located regarding cancer in humans or animals after dermal exposure to 2,3-, 2,4-, 2,5-, 

2,6-, 3,4-, or 3,5-DNT. 

3.3 GENOTOXICITY 

Data are available for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, and Tg-DNT from in vitro tests in prokaryotic 

organisms (gene mutation in Salmonella typhimurium) and in mammalian cell systems (gene mutation, 

chromosomal aberrations, DNA damage, and morphological transformation). Results of in vitro 

genotoxicity assays are presented in Table 3-9. In vivo data for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, and 

Tg-DNT, including evaluations of unscheduled DNA synthesis (UDS), DNA damage and DNA binding, 

gene mutations, and/or chromosomal aberrations, are presented in Table 3-10. Some isomers of DNT 

(2,4-DNT and especially 2,6-DNT) have tested positive in vitro and/or in vivo in other genotoxicity 

assays, with evidence of chromosomal aberrations, DNA damage and adduct formation, and 

morphological transformation. 2,3-, 2,5-, and 3,5-DNT generally tested negative in genotoxicity assays. 

Taken together, results of in vitro studies do not provide strong evidence that DNT directly induces gene 

mutations; however, there is some evidence that 2,4-DNT induces mutations in vivo. 

In vitro studies assessing genotoxicity of DNT have yielded mixed results in S. typhimurium, presumably 

due to differences in the need for metabolic activation and the sensitivity of the tester strains. 2,3-, 2,4-, 

2,5-, 2,6-, 3,4-, and 3,5-DNT have been shown to induce gene mutations in S. typhimurium in the 

presence or absence of metabolic activation in some assays, but produced negative or equivocal results in 

several others (Couch et al. 1981; Dellarco and Prival 1989; Einistö et al. 1991; Kawai et al. 1987; Mori 

et al. 1982; Neuwoehner et al. 2007; Padda et al. 2003; Sayama et al. 1989; Simmon et al. 1977; 

Spanggord et al. 1982b; Suzuki et al. 2011; Tokiwa et al. 1981; U.S. Army 1978a). Sayama et al. (1998) 

showed that 2,4-DNT was mutagenic in S. typhimurium strains possessing high levels of nitroreductase 

and O-acteyltransferase and 2,6-DNT was mutagenic in strains possessing high levels of 

O-acetyltransferase. 2,4-DNT was nonmutagenic in Escherichia coli (Dunkel et al. 1985). Mixed results 

were found for 2,4-DNT in tests of DNA damage in bacteria; positive results were found in the umu test 

using S. typhimurium and negative results were found in the SOS chromotest using E. coli (Öztürk and 

Durusoy 1999). In general, 2,3-, 2,4-, 2,5-, 2,6-, and 3,5-DNT did not induce gene mutations in 

mammalian cells (Abernathy and Couch 1982; Styles and Cross 1983); however, positive results were 

obtained for 2,4- and/or 2,6-DNT in assays that evaluated chromosomal aberrations in Chinese hamster 

lung fibroblasts or human peripheral lymphocytes (Huang et al. 1996; Suzuki et al. 2011), morphological 
 

DINITROTOLUENES 128 

3. HEALTH EFFECTS 

Table 3-9. Genotoxicity of Dinitrotoluene Isomers In Vitro 

Results 

With Without 

Species (test system) Isomer End point activation activation Reference 

Prokaryotic organisms: 

Salmonella typhimurium, 2,3-DNT Gene mutation + + Couch et al. 1981 

TA98 

S. typhimurium, TM677 2,3-DNT Gene mutation – – Couch et al. 1981 

S. typhimurium, TA98, 2,3-DNT Gene mutation – – Dellarco and Prival 

TA100 1989 

S. typhimurium, TA98; 2,3-DNT Gene mutation + NT Dellarco and Prival 

with flavin (modified assay) 1989 

mononucleotide (FMN) 

S. typhimurium, TA100; 2,3-DNT Gene mutation – NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium, TA98 2,3-DNT Gene mutation – + Spanggord et al. 

1982b 

S. typhimurium, TA100 2,3-DNT Gene mutation + + Spanggord et al. 

1982b 

S. typhimurium, TA1538 2,3-DNT Gene mutation – + Spanggord et al. 

1982b 

S. typhimurium, TA1535, 2,3-DNT Gene mutation – – Spanggord et al. 

TA1537, TA100 NR3 1982b 

S. typhimurium, TA100 2,3-DNT Gene mutation ± – Kawai et al. 1987 

S. typhimurium, TA98 2,3-DNT Gene mutation – ± Kawai et al. 1987 

S. typhimurium, TA98, 2,4-DNT Gene mutation + + Couch et al. 1981 

TM677 

S. typhimurium 2,4-DNT Gene mutation NT – Chiu et al. 1978 

S. typhimurium, TA98, 2,4-DNT Gene mutation – + Tokiwa et al. 1981 

TA100 

S. typhimurium, TA98, 2,4-DNT Gene mutation – – Dellarco and Prival 

TA100 1989 

S. typhimurium, TA98; 2,4-DNT Gene mutation + NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium, TA100; 2,4-DNT Gene mutation – NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium 2,4-DNT Gene mutation NT + Mori et al. 1982 

S. typhimurium, TA100 2,4-DNT Gene mutation – + Spanggord et al. 

1982b 

S. typhimurium, TA100 2,4-DNT Gene mutation + – Spanggord et al. 

NR3 1982b 

S. typhimurium, TA1535, 2,4-DNT Gene mutation – – Spanggord et al. 

TA1537, TA1538, TA98 1982b 

S. typhimurium, TA100 2,4-DNT Gene mutation + – U.S. Army 1978a 

S. typhimurium, TA1535 2,4-DNT Gene mutation – + U.S. Army 1978a 

S. typhimurium, TA98, 2,4-DNT Gene mutation – – U.S. Army 1978a 

1537 
 

DINITROTOLUENES 129 

3. HEALTH EFFECTS 

Table 3-9. Genotoxicity of Dinitrotoluene Isomers In Vitro 

Results 

With Without 

Species (test system) Isomer End point activation activation Reference 

S. typhimurium, TA1538 2,4-DNT Gene mutation + + U.S. Army 1978a 

S. typhimurium 2,4-DNT Gene mutation + NT Pearson et al. 1978 

S. typhimurium, TA98, 2,4-DNT Gene mutation NT + Einistö et al. 1991
 

TA98 NR, TA98/,8-DNP6, 

YG1021, YG1024
 

S. typhimurium, TA100 2,4-DNT Gene mutation ± ± Kawai et al. 1987
 

S. typhimurium, TA98 2,4-DNT Gene mutation – – Kawai et al. 1987
 

S. typhimurium, TA100 2,4-DNT Gene mutation NT – Padda et al. 2003
 

S. typhimurium, TA98, 2,4-DNT Gene mutation NT – Sayama et al. 1998
 

S. typhimurium, TA100 2,4-DNT Gene mutation NT + Sayama et al. 1998
 

YG1021, YG1024,
 

YG1041, YG1026,
 

YG1029, YG1042
 

Escherichia coli 2,4-DNT Gene mutation – – Dunkel et al. 1985
 

S. typhimurium, NM2009, 2,4-DNT DNA damage (umu NT + Öztürk and 

NM3009 test) Durusoy 1999
 

E. coli, PQ37 2,4-DNT DNA damage (SOS – – Öztürk and 

chromotest) Durusoy 1999 

S. typhimurium, TA98, 2,5-DNT Gene mutation + + Couch et al. 1981 

TM677 

S. typhimurium, TA98 2,5-DNT Gene mutation ± No data Dellarco and Prival 

1989 

S. typhimurium, TA100 2,5-DNT Gene mutation – – Dellarco and Prival 

1989 

S. typhimurium, TA98; 2,5-DNT Gene mutation + NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium, TA100; 2,5-DNT Gene mutation – NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium, TA1535, 2,5-DNT Gene mutation + + Spanggord et al. 

TA1538, TA98, TA100 1982b 

S. typhimurium, TA1537, 2,5-DNT Gene mutation – – Spanggord et al. 

TA100 NR3 1982b 

S. typhimurium, TA98, 2,6-DNT Gene mutation + + Couch et al. 1981 

TM677 

S. typhimurium, TA100, 2,6-DNT Gene mutation – – U.S. Army 1978a 

TA1535, TA1537, TA98 

S. typhimurium, TA1538 2,6-DNT Gene mutation – + U.S. Army 1978a 

S. typhimurium 2,6-DNT Gene mutation NT + Simmon et al. 1977 

S. typhimurium, TA98, 2,6-DNT Gene mutation – – Sayama et al. 1989 

TA100 

S. typhimurium, TA98, 2,6-DNT Gene mutation – + Tokiwa et al. 1981 

TA100 
 

DINITROTOLUENES 130 

3. HEALTH EFFECTS 

Table 3-9. Genotoxicity of Dinitrotoluene Isomers In Vitro 

Results 

With Without 

Species (test system) Isomer End point activation activation Reference 

S. typhimurium, TA100 2,6-DNT Gene mutation + + Spanggord et al. 

1982b 

S. typhimurium, TA1535, 2,6-DNT Gene mutation – – Spanggord et al. 

TA1537, TA1538, TA98, 1982b 

TA100 NR3 

S. typhimurium, TA98, 2,6-DNT Gene mutation – – Dellarco and Prival 

TA100 1989 

S. typhimurium, TA98; 2,6-DNT Gene mutation + NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium, TA100; 2,6-DNT Gene mutation – NT Dellarco and Prival 

with FMN (modified assay) 1989 

S. typhimurium, TA98, 2,6-DNT Gene mutation + + Suzuki et al. 2011 

TA100, TA1537 

S. typhimurium, TA98, 2,6-DNT Gene mutation NT + Einistö et al. 1991 

TA98/1,8-DNP, 

YG1021,YG1024 

S. typhimurium, TA98 NR 2,6-DNT Gene mutation + – Einistö et al. 1991 

S. typhimurium 2,6-DNT Gene mutation + NT Pearson et al. 1979 

S. typhimurium, TA98, 2,6-DNT Gene mutation ± ± Kawai et al. 1987 

TA100 

S. typhimurium, TA100 2,6-DNT Gene mutation NT – Padda et al. 2003 

S. typhimurium, TA98, 2,6-DNT Gene mutation NT – Sayama et al. 1998 

TA100 YG1021, 

S. typhimurium, YG1024, 2,6-DNT Gene mutation NT + Sayama et al. 1998 

YG1041, YG1026, 

YG1029, YG1042 

S. typhimurium, TA100 3,4-DNT Gene mutation + + Kawai et al. 1987 

S. typhimurium, TA98 3,4-DNT Gene mutation – – Kawai et al. 1987 

S. typhimurium, TA100 3,4-DNT Gene mutation + + Spanggord et al. 

1982b 

S. typhimurium, TA1535, 3,4-DNT Gene mutation – – Spanggord et al. 

TA1537, TA1538, TA98, 1982b 

TA100 NR3 

S. typhimurium, TA98 3,4-DNT Gene mutation – – Tokiwa et al. 1981 

S. typhimurium, TA100 3,4-DNT Gene mutation – + Tokiwa et al. 1981 

S. typhimurium, TA98, 3,5-DNT Gene mutation + + Couch et al. 1981 

TM677 

S. typhimurium, TA1537, 3,5-DNT Gene mutation + + Spanggord et al. 

TA1538, TA98, TA100 1982b 

S. typhimurium, TA1535, 3,5-DNT Gene mutation – – Spanggord et al. 

TA100 NR3 1982b 
 

DINITROTOLUENES 131 

3. HEALTH EFFECTS 

Table 3-9. Genotoxicity of Dinitrotoluene Isomers In Vitro 

Results 

With Without 

Species (test system) Isomer End point activation activation Reference 

S. typhimurium, TA98, Tg-DNT Gene mutation + + Couch et al. 1981 

TM677 

S. typhimurium	 Tg-DNT Gene mutation NT + Chadwick et al. 

1990 

Mammalian cells: 

CHO cells 2,4-DNT Sister chromatid + – Loveday et al. 

exchange 1989 

CHO cells 2,4-DNT Chromosomal – – Loveday et al. 

aberrations 1989 

CHO/HGPRT 2,3-DNT Gene mutation – – Abernethy and 

Couch 1982 

CHO/HGPRT 2,4-DNT Gene mutation – – Abernethy and 

Couch 1982 

CHO/HGPRT 2,4-DNT Gene mutation – – Abernethy and 

Couch 1982 

CHO/HGPRT 2,5-DNT Gene mutation – – Abernethy and 

Couch 1982 

CHO-HGPRT 2,6-DNT Gene mutation – – Abernethy and 

Couch 1982 

CHO/HGPRT 3,5-DNT Gene mutation – – Abernethy and 

Couch 1982 

CHO-HGPRT Tg-DNT Gene mutation – – Abernethy and 

Couch 1982 

P388 mouse lymphoma 2,4-DNT Gene mutation – +	 Styles and Cross 

TK	 1983 

P388 mouse lymphoma 2,6-DNT Gene mutation – –	 Styles and Cross 

TK 1983 

P388 mouse lymphoma Tg-DNT Gene mutation – – Styles and Cross 

TK 1983 

Chinese hamster lung 2,6-DNT Chromosomal + + Suzuki et al. 2011 

fibroblasts aberrations 

SHE cells 2,4-DNT Morphological NT – Holen et al. 1990 

transformation 

SHE cells 2,4-DNT Morphological NT + Engelhardt et al. 

transformation 2004 

SHE cells 2,6-DNT Morphological NT + Engelhardt et al. 

transformation 2004 

SHE cells 2,6-DNT Morphological NT – Holen et al. 1990 

transformation 

Kumming rat Sertoli cells 2,4-DNT DNA damage NT + Yang et al. 2005 

(comet assay) 
 

DINITROTOLUENES 132 

3. HEALTH EFFECTS 

Table 3-9. Genotoxicity of Dinitrotoluene Isomers In Vitro 

Results 

With Without 

Species (test system) Isomer End point activation activation Reference 

Kumming rat Sertoli cells 2,6-DNT DNA damage NT + Yang et al. 2005 

(comet assay) 

Human peripheral 2,4-DNT Chromosomal NT + Huang et al. 1996 

lymphocytes aberrations 

+ = positive; – = negative; ± = weakly positive or equivocal; CHO = Chinese hamster ovary; DNA = deoxyribonucleic 

acid; DNT = dinitrotoluene; FMN = flavin mononucleotide; NT = not tested; SHE = Syrian hamster embryo; 

Tg = technical grade 
 

 

 

DINITROTOLUENES 133 

3. HEALTH EFFECTS 

Table 3-10. Genotoxicity of Dinitrotoluene Isomers In Vivo 

Species (test system) Isomer End point Results Reference 

DNT and TNT workers Tg-DNT Chromatid-type chromosomal + Sabbioni et al. 

aberrations 2006 

Rat hepatocyte 2,4-DNT UDS + Mirsalis et al. 1989 

Rat hepatocyte 2,4-DNT UDS + Mirsalis and 

Butterworth 1982 

Rat hepatocyte (male; AP or 2,4-DNTa UDS + Ashby et al. 1985 

F344) 

Rat hepatocyte 2,4-DNT S-phase synthesis + Mirsalis et al. 1989
 

Rat hepatocyte (male; 2,3-DNT DNA damage; comet assay – Lent et al. 2012b
 

Sprague-Dawley)
 

Rat hepatocyte (male; 2,4-DNT DNA damage; comet assay – Lent et al. 2012b
 

Sprague-Dawley)
 

Rat hepatocyte (male; 2,5-DNT DNA damage; comet assay – Lent et al. 2012b
 

Sprague-Dawley)
 

Rat hepatocyte (male; 2,6-DNT DNA damage; comet assay + Lent et al. 2012b
 

Sprague-Dawley)
 

Rat hepatocyte (male; 2,6-DNT DNA damage; comet assay + Suzuki et al. 2011
 

Crl:CD(SD)
 

Rat hepatocyte (male; 2,6-DNT DNA damage; comet assay + Rothfuss et al.
 

Sprague-Dawley) 2010
 

Rat hepatocyte (male; 3,5-DNT DNA damage; comet assay – Lent et al. 2012b
 

Sprague-Dawley)
 

Rat peripheral blood (male; 2,6-DNT DNA damage; comet assay + Rothfuss et al.
 

Sprague-Dawley) 2010 

Rat hepatocyte 2,4-DNT DNA binding (adduct formation) + La and Froines 

1993 

Rat hepatocyte (male; F344) 2,4-DNT DNA binding (adduct formation) + Chadwick et al. 

1993 

Rat hepatocyte (female; 2,4-DNT DNA binding (adduct formation) + Jones et al. 2005a 

Wistar) 

Rat hepatocyte (female; 2,6-DNT DNA binding (adduct formation) + Jones et al. 2005a 

Wistar) 

Rat (male; F344) and mouse 2,6-DNT DNA binding (adduct formation) + George et al. 1996 

(male; B6C3F1) hepatocytes 

Rat peripheral blood (male; 2,3-DNT Chromosomal aberrations; – Lent et al. 2012b 

Sprague-Dawley) micronucleus assay 

Rat hepatocyte (male; F344) 2,4-DNT Chromosomal aberrations; + Suzuki et al. 2009 

micronucleus assay 

Rat hepatocyte (male; F344) 2,4-DNT Chromosomal aberrations; + Takasawa et al. 

micronucleus assay 2010 

Rat peripheral blood (male; 2,4-DNT Chromosomal aberrations; – Lent et al. 2012b 

Sprague-Dawley) micronucleus assay 

Rat peripheral blood (male; 2,5-DNT Chromosomal aberrations; – Lent et al. 2012b 

Sprague-Dawley) micronucleus assay 
 

 

 

DINITROTOLUENES 134 

3. HEALTH EFFECTS 

Table 3-10. Genotoxicity of Dinitrotoluene Isomers In Vivo 

Species (test system) Isomer End point Results Reference 

Rat peripheral blood (male; 2,6-DNT Chromosomal aberrations; – Lent et al. 2012b 

Sprague-Dawley) micronucleus assay 

Rat hepatocyte (male; F344) 2,6-DNT Chromosomal aberrations; ± Takasawa et al. 

micronucleus assay 2010 

Rat peripheral blood (male; 3,5-DNT Chromosomal aberrations; – Lent et al. 2012b 

Sprague-Dawley) micronucleus assay 

Rat bone marrow (male; 2,6-DNT Chromosomal aberrations; – Rothfuss et al. 

Sprague-Dawley) micronucleus assay 2010 

Mouse bone marrow (male; 2,4-DNTa Chromosomal aberrations; – Ashby et al. 1985 

CBA x BALBc)F1) micronucleus assay 

Mouse (female; C57BL/6J or 2,4-DNTa Gene mutation, spot test – Soares and Lock 

T stock) 1980 

aThe test substance is referred to in the publication as technical-grade 2,4-DNT. 

+ = positive result; – = negative result; ± = equivocal result; DNA = deoxyribonucleic acid; DNT = dinitrotoluene; 

Tg = technical grade; TNT = trinitrotoluene; UDS = unscheduled DNA synthesis 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 135 

3. HEALTH EFFECTS 

transformation in Syrian hamster embryo cells (Engelhardt et al. 2004; Holen et al. 1990), or DNA 

damage in rat Sertoli cells (Yang et al. 2005). 

UDS and/or S-phase synthesis (SPS) were induced in vitro in the hepatocytes of F344 rats treated with 

2,4-DNT or Tg-DNT in vivo (Ashby et al. 1985; Mirsalis et al. 1989). The mutagenicity of several of the 

metabolites of 2,6-DNT have been tested in S. typhimurium. Although neither 2,6-DNT nor its 

metabolites, 2-amino-6-nitrotoluene, 2,6-dinitrobenzylalcohol, 2-acetylamino-6-nitrobenzoic acid, and 

2-amino-6-nitrobenzoic acid, were mutagenic in this assay with or without S9 activation, other 

metabolites of 2,6-DNT were found to possess mutagenic activity (Sayama et al. 1989). The putative 

metabolite 2,6-dinitrobenzaldehyde was a direct acting mutagen (i.e., it did not require activation) 

(Sayama et al. 1989). Urine from F344 rats administered 75 mg/kg 2,6-DNT by gavage tested positive 

for mutagenicity using S. typhimurium TA98 without S9 activation (Chadwick et al. 1993). 

2,4-DNT induced lethal mutations but not reciprocal translocations in mutagenicity testing using 

Drosophilia melanogaster (Woodruff et al. 1985). Color coat mutations were also observed in the 

offspring of pregnant C57BL/6J or T stock mice administered 1,000 mg/kg technical-grade 2,4-DNT via 

gavage or intraperitoneal injection on gestation day 10 (Soares and Lock 1980). 

Micronuclei formation was observed in two assays in liver cells of 2,4-DNT-treated F344 rats (Suzuki et 

al. 2009; Takasawa et al. 2010), but were not observed in the peripheral blood of 2,4-DNT-treated 

Sprague-Dawley rats (Lent et al. 2012b). Similarly, no micronuclei were observed in the peripheral blood 

of Sprague-Dawley rats administered 2,6-DNT via gavage for 2 or 14 days (Lent et al. 2012b; Takasawa 

et al. 2010) or in the bone marrow of Sprague-Dawley rats exposed for 3 or 29 days (Rothfuss et al. 

2010); however, micronuclei were induced in the hepatocytes of 2,6-DNT-treated rats. Other isomers of 

DNT (2,3-, 2,5-, or 3,5-DNT) tested negative in micronucleus assays in rats (Lent et al. 2012b). In 

workers at a DNT and TNT manufacturing facility, significant increases in chromatid-type chromosomal 

aberrations were observed, as compared to factory controls (Sabbioni et al. 2006). When workers were 

divided into three age categories, a significant association between 2,4-DNT hemoglobin adducts and 

chromosomal frequency was observed in the youngest group, but not in the two older groups. 

The formation of DNA adducts is generally thought to indicate carcinogenic risk (La and Froines 1993). 

Both 2,4- and 2,6-DNT have induced DNA adducts in rat liver. Following treatment with 2,4-DNT at up 

to 375 mg/kg via intraperitoneal injection, three DNA adducts were found in the liver of F344 rats (La 

and Froines 1992). Four adducts, identified as 4-amino-2-nitrotoluene (4A2NT), 24TDA, and 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 136 

3. HEALTH EFFECTS 

4-acetylamino-2-aminotoluene (4AA2AT) (with 4A2NT being the predominant form) were found in the 

liver of Wistar rats administered 0.5 mmol/kg (approximately 91 mg/kg) 2,4-DNT as a single gavage dose 

(Jones et al. 2005a). Following treatment with 2,6-DNT, four DNA adducts (not identified) were found 

in the liver of rats treated with 75 mg/kg 2,6-DNT by gavage (Chadwick et al. 1993). In Wistar rats 

treated with 2,6-DNT at 0.5 mmol/kg (about 91 mg/kg) via gavage as a single dose, three adducts, 

identified as 2-amino-6-nitrotoluene (2A6NT), 26TDA, and 2-acetylamino-6-aminotoluene (2AA6AT) 

(with 2A6NT being the predominant form) were detected (Jones et al. 2005a). Two types of DNA 

adducts were detected in liver DNA of B6C3F1 mice administered 2,6-DNT at 50 mg/kg via gavage; 

these adducts differed from the four types of adducts identified in F344 rats administered 2,6-DNT via 

gavage (George et al. 1996). The formation of 4 DNA adducts was also observed after intraperitoneal 

administration of 219 mg/kg 2,6-DNT to F344 rats (La and Froines 1992, 1993). One adduct accounts for 

the majority of the radioactivity measured; about 85% of the total was in one adduct in the study using 

2,4-DNT, while in the study with 2,6-DNT, about 60% of the total adducts measured were from a single 

adduct with the other adducts constituting 10–15% of the total (La and Froines 1992, 1993). No 

quantitative or qualitative differences in adduct formation were found when treatment occurred by gavage 

or intraperitoneal injection (La and Froines 1992). The proximate DNA binding species has been 

postulated to be 2-hydroxylamino-6-nitrobenzyl alcohol (La and Froines 1993; Rickert et al. 1984). The 

DNA adducts formed after exposure to 2,4- or 2,6-DNT were persistent over time; the persistence of these 

adducts was slightly >40% in the 2 weeks after exposure (La and Froines 1992). 

Comet assays, which evaluated the ability of DNT to induce DNA damage in the hepatocytes of treated 

rats, were negative for 2,3-, 2,4-, 2,5-, and 3,5-DNT (administered via gavage at up to 275, 142, 308, or 

39 mg/kg/day, respectively), but positive in Sprague-Dawley rats treated with 2,6-DNT via gavage at 

doses from 33 to 134 mg/kg/day (Lent et al. 2012b; Suzuki et al. 2011; Rothfuss et al. 2010). DNA 

damage (as measured by comet assay) was not detected in peripheral blood of Sprague-Dawley rats 

exposed to 100 mg/kg/day for 3 days or to 33 mg/kg/day for 29 days (Rothfuss et al. 2010) 

Studies of the effects of various DNT isomers (2,4-, 2,6-, or 3,5-DNT) on sperm morphology (Soares and 

Lock 1980), spermatocyte DNA repair (Working and Butterworth 1984), and dominant lethal mutations 

(Soares and Lock 1980, U.S. Army 1979) were generally negative for these specific end points. Mice 

administered 2,4-DNT at 250 mg/kg on two consecutive days via gavage or intraperitoneal injection did 

not show evidence of an increase in morphologically aberrant sperm (Soares and Lock 1980), and UDS 

was not induced in the spermatocytes of rats treated with a single dose of 20 mg/kg 2,6-DNT via gavage 

(Working and Butterworth 1984). Dominant lethal effects were not induced in rats administered 2,4-DNT 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 137 

3. HEALTH EFFECTS 

at up to 0.07% (34 mg/kg) in the diet for 13 weeks (U.S. Army 1979) or in mice administered 250 mg/kg 

2,4-DNT or 3,5-DNT on two consecutive days via gavage or intraperitoneal injection (Soares and Lock 

1980). However, a significant increase in the number of dead implants/total implants was reported in 

females mated to males treated with 2,4-DNT at 0.2% in the diet for 13 weeks (Hodgson et al. 1976). 

3.4 TOXICOKINETICS 

3.4.1 Absorption 

3.4.1.1 Inhalation Exposure 

No studies were located regarding absorption of inhaled of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT in 

humans or laboratory animals. However, detection of DNT metabolites in the urine of workers at DNT 

manufacturing plants provides evidence that DNT may be absorbed following presumed inhalation 

exposure (Levine et al. 1985b; Turner 1986; Woollen et al. 1985). 

3.4.1.2 Oral Exposure 

Rickert et al. (1983) suggested that the rapid disappearance of radioactivity from the first quarter of the 

small intestine of rats following the oral administration of uniformly [14C]-ring-labeled 2,4- or 2,6-DNT 

indicates rapid and fairly complete absorption. 

Excretion data and observed systemic effects indicate that DNTs are absorbed following oral 

administration to experimental animals. Several strains of rats, New Zealand rabbits, beagle dogs, and 

Rhesus monkeys excreted 55–90% of the radioactivity from orally-administered radiolabeled DNTs in the 

urine, primarily within the first 24 hours (Long and Rickert 1982; Rickert and Long 1981; U.S. Army 

1978b). In mice, most of the radioactivity from 3

H-labeled 2,6-DNT was excreted in the urine (about 

50% in 8 hours) (Schut et al. 1983), whereas most of the radioactivity from 14C-labeled 2,4-DNT 

administered to mice was excreted in the feces, and only about 10% was excreted in the urine (U.S. Army 

1978b). Increased fecal excretion could be due to reduced absorption or to greater excretion via the bile. 

Studies on absorption of 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT following oral exposure of humans or animals 

were not identified. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 138 

3. HEALTH EFFECTS 

3.4.1.3 Dermal Exposure 

There were no in vivo data available specifically on the absorption of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT via the dermal route of exposure. Two studies of occupational exposure to Tg-DNT have 

suggested that dermal absorption can be a significant route of entry for DNT in humans since the levels of 

urinary metabolites of 2,4- and 2,6-DNT in loaders and operators at a DNT manufacturing plant exceeded 

those that would have resulted from the inhaled concentrations (Levine et al. 1985b; Woollen et al. 1985). 

An in vitro study examined the dermal absorption of 2,4- and 2,6-DNT in soil or acetone using excised 

pigskin (Reifenrath et al. (2002). When in acetone, 36 and 24% of the 2,4- and 2,6-DNT, respectively, 

was absorbed. The absorption of radiolabel from soil was less; 15 or 16%, respectively, was absorbed 

from a low carbon soil and 5.4 and 3.8%, respectively, was absorbed from a high carbon soil. 

3.4.2 Distribution 

3.4.2.1 Inhalation Exposure 

No studies were located regarding distribution in humans or animals following inhalation exposure to 

2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.4.2.2 Oral Exposure 

The tissue distribution of 2,4-DNT and its metabolites was studied by Rickert and Long (1980). 2,4-DNT 

was administered orally to male and female rats at doses of 10, 35, or 100 mg of 14C-labeled 2,4-DNT/kg. 

When distribution is studied solely by detecting a radioisotope label, it is the labeled atom(s) that are 

being followed, and this label may be part of either the parent DNT molecule or a metabolite. Peak 

concentrations of radioactivity in plasma, red blood cells, liver, and kidney were proportional to dose. 

Levels in liver and kidney were 5–10 times higher than those in plasma or red blood cells. Levels of 

radioactivity in other tissues were lower than those in plasma. The only clear differences between males 

and females were the higher retention of radioactivity in red blood cells of females and the concentration 

of radioactivity in livers of females, which was only half that found in males. In addition, concentrations 

of 2,4-DNT in male kidneys peaked at 4–8 hours and were 3–10 times higher than the concentrations in 

female kidneys, which peaked 1 hour after the dose. 

Rickert et al. (1983) observed that hepatic concentrations of radioactivity in male rats increased in 

two stages, with the first peak occurring 1–2 hours and a second peak occurring 8–12 hours after an oral 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 139 

3. HEALTH EFFECTS 

dose of 10 or 35 mg/kg of radiolabeled 2,4- or 2,6-DNT. The second peak was followed by a gradual 

decline up to 16 days and was thought to be the result of enterohepatic cycling. 

In mice administered 3

H-labeled 2,6-DNT, the distribution of the label was similar in the blood, liver, 

kidneys, lungs, and small and large intestines at 8 hours after administration, with very low levels 

detected in the brain, lungs, heart, and spleen (Schut et al. 1983). 

In a radioisotope labeling study in dogs and monkeys, total 2,4-DNT and its metabolites recovered in 

blood and other tissues were approximately 3.6% (dogs) and 2.2% (monkeys) of the administered dose 

(U.S. Army 1978b). Relative to blood concentrations, the liver had the highest levels of 2,4-DNT or 

metabolites. Detectable levels of 2,4-DNT or metabolites were also found in the kidney and in skeletal 

muscle (U.S. Army 1975, 1978b). 

No studies were located regarding distribution in humans or animals following inhalation exposure to 

2,3-, 2,5-, 3,4-, or 3,5-DNT. 

3.4.2.3 Dermal Exposure 

No studies were located regarding distribution in humans or animals following dermal exposure to 2,3-, 

2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.4.3 Metabolism 

The metabolism of DNTs in humans has been studied in workers exposed to Tg-DNT by the analysis of 

urinary metabolites. The routes of exposure in these studies were multiple. Since the amounts of 

metabolites excreted could not be accounted for by the inhalation exposure route alone, dermal contact 

and ingestion routes of exposure may also be of importance (Levine et al. 1985b; Woollen et al. 1985). 

Woollen et al. (1985) found that the major metabolite excreted in the urine of workers exposed to 

Tg-DNT was 2,4-dinitrobenzoic acid (conjugates were hydrolyzed before analysis). There were wide 

variations in the excretion of the metabolites in different workers. Concentrations of 2,4-dinitrobenzoic 

acid in end-of-shift urine samples from 20 male and 8 female workers, however, did not suggest a 

difference in the excretion of this metabolite between males and females. The study authors stated that 

lesser amounts of the following metabolites were also found in the urine: 2-amino-4-nitro-, 4-amino2-nitro-, and 2-amino-6-nitrobenzoic acids, and 4-(N-acetyl)amino-2-nitrobenzoic acid. Trace levels of 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 140 

3. HEALTH EFFECTS 

DNTs were also detected. Dinitrobenzyl alcohols were not detected. Neither amounts nor relative 

percentages of metabolites were reported. 

Studies of workers at a Tg-DNT manufacturing plant (Levine et al. 1985b; Turner et al. 1985) provide 

more detailed information regarding the metabolism of Tg-DNT in occupationally exposed men and 

women. The principal metabolites detected in the urine of 14 men were dinitrobenzoic acids (2,4- and 

2,6-) and 2-amino-4-nitrobenzoic acid. In the urine of three women, these metabolites were detected 

together with dinitrobenzyl alcohol glucuronides (2,4- and 2,6-). Expressed as percent of total urinary 

metabolites, the dinitrobenzoic acids, 2-amino-4-nitrobenzoic acid, and the dinitrobenzyl glucuronides 

constituted 52.5, 37.2, and 9.5%, respectively, of the total urinary DNT metabolites in men and 28.8, 

37.6, and 33.3%, respectively, of the total urinary DNT metabolite in women. 2,4-DNT and 2,6-DNT 

metabolites were present in roughly the same proportions as in the Tg-DNT. Both men and women 

excreted relatively small amounts (<1% of urinary metabolites) of 2-(N-acetyl)amino-4-nitrobenzoic acid 

(Levine et al. 1985b). 

In contrast to the findings in the Levine et al. (1985b) study, 2,4-dinitrobenzoic acid was not one of the 

primary urinary metabolites in Chinese workers exposed to Tg-DNT and mononitrotoluenes (Jones et al. 

2005b). In these workers, 2,6-dinitrobenzoic acid, 4-amino-2-nitrobenzoic acid, 2-amino-4-nitrobenzoic 

acid, and 2,6-dinitrobenzl alcohol comprised 27.3, 26.0, 21.6, and 18.3%, respectively, of the total DNT 

metabolites. 

Studies in rats have identified a complex pathway for the metabolism of 2,4-DNT (Figures 3-7 and 3-8) 

and 2,6-DNT (Figure 3-9). Metabolism occurs in the liver and also in the intestine by microflora (Long 

and Rickert 1982; Rickert et al. 1981). Both oxidized and reduced metabolites are excreted in the urine 

after oral administration of the compounds. The main urinary metabolites of 2,4- and 2,6-DNT are the 

corresponding dinitrobenzyl alcohol glucuronide, dinitrobenzoic acid, and aminonitrobenzoic acid (Long 

and Rickert 1982). An additional urinary metabolite of 2,4-DNT is 4-(N-acetyl)amino-2-nitrobenzoic 

acid (Rickert et al. 1981). Although very little information on metabolism of 2,3-, 2,5-, or 3,5-DNT was 

identified, it is anticipated that metabolism of these isomers would follow similar pathways as those 

identified for 2,4- and 2,6-DNT. Results of an in vitro metabolism study using rat liver homogenate show 

that 2,3- and 2,5-DNT are reduced to monoaminonitrotoluenes and hydroxylaminonitrotoluenes (Kozuka 

et al. 1978). 
 

 

CH OH O 3 CH3
 

NH2
 NO2 NO2 

alcohol 

dehydrogenase Nitroreductase P-450 NO2

(MINOR) (MAJOR)
 

NO2 NO2
 NO2 NO2 

2-amino-4­ 2,4-dinitrotoluene 2,4-dinitrobenzyl 2,4-dinitrobenzaldehyde nitrotoluene 

alcohol 

Nitroreductase alcohol N-acetyltransferase UDP-glucuronosyl dehydrogenase transferase 

CH3 CH3 O G O OH 

H 

N O NO2 NO2 NO2

CH3
 

NO2 NH2
 NO2 NO2 

2-(n-acetyl)amino 4-amino-2­ 2,4-dinitrobenzyl 

Nitroreductase 

2,4-dinitrobenzoic acid 4-nitrotoluene nitrotoluene alcohol glucuronide 

Nitroreductase N-acetyltransferase 

CO2H CO2H CH3
 

NO NH2
 NO2 2 

HN O NO2 NH2

2-amino-4-nitrobenzoic 4-amino-2-nitrobenzoic 

CH3 acid acid 

4-(n-acetyl)amino-2­

nitrotoluene N-acetyltransferase N-acetyltransferase 

CO2H H CO2H 

N O NO2 

CH3 

O NO2 N 

H 

2-(N-acetyl)amino-4­ CCH3

nitrobenzoic acid 4-(N-acetyl)amino-2­

nitrobenzoic acid 

DINITROTOLUENES 141 

3. HEALTH EFFECTS 

Figure 3-7. Proposed Metabolic Pathways for the Hepatic Metabolism of 

2,4-Dinitrotoluene 

Sources: Bond and Rickert 1981; Bond et al. 1981; Smith et al. 1995 
 

 

 

CH3 CH3 CH3	 CH3 CH3 H NO2 NO N 2 NO2 N O OH 

NH N	 NO2 NO2 NO2 

HO	 O 

2-nitro-4-hydroxy- 2-nitro-4- 2-nitroso-4- 2-hydroxylamino­ 2,4-dinitrotoluene 

aminotoluene nitrosotoluene	 nitrotoluene 4-nitrotoluene 

CH3 CH3 CH3 

NO2 NH2 NH2

NH2 NH2 NO2
 

2-nitro-4­ 2-amino-4­

aminotoluene	 2,4-diaminotoluene nitrotoluene 

CH3

H 

N 

CH3	 CH CH3 3 

NH2 O 

N NH2 OH 

N 

HO 

NH NH NH2 2

O 

2-nitroso-4- 2-hydroxylamino- 2-amino-4-hydroxyl- 2-amino-4­

aminotoluene 4-aminotoluene aminotoluene nitrosotoluene 

= nitroreductase reaction 

ources: Guest et al. 1982; Mori et al. 1985 

DINITROTOLUENES 142 

3. HEALTH EFFECTS 

Figure 3-8. Proposed Pathways for the Anaerobic Metabolism of 

2,4-Dinitrotoluene in Rat Intestinal Microflora 

S
 

 

DINITROTOLUENES 143 

3. HEALTH EFFECTS 

Figure 3-9. Proposed Pathways for Metabolism of 2,6-Dinitrotoluene 

Urine 

CH3 

O2N NO2 

CH2OH 

O2N NO2 

CH2O-glucuronide 

O2N NO2 

CH2OH 

O2N NHOH 

O2N NH2 

CO2H 

O2N NO2 

CO2H 

CH2O-glycuronide 

O2N NO2

CH2OH 

H2

CH2OH 

O2N 

CH2OH 

N NH2 

NO2 O2N NH2 

Liver Intestine 

2,6-dinitro- 2,6-dinitro- 2,6-dinitro- 2-amino-6-nitrotoluene benzyl alcohol 

UDPglucuronosylbenzyl alcohol benzyl alcohol 

transferase 

2,6-dinitrobenzyl 

alcohol glucuronide 

Bile 

2-hydroxyl/amino-6-

nitrobenzyl alcohol 

Nitroreductase 

2-amino-6-nitro- 2,6-dinitroglucuronidase 

benzoic acid benzoic acid 

Nitroreductase 

beta- Nitroreductase 

2,6-dinitrobenzyl 

alcohol glucuronide 

2,6-diaminobenzyl 

alcohol 

Urine Urine 

[Covalent 

binding] 

[Hepatic 

macromolecules] 

[Activated 

metabolites] 

P-450 

Sources: Chapman et al. 1993; La and Froines 1993; Rickert et al. 1984; Smith et al. 1995 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 144 

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Oxidative metabolism by cytochrome P450 predominates in the liver of experimental animals, leading to 

the formation of dinitrobenzyl alcohol, which is either converted to glucuronide conjugate or further 

oxidized to dinitrobenzoic acid. Dinitrobenzyl alcohol glucuronide is partially excreted into the bile, 

followed by metabolism by gut microflora and enterohepatic cycling (Long and Rickert 1982; Medinsky 

and Dent 1983; Mori et al. 1997; Rickert and Long 1981). Thus, DNTs appear to be first metabolized by 

the liver with the metabolites being excreted into the bile; the biliary metabolites are hydrolyzed and 

further metabolized in the intestine; after reabsorption and circulation back to the liver, the metabolites 

are activated and bound to macromolecules (Chadwick et al. 1993; Long and Rickert 1982). 

2,4- and 2,6-dinitrobenzyl glucuronide have been detected directly in the bile following administration of 

2,4- and 2,6-DNT to the male Wistar rat (Mori et al. 1997), accounting for about 35 and 51% of the dose 

respectively. Four other metabolites, 2-amino-4-nitrotoluene, 4-amino-2-nitrotoluene, 2,4-diaminotoluene, and 4-acetylamino-2-nitrobenzoic acid accounted for 0.02–0.12% of the dose; in addition to 

2,4-dinitrobenzyl alcohol, 2,4-dinitrobenzaldehyde, and 2,4-dinitrobenzoic acid (0.09–0.14%) were 

detected in the bile of rats given 2,4-DNT. 2,6-Dinitrobenzyl alcohol, 2-amino-6-nitrotoluene, and 

2,6-dinitrobenzaldehyde were detected in the bile of rats given 2,6-DNT. 

Reductive metabolism of 2,3-, 2,4-, 2,5, and 2,6-DNT occurs by Escherichia coli isolated from human 

intestine (Mori et al. 1984). For all isomers, E. coli reduced DNTs to monoaminonitrotoluenes and 

hydroxylaminonitrotoluenes. Results are consistent with E. coli reduction of DNTs via hydroxylaminonitrotoluenes to monoaminonitrotoluenes. Study authors suggest that reduced metabolites of DNT may 

play a role in the development of DNT-induced methemoglobinemia and anemia observed in humans and 

animals. 

Studies of the metabolism of 2,4-DNT by intestinal microflora in rats and mice (Guest et al. 1982; Mori et 

al. 1985) and studies in germ-free rats (Rickert et al. 1981) have shown that intestinal microflora are 

responsible for reductive metabolism of DNTs. Intestinal microorganisms hydrolyze and reduce 2,4- and 

2,6-dinitrobenzyl alcohol glucuronide to the corresponding aminonitrotoluenes, probably through nitroso 

derivatives and hydroxylamino derivatives (Mori et al. 1997). The deconjugated metabolites are 

reabsorbed and transported back to the liver by enterohepatic circulation (Medinsky and Dent 1983). In 

the liver, the newly formed amine group is N-hydroxylated by cytochrome P450 and conjugated with 

sulfate (Kedderis et al. 1984). The sulfate conjugate is unstable and can be decomposed to form a 

carbonium or nitrenium ion that can be bound to hepatic macromolecules; this ostensibly leads to 

mutations and the formation of liver tumors. Thus, sulfation may be involved in the initiation stage of 
 

 

 

 

 

 

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hepatocarcinogenesis by 2,6-DNT. Metabolism by intestinal microflora appears to be essential for the 

production of metabolites that bind covalently to liver macromolecules. 

The intestinal biotransformation of 2,6-DNT was investigated in vitro using suspended microflora 

preparation from the intestinal contents of male Wistar rats (Sayama et al. 1993). It was determined that 

the metabolites formed with the incubation of 2,6-DNT were 2-nitroso-, 2-hydroxyl amino-, and 2-amino6-nitrotoluene and 2,6-diaminotoluene. Since no metabolites were detected when 2,6-diaminotoluene was 

incubated and the recovery of 2,6-diaminotoluene was about 95%, it appears that 2,6-diaminotoluene is 

the terminal intestinal metabolite of 2,6-DNT (Sayama et al. 1993). When 2,4-DNT was examined in this 

system, two nitroazoxy compounds (2,2'-dimethyl-5-5'-dinitroazoxybenzene and 4,4'-dimethyl3,3'-dinitro azoxybenzene) were detected in addition to other known metabolites, such as nitrosonitrotoluenes, hydroxyl aminonitrotoluenes, aminonitrotoluenes, and diaminotoluene (Sayama et al. 1993). 

The nitroazoxy compounds were believed to be non-enzymatic products (Sayama et al. 1993). 

The metabolites formed by the anaerobic incubation of potassium 2,4-dinitrobenzyl glucuronide or 

potassium 2,6-dinitrobenzyl glucuronide with rat intestinal microflora have been examined (Mori et al. 

1997). Metabolites transformed from 2,4-dinitrobenzyl glucuronide were 2,4-dinitrobenzyl alcohol, 

4-amino-2-nitrobenzyl alcohol, and 2-amino-4-nitrobenzyl alcohol, which peaked at 30, 75, and 

120 minutes of the incubation. 2,6-Dinitrobenzyl alcohol and 2-amino-6-nitrobenzyl alcohol were 

detected from potassium 2,6-dinitrobenzyl glucuronide incubation. Thus, intestinal metabolism includes 

the deconjugation of the glucuronide and the reduction of the nitro compound. 

In rats, sex differences in the metabolism of 2,4-DNT have been observed. A larger percentage of the 

administered dose is excreted in the bile of male rats than is excreted in the bile of females. In females, a 

greater percentage of the dose is excreted in urine as the dinitrobenzyl alcohol glucuronide (Medinsky and 

Dent 1983; Rickert and Long 1981). The quantitative differences in urinary versus biliary excretion of 

the glucuronide conjugates by females may account for the sex differences in the susceptibility of the rat 

to the hepatocarcinogenic effects of 2,4-DNT (U.S. Army 1979). Greater urinary excretion may decrease 

the amount of the glucuronide available to the intestinal microflora for metabolism to a carcinogenic 

metabolite. 

Metabolism studies in rats, rabbits, dogs, and monkeys with 2,4-DNT revealed the major urinary 

metabolites as glucuronide conjugates of 2,4-dinitrobenzyl alcohol (20–33% of the dose) and 

2,4-aminonitro-benzyl alcohols (8–19% of the dose). Lesser amounts of aminonitrotoluene, 2,4-diamino­
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 146 

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toluene, 2,4-aminobenzyl alcohol, and 2,4-dinitrobenzoic acid were also identified in all four species. 

Mice were also evaluated in this same group of studies. In mice, approximately 3% of the administered 

dose was excreted in the urine as the glucuronide conjugate of the 2,4-dinitrobenzyl alcohol and 

approximately 3% as the glucuronide conjugates of 2,4-aminonitrobenzyl alcohol (U.S. Army 1978b). 

Another study using rats dosed with either 2,4- or 2,6-DNT also demonstrated that the primary urinary 

conjugate was the respective dinitrobenzyl alcohol glucuronide (11–17% of administered dose) (Mori et 

al. 1996). Other metabolites in rats administered 2,4-DNT included 2-amino-4-nitrobenzoic acid 

(0.71%), 4-amino-2-nitrobenzoic acid (0.52%), 4-acetylamino-2-nitrobenzoic acid (3.9%), 4-amino2-nitrotoluene (0.04%), 2,4-dinitrobenzyl alcohol (0.25%), 2,4-dinitrobenzoic acid (6.9%), and 

4-acetylamino-2-aminobenzoic acid (3.4%). After administration of 2,6-DNT, other metabolites in urine 

included 2,6-dinitrobenzoic acid (0.17%), 2-amino-6-nitrotoluene (0.44%), and 2,6-dinitrobenzyl alcohol 

(0.53%) (Mori et al. 1996). 

The urinary metabolites of DNTs, and probably the glucuronides resulting from occupational exposure of 

humans, are qualitatively the same as those resulting from oral administration to rats, but the proportions 

of nitro-reduced metabolites were lower relative to oxidized metabolites in the urine from humans (Turner 

et al. 1985). These differences may be due more to the particular routes of exposure (inhalation and 

dermal for humans; oral for rats) than differences in species. As seen in experimental animals, female 

subjects excreted a higher proportion of urinary metabolites as dinitrobenzyl alcohol glucuronides than 

did males. 

Metabolism of DNTs has not been studied in children. However, fetuses and neonates have been shown 

to be limited in their ability to biotransform xenobiotics. Although the cytochrome P450 isoforms 

responsible for DNT metabolism have not been identified, cytochromes CYP2E1, CYP2B1/2, and 

CYP2C11/6 are known to contribute to the side-chain oxidation of toluene by the rat liver, and multiple 

cytochrome P450 isoforms may contribute to the side-chain oxidation of DNTs (Chapman et al. 1993). In 

humans, CYP2E1 protein is absent from fetal and neonatal livers, but steadily increases during the first 

year of life (Vieira et al. 1996). Other isoforms’ expression in fetuses and neonates is also qualitatively 

and quantitatively different from the expression observed in adults (Komori et al. 1990; Leeder and 

Kearns 1997). In rats, while sulfotransferase (the enzyme that catalyzes sulfation) activity is almost at 

adult levels at birth, UDP-glucuronosyltransferase (the enzyme that produces glucuronide conjugates) 

activity towards different xenobiotics varies with maturation (Young and Lietman 1978). Similarly, in 

humans, sulfation capabilities develop faster than glucuronidation capabilities (Leeder and Kearns 1997). 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 147 

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While the activity of some isoforms of sulfotransferase may exceed those seen in adults during infancy 

and early childhood, the activity of UDP-glucuronosyltransferase depends on the specific isoforms of the 

enzyme, and adult levels are generally attained by 6–18 months (Leeder and Kearns 1997). Since DNTs 

undergo bioactivation in the liver and by the intestinal microflora, the toxicity of DNTs may be different 

in children. Newborns have a transient deficiency in methemoglobin reductase (Gruener 1976) and have 

a high concentration of fetal hemoglobin in their erythrocytes. Consequently, they are highly sensitive to 

methemoglobin-generating chemicals and to methemoglobinemia generated by DNTs. 

3.4.4 Elimination and Excretion 

3.4.4.1 Inhalation Exposure 

In occupational settings, in addition to inhalation, some oral and dermal exposure can occur. The 

elimination of DNTs in the urine of workers exposed to Tg-DNT has been studied by several investigators 

(Levine et al. 1985b; Turner et al. 1985; Woollen et al. 1985). 

Woollen et al. (1985) observed that the highest rates of excretion of 2,4-dinitrobenzoic acid occurred near 

the end of the work shift. The half-life for urinary excretion of 2,4-dinitrobenzoic acid was calculated to 

be 2–5 hours. This estimate appears to be the initial phase of a biphasic elimination profile since even 

3 days after the last exposure, detectable levels of 2,4-dinitrobenzoic acid were present in urine. 

Turner et al. (1985) determined the metabolic profiles in workers exposed to DNTs. The half-life for 

excretion of DNT metabolites in urine ranged from 0.8 to 4.5 hours. The half-lives for 2,4-dinitrobenzoic 

acid and 2,4-dinitrobenzyl alcohol glucuronide tended to be shorter than those for the metabolites that 

resulted from both oxidative and reductive metabolism. 

No studies investigating elimination and excretion of 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT following 

inhalation exposure of humans or animals were located. 

3.4.4.2 Oral Exposure 

No studies were located regarding excretion in humans following oral exposure to 2,3-, 2,4-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 148 

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Schut et al. (1983) reported that in mice, urine was the main route of elimination of 3H-labeled 2,6-DNT, 

with about 50% excreted after 8 hours. U.S. Army (1978b) observed that most of the radioactivity from 

14C-labeled 2,4-DNT administered to mice was excreted in the feces and only about 10% in the urine. 

Differences between these two studies could be due, in part, to the use of different species of mice. 

Male and female rats excreted 55–90% of the radioactivity from 14C-2,4-DNT or 14C-2,6-DNT in the 

urine, and 15–30% in the feces, within 72 hours after dosing (Long and Rickert 1982; Rickert and Long 

1981). With 2,4-DNT, the females excreted a greater percentage of the dose in the urine as 

2,4-dinitrobenzylalcohol glucuronide than did the males (except at the highest dose), but with 2,6-DNT, 

no sex-related difference in urinary excretion was seen. 

In experiments with bile duct-cannulated rats, male rats excreted 25% of the radioactivity from 

14C-2,4-DNT into the bile over a 36-hour period, whereas female rats excreted 18% (Medinsky and Dent 

1983). Biliary excretion of radioactivity was linearly related to dose in males; females were evaluated 

only at one dose. Biliary excretion of radioactivity was virtually complete within 24 hours for males and 

12 hours for females. Mean half-times of biliary excretion ranged from 3.3 to 5.3 hours. Urinary 

excretion was also significant, with greater amounts of radioactivity excreted in the urine of rats from 

which bile was not collected (60–90% of the dose) than in the urine of rats from which bile was collected 

(20–60% of the dose). This finding indicates that biliary metabolites were absorbed from the intestines 

(enterohepatic cycling). Whether or not bile was collected, female rats excreted more radioactivity in 

urine than did male rats. Greater than 90% of the urinary excretion of labeled metabolites appeared in 

urine collected during the first 24 hours. At the end of 36 hours, only 0.02–0.05% of the radioactivity 

was detectable in the livers; 20–60% of this was covalently bound. 

No studies investigating elimination and excretion of 2,3-, 2,5-, 3,4-, or 3,5-DNT following oral exposure 

of humans or animals were located. 

3.4.4.3 Dermal Exposure 

There are no kinetic data in humans in which the route of exposure was specifically dermal. Occupational 

exposure studies available for Tg-DNT involved multiple routes of exposure (Levine et al. 1985b; Turner 

et al. 1985; Woollen et al. 1985). The major routes of exposure in these studies were considered to be 

inhalation and dermal. The results were discussed previously in Section 3.4.4.1. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 149 

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No studies were located regarding excretion in animals following dermal exposure to 2,3-, 2,4-, 2,5-, 2,6-, 

3,4-, or 3,5-DNT. 

3.4.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models 

Physiologically based pharmacokinetic (PBPK) models use mathematical descriptions of the uptake and 

disposition of chemical substances to quantitatively describe the relationships among critical biological 

processes (Krishnan et al. 1994). PBPK models are also called biologically based tissue dosimetry 

models. PBPK models are increasingly used in risk assessments, primarily to predict the concentration of 

potentially toxic moieties of a chemical that will be delivered to any given target tissue following various 

combinations of route, dose level, and test species (Clewell and Andersen 1985). Physiologically based 

pharmacodynamic (PBPD) models use mathematical descriptions of the dose-response function to 

quantitatively describe the relationship between target tissue dose and toxic end points. 

PBPK/PD models refine our understanding of complex quantitative dose behaviors by helping to 

delineate and characterize the relationships between: (1) the external/exposure concentration and target 

tissue dose of the toxic moiety, and (2) the target tissue dose and observed responses (Andersen and 

Krishnan 1994; Andersen et al. 1987). These models are biologically and mechanistically based and can 

be used to extrapolate the pharmacokinetic behavior of chemical substances from high to low dose, from 

route to route, between species, and between subpopulations within a species. The biological basis of 

PBPK models results in more meaningful extrapolations than those generated with the more conventional 

use of uncertainty factors. 

The PBPK model for a chemical substance is developed in four interconnected steps: (1) model 

representation, (2) model parameterization, (3) model simulation, and (4) model validation (Krishnan and 

Andersen 1994). In the early 1990s, validated PBPK models were developed for a number of 

toxicologically important chemical substances, both volatile and nonvolatile (Krishnan and Andersen 

1994; Leung 1993). PBPK models for a particular substance require estimates of the chemical substancespecific physicochemical parameters, and species-specific physiological and biological parameters. The 

numerical estimates of these model parameters are incorporated within a set of differential and algebraic 

equations that describe the pharmacokinetic processes. Solving these differential and algebraic equations 

provides the predictions of tissue dose. Computers then provide process simulations based on these 

solutions. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 150 

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The structure and mathematical expressions used in PBPK models significantly simplify the true 

complexities of biological systems. If the uptake and disposition of the chemical substance(s) are 

adequately described, however, this simplification is desirable because data are often unavailable for 

many biological processes. A simplified scheme reduces the magnitude of cumulative uncertainty. The 

adequacy of the model is, therefore, of great importance, and model validation is essential to the use of 

PBPK models in risk assessment. 

PBPK models improve the pharmacokinetic extrapolations used in risk assessments that identify the 

maximal (i.e., the safe) levels for human exposure to chemical substances (Andersen and Krishnan 1994). 

PBPK models provide a scientifically sound means to predict the target tissue dose of chemicals in 

humans who are exposed to environmental levels (for example, levels that might occur at hazardous waste 

sites) based on the results of studies where doses were higher or were administered in different species. 

Figure 3-10 shows a conceptualized representation of a PBPK model. 

If PBPK models for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT exist, the overall results and individual 

models are discussed in this section in terms of their use in risk assessment, tissue dosimetry, and dose, 

route, and species extrapolations. 

A PBPK model has not been developed for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. 

3.5 MECHANISMS OF ACTION 

3.5.1 Pharmacokinetic Mechanisms 

No information was located regarding the mechanism of absorption of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 

3,5-DNT. It is known that absorption occurs after inhalation exposure based on the metabolites found in 

the urine of workers at DNT manufacturing plants (Levine et al. 1985b; Turner 1986; Woollen et al. 

1985). In studies of rats, rabbits, dogs, and monkeys, most orally administered 2,4- or 2,6-DNT has been 

shown to be absorbed (Long and Rickert 1982; Rickert and Long 1981; U.S. Army 1978b). There 

appears to be minimal accumulation of these compounds after a single exposure. After repeated oral 

exposure in rats, 2,4-DNT and its metabolites were preferentially distributed to the liver, kidney, brain, 

lung, and skeletal muscle. The primary metabolite of 2,4-DNT excreted by humans exposed via 

inhalation and dermal routes of exposure in occupational studies or animals exposed via the oral route is 

2,4-dinitrobenzyl alcohol and/or its glucuronide (EPA 1992). In addition to this, humans also excrete 
 

 

DINITROTOLUENES 151 

3. HEALTH EFFECTS 

Figure 3-10. Conceptual Representation of a Physiologically Based 

Pharmacokinetic (PBPK) Model for a 

Hypothetical Chemical Substance
 

Inhaled chemical Exhaled chemical 

Lungs 

Liver 

Fat 

Slowly 

perfused 

tissues 

Richly 

perfused 

tissues 

Kidney 

Skin 

V 

E 

N 

O 

U 

S 

B 

L 

O 

O 

D 

A 

R 

T 

E 

R 

I 

A 

L 

B 

L 

O 

O 

D 

Vmax 

Km 

Ingestion 

GI 

Tract 

Feces 

Urine

 Chemicals 

contacting skin 

Note: This is a conceptual representation of a physiologically based pharmacokinetic (PBPK) model for a 

hypothetical chemical substance. The chemical substance is shown to be absorbed via the skin, by inhalation, or by 

ingestion, metabolized in the liver, and excreted in the urine or by exhalation. 

Source: Krishnan and Andersen 1994 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 152 

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2-amino-4-nitrobenzyl alcohol in the urine. 2,4-Dinitrobenzoic acid is another major metabolite (EPA 

1992). Both 2-nitroso-4-nitrotoluene and 2-amino-4-nitrotoluene, metabolites of 2,4-DNT in humans, 

have been shown to be mutagenic in vitro (EPA 1992). It has been suggested that these intermediates 

may bind covalently to hepatic macromolecules, such as DNA and RNA (EPA 1992). 

3.5.2 Mechanisms of Toxicity 

Effects of Metabolism on Toxicity. The primary mechanism of toxicity for DNTs involves bioactivation 

to form reactive intermediates (Kedderis et al. 1984; Sayama et al. 1989). Detailed information on the 

biotransformation of DNTs is presented in Section 3.4.3. Briefly, metabolism of DNTs begins in the 

liver, where it is oxidized by cytochrome P450 and conjugated with glucuronic acid to form the major 

metabolite, dinitrobenzyl alcohol glucuronide, and is excreted in bile or urine (Long and Rickert 1982; 

Medinsky and Dent 1983). The glucuronide excreted in bile undergoes biotransformation by intestinal 

microflora, where the conjugate is hydrolyzed and subsequently reduced by nitroreductase to the 

corresponding aminonitrobenzyl alcohol (Chadwick et al. 1993; Guest et al. 1982; Mori et al. 1985), 

probably through nitroso derivatives and hydroxylamino derivatives. The deconjugated metabolites are 

reabsorbed and transported back to the liver by enterohepatic circulation (Medinsky and Dent 1983). In 

the liver, the newly formed amine group is N-hydroxylated by cytochrome P450 and conjugated with 

sulfate (Kedderis et al. 1984). The sulfate conjugate is unstable and can be decomposed to form a 

carbonium or nitrenium ion that can be bound to hepatic macromolecules; this ostensibly leads to 

mutations and the formation of liver tumors. Thus, sulfation may be involved in the initiation stage of 

hepatocarcinogenesis by 2,6-DNT. 

Target Organ Toxicity. The mechanism of toxicity of the hematological effects of DNTs is described by 

U.S. Army (1979). The effect of DNTs on the blood is also produced by aromatic amines and most 

organic and inorganic nitrates. These compounds or their metabolites oxidize the ferrous ion in 

hemoglobin and produce methemoglobin. Hydroxylamine is probably the oxidizing species, because it is 

an intermediate in the reduction of nitro to amines. Within limits, the body can correct methemoglobinemia, but the corrective measures can be overwhelmed, producing numerous secondary effects including 

anoxia. The presence of methemoglobin leads to the formation of aggregates of hemoglobin degradation 

products called Heinz bodies. The presence of Heinz bodies is a sensitive indicator of blood toxicity as it 

indicates that some hemoglobin has been destroyed. High levels of methemoglobin are removed by 

catabolism, leading to the development of anemia. The body compensates for the destruction of red blood 

cells by increasing erythrocyte production, resulting in large numbers of immature erythrocytes, called 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 153 

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reticulocytes, in the blood. If the toxic dose is not too severe, these compensatory mechanisms suffice. 

Thus, "compensated anemia," normal erythrocyte levels with reticulocytosis, may exist in exposed 

individuals. When the production of red blood cells can no longer keep pace with the hemolysis, frank 

anemia may be present (U.S. Army 1979). 

As discussed in Section 3.2.2.7 (Oral Exposure; Cancer), 2,4-, 2,6-, or Tg-DNT have been shown to 

induce hepatocellular carcinoma following chronic-duration oral exposure. Thus, hepatic effects 

observed at less than chronic exposure durations or at lower doses may represent early stages of 

progressive development to hepatic cancer or threshold level effects for development of cancer; however, 

the mechanism of toxicity for hepatic effects has not been elucidated. A study examining changes in gene 

expression in the livers of female rats administered 2,4-DNT at 5–398 mg/kg/day or 2,6-DNT at 5– 

199 mg/kg/day via gavage as a single dose showed that treatment with these compounds perturbed 

pathways involved in DNA damage response, cell death signaling, detoxification and lipid metabolism, 

the oxidative stress response, and the immune response 24 and 48 hours after dosing (Deng et al. 2011). 

There are limited data on the mechanisms of hepatotoxicity for the six isomers. Spanggord et al. (1990) 

exposed rat hepatocytes to each of the six isomers of DNT in vitro and observed greater toxicity to 

hepatocytes (based on dose-response relationships for protein synthesis and LDH release) from 2,3-, 2,5-, 

and 3,4-DNT than from 2,4-, 2,6-, or 3,5-DNT. 

The mechanisms of toxicity for reproductive effects (testicular atrophy, degeneration of the seminal 

vesicles, and decreased sperm production) observed in animal studies (Bloch et al. 1988; U.S. Army 

1976, 1978b, 1979) have not been elucidated. However, data from in vitro studies indicate that testicular 

degeneration may be associated with structural changes in Sertoli cells (Reader and Foster 1990). Bloch 

et al. (1988) reported that diminished sperm counts in rats acutely exposed to 2,4-DNT were accompanied 

by fine structural alterations of Sertoli cells. Increased serum levels of FSH, also indicative of Sertoli cell 

malfunction, were also reported (Bloch et al. 1988). Reader and Foster (1990) reported that Sertoli cell 

cultures prepared from the testes of Wistar rats and treated with up to 100 μM 2,3-, 2,4- or 2,6-DNT 

remained intact; however, cells treated with 2,3-, 2,4-, or 2,6-DNT showed some evidence of damage 

(absence of germ cells and some Sertoli cells containing cytoplasmic vacuoles). Treatment of the Sertoli 

cell cultures with 100 μM 3,4-DNT, in contrast, resulted in subconfluent monolayers with a significant 

decrease in total cellular protein, as well as stellate appearance and prominent cytoplasmic vacuolation. 

Germ cell detachment from Sertoli germ cell cocultures was significantly increased (p<0.05) compared to 

controls at 10 μM 2,4- or 2,6-DNT and at 0.01 μM 2,3- or 3,4-DNT (Reader and Foster 1990). Further 

evidence of disruption of Sertoli cell function was observed as increased production of lactate and 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 154 

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pyruvate (including marked increases in pyruvate production with exposure to 3,4-DNT), although 

increases in pyruvate production were minimal with 2,6-DNT (Reader and Foster 1990). Another study 

showed that adult immortalized rat Sertoli cells exposed to 2,4-DNT at 25–200 μmol/L showed increased 

numbers of autophagosomes and autophagic lysosomes, which may signal the removal of cellular 

components damaged by exposure or impending apoptosis (Sorenson and Brabec 2003). Thus, it appears 

that structural changes in the Sertoli cells may be precipitating events responsible for reproductive effects 

observed in animals exposed to DNTs. 

The effects of 2,4-DNT on body weights of animals may be mediated through PPARα signaling. 

Wilbanks et al. (2014) observed decreased body weight and lower swim-to-exhaustion times in wild type 

C57Bl/6N mice exposed to 134 mg/kg/day 2,4-DNT via gavage for 14 days, while these effects were 

much less severe in exposed PPARα-null mice. Additional support for the role of PPARα signaling was 

shown by the decreases in expression of genes downstream of this signaling pathway (especially those 

involved in fatty acid transport) in wild type mice. Wilbanks et al. (2014) proposed that impairment of 

the PPARα signaling pathway was a key event in the mechanism of 2,4-DNT-induced effects on energy 

metabolism and weight loss. 

Carcinogenesis. The mechanisms of DNT-induced carcinogenicity have not been described. However, 

genotoxicity assays in vitro and in vivo indicate that DNT isomers have the potential to induce gene 

mutations and other forms of DNA damage (DNA adduct formation, chromosomal aberrations, and 

UDS). The mechanism underlying the development of renal tumors in CD-1 male mice exposed to oral 

2,4-DNT at 95 mg/kg/day for 2 years (U.S. Army 1979) has not been determined. However, workers 

exposed occupationally to explosives containing 30% Tg-DNT with renal cancer typically showed 

pathological patterns of urinary excretion, indicated by the molecular weight of excreted proteins 

(i.e., increased excretion of tubular proteins of low molecular weight and glomerular leakage with 

excretion of high molecular weight proteins), and/or specific biomarkers of damage to the proximal 

tubule of the nephron (α1-microglobulin and glutathione-S-transferase α). These data are consistent with 

the hypothesis that renal tumors are initiated by DNT isomers and that promotion occurs via damage to 

the proximal tubule of the nephron (Brüning et al. 2001). 

In hepatic tumor initiation-promotion experiments, Tg-DNT and 2,6-DNT were found to have tumorpromoting and tumor-initiating activity; 2,4-DNT showed only tumor-promoting activity (Leonard et al. 

1983, 1986; Mirsalis and Butterworth 1982; Popp and Leonard 1982). 2,6-DNT was indicated to be a 

complete hepatocarcinogen and is primarily responsible for the carcinogenic activity of Tg-DNT. Hepatic 
 

 

 

 

 

 

 

 

 

 

 

 

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DNA adducts have been detected by 32P-postlabeling technique in 2,6-DNT-treated B6C3F1 mice and 

F344 rats (George et al. 1996). In male Wistar rats administered 2,4- or 2,6-dinitrobenzyl glucuronide, 

the major compounds excreted in the bile after treatment with 2,4- or 2,6-DNT, secondary metabolism led 

to the production of genotoxic agents in the urine and bile (Mori et al. 2000). 2,4-Dinitrobenzaldehyde 

(mutagenic in the Ames assay using S. typhimurium strains TA98 and TA100) and 2,4-diaminotoluene 

(carcinogenic) were detected in the urine and bile of 2,4-dintrobenzyl glucuronide-treated rats; 2-amino6-nitrobenzyl alcohol (genotoxic) was detected in 2,6-dintrobenzyl glucuronide-treated rats. The 

formation of these metabolites may contribute to the hepatocarcinogenesis induced by 2,4- and 2,6-DNT. 

2,6-Dinitrobenzaldehyde, another metabolite of 2,6-DNT, was found to be a direct-acting mutagen in the 

S. typhimurium strain TA98 and TA100 systems, not requiring metabolic activation by the S9 mix. 

4-Amino-2-nitrobenzyl alcohol, 2-amino 4 nitrobenzyl alcohol, and 2-amino-6-nitrobenzyl alcohol are 

also mutagenic metabolites of 2,4- and 2,6-DNT, with their mutagenicity requiring metabolic activation 

(Mori et al. 1982; Sayama et al. 1989). Kedderis et al. (1984) proposed a bioactivation mechanism 

relating to the genotoxicity of 2,6-DNT in male F344 rats. They showed that the active metabolite of 

2,6-DNT in the male F344 rat is the hydroxylamino sulfate of aminonitrobenzyl alcohol formed by the 

intestinal metabolism of benzyl glucuronide of 2,6-dinitrobenzyl alcohol excreted in bile. The sulfate 

conjugate is unstable, and the formation of electrophilic carbonium or nitrenium ions from these 

conjugates leads to subsequent binding to DNA. 

3.5.3 Animal-to-Human Extrapolations 

Correlation of toxic effects between humans and animals for 2,4- and 2,6-DNT with regard to hematologic and neurological effects has been noted (Ellis et al. 1985; Hong et al. 1985; Lane et al. 1985; Lee et 

al. 1985; McGee et al. 1947; U.S. Army 1978b, 1979). Other effects for 2,4- and 2,6-DNT, such as 

reproductive, hepatic, renal, and cancer have been noted in animals (Ellis et al. 1985; Hong et al. 1985; 

Lee et al. 1985; Leonard et al. 1983, 1986; McGown et al. 1983; Stoner et al. 1984; U.S. Army 1976, 

1978b, 1979), but insufficient data are available to state definitively whether they are effects in humans. 

Two mutagenic metabolites of 2,4-DNT have been found in humans, mice, and rats (EPA 1992). 

Although rats appear to be more sensitive to the effects of 2,4-, 2,5-, 2,6-, and 3,4-DNT than mice (Hong 

et al. 1985; Lane et al. 1985; U.S. Army 1975, 1978a, 1978b; Vernot et al. 1977), dogs appear to be the 

most sensitive of the three species (Ellis et al. 1985; U.S. Army 1976, 1978b, 1979). However, limited 

intermediate-duration data using 2,6-DNT have shown mice to be more sensitive than rats (U.S. Army 

1976). It should be noted that dogs were fed DNTs by capsule in experimental studies, whereas the 
 

 

 

 

 

 

 

 

 

 

 

 

 

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rodents received the test chemical in feed (Ellis et al. 1985; Hong et al. 1985; Lee et al. 1985; U.S. Army 

1975, 1976, 1978b, 1979). 

Extrapolating animal toxicity data to predict human risk from exposure to 2,4- and 2,6-DNT appears to be 

reasonable because of qualitative similarities in metabolism and known toxic effects. Based on what is 

known about these two isomers, extrapolation from animal toxicity data to predict human risk from 

exposure to the remaining isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT) is also assumed to be reasonable. 

3.6 TOXICITIES MEDIATED THROUGH THE NEUROENDOCRINE AXIS 

Recently, attention has focused on the potential hazardous effects of certain chemicals on the endocrine 

system because of the ability of these chemicals to mimic or block endogenous hormones. Chemicals 

with this type of activity are most commonly referred to as endocrine disruptors. However, appropriate 

terminology to describe such effects remains controversial. The terminology endocrine disruptors, 

initially used by Thomas and Colborn (1992), was also used in 1996 when Congress mandated the EPA to 

develop a screening program for “...certain substances [which] may have an effect produced by a 

naturally occurring estrogen, or other such endocrine effect[s]...”. To meet this mandate, EPA convened a 

panel called the Endocrine Disruptors Screening and Testing Advisory Committee (EDSTAC), and in 

1998, the EDSTAC completed its deliberations and made recommendations to EPA concerning endocrine 

disruptors. In 1999, the National Academy of Sciences released a report that referred to these same types 

of chemicals as hormonally active agents. The terminology endocrine modulators has also been used to 

convey the fact that effects caused by such chemicals may not necessarily be adverse. Many scientists 

agree that chemicals with the ability to disrupt or modulate the endocrine system are a potential threat to 

the health of humans, aquatic animals, and wildlife. However, others think that endocrine-active 

chemicals do not pose a significant health risk, particularly in view of the fact that hormone mimics exist 

in the natural environment. Examples of natural hormone mimics are the isoflavinoid phytoestrogens 

(Adlercreutz 1995; Livingston 1978; Mayr et al. 1992). These chemicals are derived from plants and are 

similar in structure and action to endogenous estrogen. Although the public health significance and 

descriptive terminology of substances capable of affecting the endocrine system remains controversial, 

scientists agree that these chemicals may affect the synthesis, secretion, transport, binding, action, or 

elimination of natural hormones in the body responsible for maintaining homeostasis, reproduction, 

development, and/or behavior (EPA 1997). Stated differently, such compounds may cause toxicities that 

are mediated through the neuroendocrine axis. As a result, these chemicals may play a role in altering, 

for example, metabolic, sexual, immune, and neurobehavioral function. Such chemicals are also thought 
 

 

 

 

 

 

 

 

 

 

 

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to be involved in inducing breast, testicular, and prostate cancers, as well as endometriosis (Berger 1994; 

Giwercman et al. 1993; Hoel et al. 1992). 

No studies were located regarding endocrine disruption in humans and/or animals after exposure to 2,3-, 

2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT. 

No in vitro studies were located regarding endocrine disruption of 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 

3,5-DNT. 

3.7 CHILDREN’S SUSCEPTIBILITY 

This section discusses potential health effects from exposures during the period from conception to 

maturity at 18 years of age in humans, when all biological systems will have fully developed. Potential 

effects on offspring resulting from exposures of parental germ cells are considered, as well as any indirect 

effects on the fetus and neonate resulting from maternal exposure during gestation and lactation. 

Relevant animal and in vitro models are also discussed. 

Children are not small adults. They differ from adults in their exposures and may differ in their 

susceptibility to hazardous chemicals. Children’s unique physiology and behavior can influence the 

extent of their exposure. Exposures of children are discussed in Section 6.6, Exposures of Children. 

Children sometimes differ from adults in their susceptibility to hazardous chemicals, but whether there is 

a difference depends on the chemical (Guzelian et al. 1992; NRC 1993). Children may be more or less 

susceptible than adults to health effects, and the relationship may change with developmental age 

(Guzelian et al. 1992; NRC 1993). Vulnerability often depends on developmental stage. There are 

critical periods of structural and functional development during both prenatal and postnatal life, and a 

particular structure or function will be most sensitive to disruption during its critical period(s). Damage 

may not be evident until a later stage of development. There are often differences in pharmacokinetics 

and metabolism between children and adults. For example, absorption may be different in neonates 

because of the immaturity of their gastrointestinal tract and their larger skin surface area in proportion to 

body weight (Morselli et al. 1980; NRC 1993); the gastrointestinal absorption of lead is greatest in infants 

and young children (Ziegler et al. 1978). Distribution of xenobiotics may be different; for example, 

infants have a larger proportion of their bodies as extracellular water, and their brains and livers are 

proportionately larger (Altman and Dittmer 1974; Fomon 1966; Fomon et al. 1982; Owen and Brozek 
 

 

 

 

 

 

 

 

 

 

 

 

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1966; Widdowson and Dickerson 1964). The fetus/infant has an immature (developing) blood-brain 

barrier that past literature has often described as being leaky and poorly intact (Costa et al. 2004). 

However, current evidence suggests that the blood-brain barrier is anatomically and physically intact at 

this stage of development, and the restrictive intracellular junctions that exist at the blood-CNS interface 

are fully formed, intact, and functionally effective (Saunders et al. 2008, 2012). 

However, during development of the blood-brain barrier, there are differences between fetuses/infants and 

adults which are toxicologically important. These differences mainly involve variations in physiological 

transport systems that form during development (Ek et al. 2012). These transport mechanisms (influx and 

efflux) play an important role in the movement of amino acids and other vital substances across the 

blood-brain barrier in the developing brain; these transport mechanisms are far more active in the 

developing brain than in the adult. Because many drugs or potential toxins may be transported into the 

brain using these same transport mechanisms—the developing brain may be rendered more vulnerable 

than the adult. Thus, concern regarding possible involvement of the blood-brain barrier with enhanced 

susceptibility of the developing brain to toxins is valid. It is important to note however, that this potential 

selective vulnerability of the developing brain is associated with essential normal physiological 

mechanisms; and not because of an absence or deficiency of anatomical/physical barrier mechanisms. 

The presence of these unique transport systems in the developing brain of the fetus/infant is intriguing; as 

it raises a very important toxicological question as to whether these mechanisms provide protection for 

the developing brain or do they render it more vulnerable to toxic injury. Each case of chemical exposure 

should be assessed on a case-by-case basis. Research continues into the function and structure of the 

blood-brain barrier in early life (Kearns et al. 2003; Saunders et al. 2012; Scheuplein et al. 2002). 

Many xenobiotic metabolizing enzymes have distinctive developmental patterns. At various stages of 

growth and development, levels of particular enzymes may be higher or lower than those of adults, and 

sometimes unique enzymes may exist at particular developmental stages (Komori et al. 1990; Leeder and 

Kearns 1997; NRC 1993; Vieira et al. 1996). Whether differences in xenobiotic metabolism make the 

child more or less susceptible also depends on whether the relevant enzymes are involved in activation of 

the parent compound to its toxic form or in detoxification. There may also be differences in excretion, 

particularly in newborns who all have a low glomerular filtration rate and have not developed efficient 

tubular secretion and resorption capacities (Altman and Dittmer 1974; NRC 1993; West et al. 1948). 

Children and adults may differ in their capacity to repair damage from chemical insults. Children also 
 

 

 

 

 

 

 

 

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have a longer remaining lifetime in which to express damage from chemicals; this potential is particularly 

relevant to cancer. 

Certain characteristics of the developing human may increase exposure or susceptibility, whereas others 

may decrease susceptibility to the same chemical. For example, although infants breathe more air per 

kilogram of body weight than adults breathe, this difference might be somewhat counterbalanced by their 

alveoli being less developed, which results in a disproportionately smaller surface area for alveolar 

absorption (NRC 1993). 

No specific health effects resulting from DNT exposure have been observed in children. Generally, 

health effects observed in adults should also be of potential concern in children. 

No direct information is available regarding the effects of DNTs on the developmental process in humans, 

and there are few developmental studies on animals. When Tg-DNT was administered by gavage to 

pregnant rats for 14 days during gestation, and pups were evaluated for developmental toxicity either at 

gestation day 20 or postpartum day 60 (Jones-Price et al. 1982), adverse effects on hematologic 

parameters and altered organ weights were observed in both dams and fetuses when dams were 

administered 100 or 150 mg/kg/day. However, the fetal toxicity was not dose-related. A decrease in 

relative liver weight was observed in the postpartum pups at the low dose of 14 mg/kg/day. Dose-related 

effects on postnatal development were not observed in pups when dams were administered 35 or 

75 mg/kg/day. 

No consistent changes were observed in the number of preimplantation losses, implantation sites, or 

living or non-living fetuses in male Sprague-Dawley rats gavaged with 2,4 DNT at 0, 60, 180, or 

240 mg/kg/day for 5 days (Lane et al. 1985). Exposure of male animals to DNTs does not cause 

dominant lethal mutation or increases in the proportion of nonviable conception (U.S. Army 1979). 

DNTs have been found to be genotoxic using in vivo test systems (Ashby et al. 1985; Huang et al. 1995; 

Mirsalis et al. 1989). Although 2,6-DNT itself showed no mutagenicity towards S. typhimurium strains 

TA98 and TA100 with or without activation by S9 mix, 2,6-dinitrobenzaldehyde, a metabolite of 

2,6-DNT, was found to be a direct-acting mutagen, not requiring metabolic activation (Sayama et al. 

1989). The reason that DNTs are not shown to bind to DNA and cause mutations in most of the shortterm in vitro assays for genotoxicity is that the formation of DNA-reactive DNT metabolites involved 

several different biotransforming enzymes in the intestinal microflora and in the liver. However, DNTs 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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did not cause dominant lethal mutation or increases in the proportion of nonviable conceptions following 

exposure of male animals (U.S. Army 1979), so it is not clear if the genotoxic form of DNT might 

potentially reach the germ cells following oral, inhalation, or dermal exposure. 

It is unlikely that DNTs and its metabolites will accumulate in maternal tissues because of its low octanolwater partition coefficient. No studies are available that demonstrate DNTs or their metabolites cross the 

placenta or get into breast milk. Thus, it is unlikely that the developing fetus or nursing infant would be 

exposed to DNTs as a consequence of maternal exposure prior to gestation. However, developmental 

toxicity from DNTs could potentially occur because of its ability to deplete the amount of oxygen 

available to the developing fetus. Pregnant women and their fetuses may be susceptible to the oxygen 

depletion implied by the hematotoxicity of DNTs based on a study of rats (Jones-Price et al. 1982). 

Newborns have a transient deficiency in methemoglobin reductase which reduces methemoglobin back to 

hemoglobin (Gruener 1976). They also have a high concentration of fetal hemoglobin in their 

erythrocytes (Smith 1996). Thus, newborns are unusually sensitive to methemoglobin-generating 

chemicals such as DNT. The metabolism of DNTs has not been studied in children or appropriate animal 

models. However, while some of the enzymes involved in DNT metabolism reach or exceed adult levels 

during infancy and early childhood, other enzymes such as UDP-glucuronosyltransferase may attain adult 

levels by 6–18 months of age (Leeder and Kearns 1997). Thus, the toxicity of DNTs may be different in 

children. 

3.8 BIOMARKERS OF EXPOSURE AND EFFECT 

Biomarkers are broadly defined as indicators signaling events in biologic systems or samples. They have 

been classified as markers of exposure, markers of effect, and markers of susceptibility (NAS/NRC 

1989). 

A biomarker of exposure is a xenobiotic substance or its metabolite(s) or the product of an interaction 

between a xenobiotic agent and some target molecule(s) or cell(s) that is measured within a compartment 

of an organism (NAS/NRC 1989). The preferred biomarkers of exposure are generally the substance 

itself, substance-specific metabolites in readily obtainable body fluid(s), or excreta. However, several 

factors can confound the use and interpretation of biomarkers of exposure. The body burden of a 

substance may be the result of exposures from more than one source. The substance being measured may 

be a metabolite of another xenobiotic substance (e.g., high urinary levels of phenol can result from 

exposure to several different aromatic compounds). Depending on the properties of the substance (e.g., 
 

 

 

 

 

 

 

 

 

 

 

 

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biologic half-life) and environmental conditions (e.g., duration and route of exposure), the substance and 

all of its metabolites may have left the body by the time samples can be taken. It may be difficult to 

identify individuals exposed to hazardous substances that are commonly found in body tissues and fluids 

(e.g., essential mineral nutrients such as copper, zinc, and selenium). Biomarkers of exposure to 2,3-, 

2,4-, 2,5-, 2,6-, 3,4-, and 3,5-DNT are discussed in Section 3.8.1. 

Biomarkers of effect are defined as any measurable biochemical, physiologic, or other alteration within an 

organism that, depending on magnitude, can be recognized as an established or potential health 

impairment or disease (NAS/NRC 1989). This definition encompasses biochemical or cellular signals of 

tissue dysfunction (e.g., increased liver enzyme activity or pathologic changes in female genital epithelial 

cells), as well as physiologic signs of dysfunction such as increased blood pressure or decreased lung 

capacity. Note that these markers are not often substance specific. They also may not be directly 

adverse, but can indicate potential health impairment (e.g., DNA adducts). Biomarkers of effects caused 

by DNTs are discussed in Section 3.8.2. 

A biomarker of susceptibility is an indicator of an inherent or acquired limitation of an organism's ability 

to respond to the challenge of exposure to a specific xenobiotic substance. It can be an intrinsic genetic or 

other characteristic or a preexisting disease that results in an increase in absorbed dose, a decrease in the 

biologically effective dose, or a target tissue response. If biomarkers of susceptibility exist, they are 

discussed in Section 3.10, Populations That Are Unusually Susceptible. 

There are no biomarkers of exposure or effects that have been validated in children or in adults exposed 

as children. 

3.8.1 Biomarkers Used to Identify or Quantify Exposure to DNTs 

Workers exposed to DNTs in a manufacturing plant excreted 2,4-DNT, 2,6-DNT, and their metabolites in 

the urine (Levine et al. 1985b). The concentrations of DNT in air ranged from 0.1 to 5.9 mg/m3

. 

Concentrations of DNTs and metabolites ranged from 1.68 to 16.74 mg/day (or 1.74–17.31 mg/L, based 

on an average daily urine volume of 967 mL), with widespread daily variations. Estimates of inhaled 

DNTs ranged from 0.5 to 4.9 mg/day, less than the total excreted suggesting that dermal exposure, and 

possibly oral exposure, contributed to the body burden of DNTs. Jones et al. (2005b) did not find a 

significant correlation between the levels of air concentrations of 2,4- and 2,6-DNT and the sum of urine 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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metabolites among workers at a DNT and mononitrotoluene manufacturing facility. The 8-hour TWA air 

concentrations were 0.043 and 0.014 mg/m3 for 2,4- and 2,6-DNT, respectively. 

Woollen et al. (1985) determined that the urinary concentration of a Tg-DNT metabolite, 2,4-dinitrobenzoic acid, was <1 mg/L at the beginning of the work week and ranged from 3.4 to 41 mg/L at the end 

of the shift. Atmospheric DNT levels of undetectable to 0.03 mg/m3 were monitored with personal air 

samples. Static samples near dusty process areas monitored were 0.02–2.68 mg/m3

. The study authors 

estimate that inhalation exposures ranged from 1 to 14 mg/day. As in Levine et al. (1985b), inhalation 

exposure does not account for the entire amount of DNT metabolites excreted in urine. Dermal and 

ingestion exposures are, therefore, likely to have occurred. 

Significant correlations between urinary levels of hemoglobin adducts and urinary metabolites were found 

for 2,4- and 2,6-DNT in workers exposed to Tg-DNT (Sabbioni et al. 2006). The hemoglobin adducts 

resulting in exposure to 2,4-DNT were 4-amino-2-nitrotoluene and 2,4-toluenediamine; the hemoglobin 

adducts from 2,6-DNT exposure were 2-amino-6-nitrotoluene and 2,6-toluenediamine. The best 

correlations between urinary metabolites and hemoglobin adducts were 2,4-dinitrobenzyl alcohol and 

2,6-dinitrobenzyl alcohol with hemoglobin 2-4-nitrotoluene and hemoglobin-2-amino-6-nitrotoluene, 

respectively. 

3.8.2 Biomarkers Used to Characterize Effects Caused by DNTs 

After exposure to DNTs, methemoglobin levels in the blood may be elevated (Bentur and Keyes 2004). 

The methemoglobinemia present may be quite profound and its onset is often delayed by up to 4 hours 

(Bentur and Keyes 2004). Another hematological change that might be present in individuals who have 

undergone repeated or prolonged exposure to DNTs is that which is consistent with Heinz bodies and 

hemolytic anemia. 

DNA adducts have been found in the livers of rats treated orally with either 2,4- or 2,6-DNT (La and 

Froines 1992, 1993). The formation of DNA adducts is believed to be indicative of carcinogenic risk. 

Decreased spermatogenesis has been reported in treated rats, mice, and dogs (Bloch et al. 1988; U.S. 

Army 1979). However, decrease in sperm counts in workers exposed to DNTs has been reported in only 

one study (CDC 1981). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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3.9 INTERACTIONS WITH OTHER CHEMICALS 

Perkins (1919) reported that “alcoholic subjects have very little resistance to DNT.” 

Exposure of male rats to 2,6-DNT for 5 days reduced the rate of metabolism of phenobarbital; exposure to 

2,6-DNT for 4 weeks increased phenobarbital metabolism (Short and Lee 1980). Exposure of rats to 

2,4-DNT did not affect the rate of phenobarbital metabolism. 

The effects of the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), on 2,6-DNT genotoxicity were 

examined in male weanling F344 rats (George et al. 1992). The rats were treated orally with 54.4 mg/kg 

2,4,5-T for 4 weeks, then with 75 mg/kg 2,6-DNT, 1, 2, or 4 weeks after the first dose of 2,4,5-T; urine 

was then collected for 24 hours. In animals treated for 1 week with 2,4,5-T, there was a decrease in 

transformation of 2,6-DNT to mutagenic metabolites in the urine, but there were no changes in intestinal 

enzyme activities (George et al. 1992). Longer treatments with 2,4,5-T did not alter urine genotoxicity 

compared to controls, and there was a transient increase in cecal azo reductase and nitroreductase after 

2 weeks with a decrease in intestinal β-glucuronidase activity, but all levels were normal after 4 weeks. 

Pretreatment of rats with alachlor for 3 weeks prior to a single oral administration of 75 mg/kg 2,6-DNT 

resulted in the production of genotoxic metabolites, as evaluated in a S. typhimurium histidine reversion 

bioassay in the absence of S9 activation (George et al. 1998). Significant increases, compared to 

2,6-DNT-only exposed rats, were not observed in rats exposed to alachlor for 1 or 5 weeks. 

Interaction of DNTs with other chemicals has not been observed in children. 

3.10 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE 

A susceptible population will exhibit a different or enhanced response to DNTs than will most persons 

exposed to the same level of DNTs in the environment. Reasons may include genetic makeup, age, health 

and nutritional status, and exposure to other toxic substances (e.g., cigarette smoke). These parameters 

result in reduced detoxification or excretion of DNTs, or compromised function of organs affected by 

DNTs. Populations who are at greater risk due to their unusually high exposure to DNTs are discussed in 

Section 6.7, Populations with Potentially High Exposures. 

Several studies have examined the possible association between DNT toxicity and genetic 

polymorphisms. Sabbioni et al. (2006) found that workers exposed to DNTs and TNT with the 
 

 

 

 

 

 

 

 

 

 

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glutathione S-transferase T1 null genotype or N-acetyltransferase 1 fast acetylators genotype had 

significantly more chromatid-type chromosomal aberrations than controls of the same genotypes. 

Workers with the sulfotransferase 1A1 Arg/Arg or sulfotransferase 1A2 Asn/Asn genotypes or 

N-acetyltransferase 1 fast acetylators had significantly more total aberrations than controls with the same 

genotypes. These results suggest differences in susceptibility to DNT toxicity. In contrast, Brüning et al. 

(1999) found that all of the workers at a copper mine using Tg-DNT explosives with urothelial cancer 

were slow acetylators. 

Humans sensitive to DNTs may include individuals with cardiovascular problems. Hematological effects 

associated with exposure to 2,4-DNT may place persons with anemia, including sickle cell anemia or 

other diseases of the blood, at an increased risk. 

Persons with chronic neurological disorders may also have an increased sensitivity to DNT exposure. 

Although there are insufficient data available to draw firm conclusions, it appears that pregnant women 

and their fetuses may be susceptible to the oxygen depletion implied by the hematotoxicity of DNTs 

based on a study on rats (Jones-Price et al. 1982). Although it has been reported that alcoholics may have 

a decreased resistance to the effects of Tg-DNT (Perkins 1919), the extent of this compromise has not 

been determined. 

The susceptibility of children to the health effects of DNTs may be different from that of adults, as 

discussed in Section 3.7. 

3.11 METHODS FOR REDUCING TOXIC EFFECTS 

This section will describe clinical practice and research concerning methods for reducing toxic effects of 

exposure to DNTs. However, because some of the treatments discussed may be experimental and 

unproven, this section should not be used as a guide for treatment of exposures to DNTs. When specific 

exposures have occurred, poison control centers and medical toxicologists should be consulted for 

medical advice. The following texts provide specific information about treatment following exposures to 

DNTs: 

Bentur Y, Keyes DC. 2004. Explosives. In: Dart RC, ed. Medical Toxicology. 3rd ed. Philadelphia, 

PA: Lippincott Williams & Wilkins, 1258-1279. 

Currance PL, Clements B, Bronstein AC. 2007. Dinitrophenol and related compounds. In: Emergency 

care for hazardous materials exposure. St. Louis, MO: Mosby Jems, 335-338. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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There are no known pediatric-specific methods for reducing peak absorption following exposure, 

reducing burden, or interfering with the mechanism of action for toxic effects. 

3.11.1 Reducing Peak Absorption Following Exposure 

Limited information from humans indicates that DNTs is absorbed after inhalation exposure, while 

animal data suggest that DNTs are rapidly and completely absorbed after oral exposure. Efforts to reduce 

absorption following acute exposure to DNTs should focus on removing the individual from the site of 

exposure and decontaminating exposed areas of the body. Contaminated clothing and jewelry should be 

removed and skin should be washed with soap and water; eyes exposed to DNTs should be immediately 

irrigated with water and normal saline (Bentur and Keyes 2004; Currance et al. 2007). If ingestion of 

DNTs occurs, emetics are not recommended due to the potential for central nervous system depression; 

the mouth should be rinsed, and water can be administered for dilution if the patient can swallow, has a 

good gag reflex, and is not drooling (Bentur and Keyes 2004; Currance et al. 2007). Gastric lavage and 

administration of activated charcoal may be considered shortly following ingestion provided airways are 

protected (Bentur and Keyes 2004; Currance et al. 2007). 

3.11.2 Reducing Body Burden 

There are no data to support the use of hemodialysis, forced diuresis, hyperbaric oxygen, or 

hemoperfusion for treatment of methemoglobinemia alone, but these treatments may provide adjunctive 

care after DNT ingestion when supportive care is inadequate (Bentur and Keyes 2004). 

3.11.3 Interfering with the Mechanism of Action for Toxic Effects 

Methods for reducing DNT-induced methemoglobinemia (Bentur and Keyes 2004) are the same as those 

recommended for treating methemoglobinemia caused by overexposure to nitrate and/or nitrite (Agency 

for Toxic Substances and Disease Registry 2013; Leikin and Paloucek 2008; Seifert 2004). Severe 

methemoglobinemia (methemoglobin levels generally >30% of total hemoglobin) can be reduced by 

intravenous administration of methylene blue (1–2 mg/kg). Exchange transfusions may be considered for 

patients who do not respond to methylene blue (particularly patients with glucose-6-phosphate 

dehydrogenase deficiency or hemoglobin M), and patients where methylene blue is contraindicated (e.g., 

patients on serotonin uptake inhibitors). In symptomatic patients, 100% oxygen and assisted ventilation 

should be considered; seizures can be treated with oxygen and benzodiazepines, followed by 
 

 

 

 

 

 

 

 

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phenobarbital (Seifert 2004). Hyperbaric oxygen therapy may be of some benefit, but has not been 

demonstrated in controlled studies (Leikin and Paloucek 2008; Seifert 2004). 

3.12 ADEQUACY OF THE DATABASE 

Section 104(I)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the 

Administrator of EPA and agencies and programs of the Public Health Service) to assess whether 

adequate information on the health effects of DNTs is available. Where adequate information is not 

available, ATSDR, in conjunction with the National Toxicology Program (NTP), is required to assure the 

initiation of a program of research designed to determine the health effects (and techniques for developing 

methods to determine such health effects) of DNTs. 

The following categories of possible data needs have been identified by a joint team of scientists from 

ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would 

reduce the uncertainties of human health assessment. This definition should not be interpreted to mean 

that all data needs discussed in this section must be filled. In the future, the identified data needs will be 

evaluated and prioritized, and a substance-specific research agenda will be proposed. 

3.12.1 Existing Information on Health Effects of DNTs 

The existing data on health effects of inhalation, oral, and dermal exposure of humans and animals to 

2,4-DNT; 2,6-DNT; 2,3-, 2,5-, 3,4-, and 3,5-DNT; and Tg-DNT are summarized in Figures 3-11, 3-12, 

3-13, and 3-14, respectively. The purpose of these figures is to illustrate the existing information 

concerning the health effects of 2,4-, 2,6-, 2,3-, 2,5-, 3,4-, 3,5-, and Tg-DNT. Each dot in the figure 

indicates that one or more studies provide information associated with that particular effect. The dot does 

not necessarily imply anything about the quality of the study or studies, nor should missing information in 

this figure be interpreted as a “data need”. A data need, as defined in ATSDR’s Decision Guide for 

Identifying Substance-Specific Data Needs Related to Toxicological Profiles (Agency for Toxic 

Substances and Disease Registry 1989), is substance-specific information necessary to conduct 

comprehensive public health assessments. 

Generally, ATSDR defines a data gap more broadly as any substance-specific information missing from 

the scientific literature. 
 

DINITROTOLUENES 167 

3. HEALTH EFFECTS 

Figure 3-11. Existing Information on Health Effects of 2,4-Dinitrotoluene 

Inhalation 

Oral 

Dermal 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer 

Systemic 

Inhalation 

Oral 

Dermal 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer 

Systemic 

Human 

Animal 

Existing Studies 
 

DINITROTOLUENES 168 

3. HEALTH EFFECTS 

Figure 3-12. Existing Information on Health Effects of 2,6-Dinitrotoluene 

Inhalation 

Oral 

Dermal 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer 

Systemic 

Inhalation 

Oral 

Dermal 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer 

Systemic 

Human 

Animal 

Existing Studies 
 

DINITROTOLUENES 169 

3. HEALTH EFFECTS 

Figure 3-13. Existing Information on Health Effects of 2,3-, 2,5-, 3,4-, and 

3,5-Dinitrotoluene 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic 

Neurologic

Reproductive

Developmental 

Genotoxic

Cancer 

Syste mic 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic 

Neurologic

Reproductive

Developmental 

Genotoxic

Cancer 

Syste mic 

Inhalation 

Oral 

Dermal 

Human 

Inhalation 

Oral 

Dermal 

Animal 

Existing Studies 
 

DINITROTOLUENES 170 

3. HEALTH EFFECTS 

Figure 3-14. Existing Information on Health Effects of Technical-Grade 

Dinitrotoluene
 

Inhalation 

Oral 

Dermal 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer 

Systemic 

Death 

Acute 

Intermediate

Chronic 

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer 

Systemic 

Animal 

Human 

Inhalation 

Oral 

Dermal 

Existing Studies 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 171 

3. HEALTH EFFECTS 

3.12.2 Identification of Data Needs 

There are no data on the health effects of 2,3-, 2,5-, 3,4-, or 3,5-DNT in humans by any route of exposure. 

As shown in Figures 3-11 (2,4-DNT), 3-9 (2,6-DNT), and 3-11 (Tg-DNT), there are limited data on 

health effects in humans, primarily for Tg-DNT, following inhalation exposure. With the exception of 

animal oral LD50 values, the animal data for 2,3-, 2,5-, 3,4-, and 3,5-DNT are limited to a single 14-day 

gavage study in rats (Figure 3-13). 

The available reports generally lack quantitative information on exposure levels. Human data are 

particularly sparse. Most toxicity studies have focused on the main systemic effects of obvious clinical 

significance, as described in the previous sections. 

The toxicity of these chemicals has been extensively investigated in animals after oral exposure, but not 

after inhalation exposure, and only in a very limited way after dermal exposure. The potential 

carcinogenicity of these chemicals has been investigated following oral exposure in typical chronic 

bioassays as well as in less-than-lifetime studies. 

Acute-Duration Exposure. Although there are no human data available from acute-duration oral 

exposure to 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-DNT, data currently available from animal studies of 2,3-, 

2,4-, 2,5-, 2,6-, and 3,4-DNT are appropriate for evaluation of oral toxicity (Lent et al. 2012a; USAPHC 

2011a, 2011b, 2011c, 2011d, 2011e, 2011f; U.S. Army 1975, 1978a; Vernot et al. 1977). In gavage 

studies in rats and mice, LD50 values of 1,120 and 1,070 mg/kg, respectively, were identified for 2,3-DNT 

(Vernot et al. 1977). The LD50 values for 2,4-DNT determined after gavage dosing ranged from 270 to 

650 mg/kg in rats and from 1,340 to 1,954 mg/kg in mice (U.S. Army 1975, 1978a; Vernot et al. 1977). 

Gavage dosing of 2,5-DNT identified LD50 values of 710 mg/kg in rats and 1,230 mg/kg in mice (Vernot 

et al. 1977). After oral administration of 2,6-DNT, LD50 values ranged from 180 to 795 mg/kg in rats and 

from 621 to 807 mg/kg in mice (U.S. Army 1975, 1978a; Vernot et al. 1977). Ataxia was observed in 

these animals before death. Oral LD50 values of 1,070 and 1,410 mg/kg 3,4-DNT in rats and mice were 

determined after gavage dosing (Vernot et al. 1977). Decreased body weight gain was noted in rats 

administered 2,4-DNT via gavage at 398 mg/kg and observed for 48 hours and in rats administered 

2,6-DNT at ≥50 mg/kg and observed for 48 hours; body weight loss occurred at 199 mg/kg 2,6-DNT 

(Deng et al. 2011). After 14 days of gavage administration of 2,4-, 2,6-, or 3,4-DNT, decreased body 

weights were observed at doses of 142, 35, and 227 mg/kg/day, respectively (Lent et al. 2012a USAPHC 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 172 

3. HEALTH EFFECTS 

2011b, 2011d, 2011e). Slight cyanosis was observed in rats administered 60 mg/kg 2,4-DNT by gavage 

for 5 days (Lane et al. 1985), but no changes in hematological parameters were found in rats fed up to 

273 mg/kg/day in the diet for 14 days (McGown et al. 1983). Evidence of erythrocytosis (increased red 

blood cells, hemoglobin, and hematocrit) was observed in rats administered 2,4-DNT at ≥99 mg/kg and in 

rats administered 2,6-DNT at 199 mg/kg at 24 and/or 48 hours after dosing (Deng et al. 2011). Increased 

incidences of splenic extramedullary hematopoiesis were seen in male Sprague-Dawley rats exposed by 

gavage for 14 days to DNT isomers; the LOAELs for this effect were 275, 71, 39, 68, and 57 mg/kg/day 

for 2,3-, 2,4-, 2,5-, 2,6-, and 3,4-DNT; 3,4-DNT did not induce this effect at doses up to 39 mg/kg/day 

(Lent et al. 2012a USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f). Hepatic effects, including 

increased blood cholesterol and alanine aminotransferase levels, and renal effects, such as hyaline droplet 

accumulation, were observed in rats fed 2,4-DNT in the diet for 14 days (McGown et al. 1983). Sinusoid 

congestion was noted in the livers of rats treated with 2,4-DNT at 398 mg/kg and in rats treated with 

2,6-DNT at 199 mg/kg and evaluated at 24 or 48 hours; levels of albumin in the serum were also 

increased in rats treated with 2,4-DNT at 99 mg/kg (Deng et al. 2011). In studies by Lent et al. (2012a; 

USAPHC 2011a, 2011b, 2011c, 2011d, 2011e, 2011f) of male rats exposed by gavage to DNT isomers 

for 14 days, single cell necrosis and increased glycogen deposition were observed at 36 mg/kg/day 

2,4-DNT; hepatocellular and oval cell hyperplasia, along with hepatocellular hypertrophy were observed 

at 35 mg/kg/day 2,6-DNT. The other isomers did not induce hepatic effects at the doses up to those 

causing lethality. Effects on the male reproductive tract, including decreased organ weights and testicular 

lesions, were seen in rats exposed for 14 days by gavage to doses of 142, 68, and 19 mg/kg/day of 2,4-, 

2,6-, and 3,5-DNT, respectively (Lent et al. 2012a; USAPHC 2011b, 2011d, 2011f). 

There were no acute-duration inhalation or dermal studies in humans available for evaluation. The LD50 

value determined after exposure to 2,6-DNT as an aerosol in rats (combined males and females) was 

0.43 mg/L (0.24 mg/L for males and 0.66 mg/L for females) (CMA 1991). Treated rats showed evidence 

of respiratory distress (exaggerated breathing, ataxia, and lethargy); rats that died experienced lung 

congestion and increased relative lung weights. Both 2,4- and 2,6-DNT were shown to be mild primary 

dermal irritants in rabbits (U.S. Army 1975, 1978a). Additional acute inhalation studies and dermal 

studies would be useful for determining route-specific toxicity. 

Intermediate-Duration Exposure. Currently available animal studies using repeated-dose exposure 

are appropriate for evaluation of oral toxicity for both 2,4- and 2,6-DNT (Ellis et al. 1985; Hazleton 

Laboratories 1977, 1982; Hong et al. 1985; Jones-Price et al. 1982; Lee et al. 1985; McGown et al. 1983; 

U.S. Army 1976, 1978b, 1979); no data were located regarding repeated-dose toxicity for 2,3-, 2,5-, 3,4-, 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 173 

3. HEALTH EFFECTS 

or 3,5-DNT. Methemoglobinemia and its sequelae (Heinz bodies, anemia, reticulocytosis), 

hemosiderosis, extramedullary hematopoiesis, and cyanosis have been observed in animals after oral 

treatment with 2,4-, 2,6-, or Tg-DNT (Hazleton Laboratories 1977, 1982; U.S. Army 1976, 1978b). Mild 

hepatocellular dysplasia was observed in mice fed 2,4-DNT in the diet for 13 weeks (Hong et al. 1985; 

U.S. Army 1978b), but no hepatotoxicity was observed after 2,4-DNT administration to rats or dogs for 

the same duration (U.S. Army 1978a, 1978b). However, treatment with 2,6-DNT did cause bile duct 

hyperplasia in rats and mice (U.S. Army 1976). This lesion, as well as hepatic degeneration, was 

observed in dogs dosed with 2,6-DNT (U.S. Army 1976). 

Oral administration of 2,4-DNT to rats, mice, or dogs for 13 weeks did not cause any significant adverse 

renal effects (Hong et al. 1985; U.S. Army 1978b). Administration of 2,6-DNT to dogs for 13 weeks 

caused renal inflammation and degeneration, which were not observed in rats or mice (U.S. Army 1976). 

Decreased body weight gain or weight loss was observed in rats and mice after administration of 

2,4-DNT (Hong et al. 1985; Lee et al. 1985; Leonard et al. 1987; NCI 1978; U.S. Army 1978b, 1979) and 

in rats, mice, and dogs after administration of 2,6-DNT (U.S. Army 1976). Subchronic inhalation would 

be useful for determination of toxic effects in order to derive an MRL for 2,4-DNT and to determine a 

mechanism of action from routes of exposure that are more characteristic of occupational exposure. 

Chronic-Duration Exposure and Cancer. There are no data available in humans regarding the 

carcinogenicity of 2,3-, 2,5-, 2,6-, 3,4-, or 3,5-DNT. A retrospective cohort mortality study performed 

using data from workers at ammunition plants that used 2,4-DNT or Tg-DNT found no increases in 

mortality due to either malignant neoplasms as a whole or from particular cancers (Levine et al. 1986b). 

However, the small cohort examined in this study limited its statistical power. Both 2,4- and 2,6-DNT 

have been found to cause hepatocellular carcinoma in rats (Leonard et al. 1987; U.S. Army 1979). Renal 

cancer was observed in mice after administration of 2,4-DNT in the diet (U.S. Army 1979). EPA has 

derived a cancer slope factor for oral exposure to 2,4-DNT/2,6-DNT mixture (IRIS 2003). No animal 

data pertaining to the carcinogenicity of 2,3-, 2,5-, 3,4-, or 3,5-DNT were located. 

Excessive mortality rates from ischemic heart disease and residual diseases of the circulatory system were 

observed in ammunition plant workers (Levine et al. 1986a). Because it is expected that these workers 

would have a lower incidence of cardiovascular disease due to the healthy worker effect, this finding is 

unusual. Further epidemiological studies to verify these findings are needed; newer analyses should 

control for risk factors, particularly cigarette smoking, which was not done by the Levine et al. (1986a) 

study. 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 174 

3. HEALTH EFFECTS 

No data were located regarding the chronic toxicity of 2,3-, 2,5-, 3,4-, or 3,5-DNT. The currently 

available studies in laboratory animals on the effects of 2,4- and 2,6-DNT after chronic exposure are 

appropriate for evaluation of chronic oral toxicity (Ellis et al. 1985; Hazleton Laboratories 1982; Lee et 

al. 1985; Leonard et al. 1978; NCI 1978; U.S. Army 1978b, 1979). Hematological effects, including 

anemia, compensatory anemia, methemoglobinemia, and Heinz bodies, have been observed after chronic 

administration of 2,4-DNT to dogs, mice, and rats (Ellis et al. 1985; Hong et al. 1985; U.S. Army 1978b, 

1979). Data were insufficient for the derivation of a chronic-duration oral MRL for 2,6-DNT. Severe 

hepatocellular changes, such as degeneration and vacuolation and dysplasia, were found in rats, mice, and 

dogs administered 2,4- or 2,6-DNT for chronic durations in oral exposure studies (Ellis et al. 1985; Hong 

et al. 1985; Leonard et al. 1987; U.S. Army 1979). Renal cystic dysplasia was observed in mice, but not 

rats or dogs, treated orally with 2,4-DNT for chronic-duration periods (Hong et al. 1985; U.S. Army 

1979). Chronic-duration studies have not been performed in mice using 2,6-DNT to determine whether 

these findings would also result after administration of this isomer. Although no histopathological effects 

were found in adrenal, pituitary, or thyroid glands of rats after chronic oral administration of Tg-DNT, 

increases in parathyroid hyperplasia, fatty metamorphosis, and vascular ectasia were found (Hazleton 

Laboratories 1982). Further studies may be useful to verify these findings. Effects on body weight, 

including body weight loss, were reported in almost all chronic-duration oral studies (Ellis et al. 1985; 

Hazleton Laboratories 1982; Hong et al. 1985; Leonard et al. 1987; NCI 1978; U.S. Army 1979). 

A well-controlled chronic inhalation study and dermal studies would be useful for determination of the 

potential for route-specific toxicity. In addition, for both 2,4- and 2,6-DNT, well-controlled 

epidemiological evaluations of larger occupationally exposed populations would contribute valuable 

insights regarding the human relevancy of chronic health effects observed in animal studies. 

Genotoxicity. All isomers of DNT (2,3-, 2,4-, 2,6-, 3,4-, and 3,5-DNT) have caused gene mutations in 

one or more reverse mutation assays using S. typhimurium (Couch et al. 1981; Dellarco and Prival 1989; 

Spanggord et al. 1982b; Tokiwa et al. 1981; U.S. Army 1978a). However, the test system has given 

variable results because of the need for metabolic activation and the sensitivity of the tester strains. In 

vivo assays using 2,4-DNT have shown UDS and S-phase synthesis using rat hepatocytes (Ashby et al. 

1985; Mirsalis and Butterworth 1982; Mirsalis et al. 1989), chromosomal aberrations using human 

lymphocytes (Huang et al. 1995), and DNA binding in rat hepatocytes (Chadwick et al. 1993; La and 

Froines 1993). The genotoxicity of Tg-DNT is believed to be due to the potent genotoxicity of 2,6-DNT, 

as evidenced in an in vivo-in vitro hepatocyte UDS system (Mirsalis and Butterworth 1982). Both 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 175 

3. HEALTH EFFECTS 

2,4- and 2,6-DNT have induced DNA adducts in rat liver (La and Froines 1992, 1993). Studies currently 

available for 2,4- and 2,6-DNT are considered to be appropriate for evaluation of genotoxicity. 

Reproductive Toxicity. The currently available laboratory data on reproductive toxicity are 

considered appropriate for evaluation of oral exposure of animals to both isomers. Several studies in rats, 

mice, and dogs with either isomer have shown impairment of the male reproductive system. The effects 

observed include testicular atrophy, degeneration of the seminal vesicles, and decreased sperm production 

(Bloch et al. 1988; U.S. Army 1976, 1978b, 1979). In vitro studies have shown that the testicular 

degeneration is due, at least in part, to structural changes in Sertoli cells (Reader and Foster 1990). 

Animal studies of reproductive toxicity using inhalation exposure would provide information relative to 

occupational exposure conditions. 

Several assessments of reproductive function in exposed workers have been performed that did not detect 

differences in sperm production or fertility rates as a result of exposure (Hammill et al. 1982; Levine et al. 

1985a; NIOSH 1982). However, an earlier study reported a significant reduction in the sperm counts of 

exposed workers, as well as an increase, of marginal statistical significance, in the number of spontaneous 

abortions in their wives (NIOSH 1980). These studies were all limited by the small exposure populations 

studied and the lack of historical individual exposure monitoring. Further epidemiological studies of 

larger exposed occupational populations with exposure data may be considered useful since questions of 

potential reproductive effects associated with these exposures have not yet been clearly resolved. 

Developmental Toxicity. No data are available regarding developmental effects in humans after oral 

exposure to DNTs, but animal studies that have been performed show possible developmental effects. 

The only developmental effect observed in a three-generation reproductive study in rats using 2,4-DNT 

was a decrease in pup viability. This decrease was attributed to maternal neglect and a high incidence of 

maternal death during parturition. Tg-DNT administered to pregnant dams caused a decrease in relative 

liver weight in postpartum pups and possible transient neurotoxicity (Jones-Price et al. 1982). Further 

studies may be useful to elucidate these effects. Additional animal studies using 2,4- and 2,6-DNT by 

oral and inhalation routes should analyze fetal and maternal blood for hematological parameters. This is 

recommended because any factor that could reduce the amount of oxygen to developing tissue is expected 

to have adverse consequences in the offspring. 

Immunotoxicity. Although no data are available regarding immunological or lymphoreticular effects 

in humans, some data on these end points are available in animals. The currently available information 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 176 

3. HEALTH EFFECTS 

on the potential immunotoxic effects of 2,4- and 2,6-DNT is sufficient to describe the sensitizing potential 

of DNTs. Mild sensitization has been reported in guinea pigs after dermal exposure to 2,6-DNT, but not 

2,4-DNT (U.S. Army 1975, 1978a). No effects on IgE, the antibody associated with allergic or 

hypersensitive reactions, were reported in rats or dogs exposed to either the 2,4- or the 2,6-DNT isomer 

(Ellis et al. 1985; Lee et al. 1985; U.S. Army 1976, 1978b). Studies have not been performed that would 

describe effects on immunocompetence following exposure to DNTs. A battery of immunotoxicity tests 

would provide a better assessment of possible effects in humans. 

Neurotoxicity. The nervous system has been shown to be a major target of 2,4- and 2,6-DNT toxicity 

in animals (Ellis et al. 1985; Kozuka et al. 1979; Lee et al. 1979, 1985; U.S. Army 1979). Clinical signs 

in dogs have included incoordination and stiffness of the hind legs leading to complete paralysis; 

cerebellar vacuolation, hypertrophy, and focal gliosis; and cerebellar and brain stem hemorrhage. In 

mice, depression and hyperexcitability were observed, while some rats administered 2,4-, 2,6-, 3,4-, and 

3,5-DNT showed neuromuscular symptoms (including facial twitching, ataxia, and/or limb paralysis). 

Acute-duration exposure of rats to 3,5-DNT resulted in inflammatory infiltrates in the brain. More 

systematic examination of the neurological effects of these compounds in laboratory animals would be 

useful to fully assess behavioral abnormalities and morphological damage to the nervous system. 

Although results of an in vitro study in neuroblastoma cells show that 2,4-DNT is cytotoxic and produced 

cell death (Banerjee et al. 1999), the biochemical mechanisms of DNT neurotoxicity is not known. 

Generalized symptoms of neurotoxicity, including headache, sleepiness, dizziness, and tingling pain in 

the extremities were reported in workers occupationally exposed to 2,4-DNT (McGee et al. 1947; Perkins 

1919). However, the more recent occupational studies performed failed to examine workers for 

symptoms of neurotoxicity (Hammill et al. 1982; Levine et al. 1985a; NIOSH 1980, 1982). Because the 

early reports of potential neurotoxicity in exposed workers have not been followed-up in more recent 

studies, neurological examination of workers in occupational studies could provide additional information 

regarding the potential magnitude of neurotoxic effects. 

Epidemiological and Human Dosimetry Studies. Epidemiology studies of workers exposed to 

DNTs suggest a potential for heart disease in exposed populations (Levine et al. 1986a). Doses of DNTs 

associated with heart disease in humans have not been determined. Further studies with historical cohort 

monitoring data and control for potentially confounding factors such as concomitant exposure to other 

chemicals and cigarette smoking would be useful to verify these findings. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 177 

3. HEALTH EFFECTS 

Animal studies have indicated that the male reproductive system is a target of DNT toxicity. 

Epidemiological studies have provided only suggestive evidence of a reproductive effect in workers 

exposed to DNTs. Studies of larger worker populations may help to determine more conclusively the 

magnitude of the potential for reproductive toxicity in exposed humans. 

Other effects that were observed in animal studies but not confirmed in human populations include liver 

and kidney toxicity, neurotoxicity, and cancer. Well-controlled epidemiological studies examining these 

end points in humans would be useful. 

Biomarkers of Exposure and Effect 

Exposure. A rapid, accurate method for determining exposure to DNTs has been developed using 

spectrophotometric analysis of complexes of primary arylamines, which result from the reduction of 

DNTs and its metabolites (Smith et al. 1995). 

Effect. Epidemiological studies that correlate quantitative estimates of exposure with disease outcomes 

would be useful. Studies that identify subtle physiological changes, such as altered blood chemistry 

indices, associated with a particular disease state are not available. 

A disease registry is not currently available. The development of a registry of exposures and diseases 

would provide a useful reference tool for assessing the variations in exposure concentrations and health 

effects from, for example, geography, season, regulatory actions, presence of hazardous waste landfills, or 

manufacturing and use facilities. These assessments, in turn, would provide a better understanding of the 

needs for some types of research or data acquisition based on the current exposure concentrations. 

Absorption, Distribution, Metabolism, and Excretion. The toxicokinetics of 2,4- and 2,6-DNT 

in rats by the oral route have been extensively studied. That Tg-DNT is absorbed and excreted in the 

urine by humans in an occupational setting (where the main routes of absorption are considered to be 

inhalation and dermal) has also been documented. There are no data available in animals on the 

toxicokinetics of DNTs by the dermal or inhalation routes. Toxicokinetics studies in rats administered the 

test materials by the inhalation and dermal routes would be critical in understanding possible differences 

in the toxicity of DNTs by different routes of administration. The main routes of exposure of humans are 

dermal and inhalation. Understanding the possible differences in toxicity in animals by different routes 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 178 

3. HEALTH EFFECTS 

would be valuable in determining the significance of findings in humans who may be exposed by 

inhalation or dermal routes. 

Comparative Toxicokinetics. Absorption and excretion studies in several species indicate that there 

are considerable differences between mice and the other species evaluated. More detailed study of the 

metabolism of DNTs by mice, including the role of biliary excretion and enterohepatic cycling, would 

assist in understanding why the metabolism in mice is different from other species and which species may 

be the most appropriate model for evaluating hazards and risks to humans. 

Methods for Reducing Toxic Effects. The most important method for reducing the toxic effects of 

DNTs is removal of the person from the area of exposure. Skin and eyes should be rinsed copiously 

(Bronstein and Currance 1994), although absorption through the skin has not been adequately examined. 

Gastric lavage, with subsequent administration of activated charcoal, and cathartics may be of some 

benefit in reducing peak absorption after oral exposure to DNTs. Methylene blue treatment is used with 

patients presenting with serious methemoglobinemia (Bentur and Keyes 2004). No additional studies are 

considered necessary at this time to examine further methods for reducing body burden of DNTs. Further 

studies on supportive therapy after DNT exposure, such as the use of hemodialysis, forced diuresis, 

hyperbaric oxygen, or hemoperfusion might be useful. 

Children’s Susceptibility. Data needs relating to both prenatal and childhood exposures, and 

developmental effects expressed either prenatally or during childhood, are discussed in detail in the 

Developmental Toxicity subsection above. 

There is inadequate experimental evidence to evaluate if the pharmacokinetics of DNTs are different in 

children. There are no studies on whether DNTs or their active metabolites can cross the placenta or be 

excreted in breast milk, so it cannot be determined if fetuses may be exposed in utero or if infants may be 

exposed via breast milk ingestion. There are also no data to show if DNTs and their metabolites are 

stored in maternal tissues and thus might be later mobilized during gestation or lactation; however, DNTs 

and their metabolites are not likely to be stored because of their low octanol-water partition coefficient. 

There is little experimental evidence to evaluate whether the metabolism of DNTs or their mechanisms of 

action are different in children. As discussed in Section 3.7, newborns are highly sensitive to the 

methemoglobin-generating effect of DNTs because of their deficiency in methemoglobin reductase 

(Gruener 1976), which reduces methemoglobin back to hemoglobin. In addition, newborns have a high 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 179 

3. HEALTH EFFECTS 

concentration of fetal hemoglobin in their erythrocytes. It will be useful to determine if fetal hemoglobin 

is more sensitive to the methemoglobin-generating effect of DNTs. It will also be helpful to have data on 

the metabolism and mechanism of action of DNTs on children to determine if children are more 

vulnerable than adults to health effects from exposure to DNTs, as some enzymes involved in DNT 

metabolism are known to have developmental regulation. There are no biomarkers of exposure or effect 

that have been validated in children or in adults exposed as children. There are no data to determine 

whether there are any interactions with other chemicals unique to children, or whether interactions 

observed in adults also occur in children. Although DNTs are shown to be genotoxic, it is not known if 

parental exposure to DNT may affect children via parental germ cells, or if DNTs may indirectly affect 

the fetus during maternal exposure. 

Child health data needs relating to exposure are discussed in Section 6.8.1, Identification of Data Needs: 

Exposures of Children. 

3.12.3 Ongoing Studies 

No ongoing studies were identified for DNTs. 
 

DINITROTOLUENES 180 

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DINITROTOLUENES 181 

4. CHEMICAL AND PHYSICAL INFORMATION 

4.1 CHEMICAL IDENTITY 

Information regarding the chemical identity of the individual isomers of DNT is located in Table 4-1. 

4.2 PHYSICAL AND CHEMICAL PROPERTIES 

Information regarding the physical and chemical properties of the individual isomers of DNT is located in 

Table 4-2. Data regarding specific isomers of DNT have been provided whenever possible. The isomers 

of DNT have many similar traits, including identical molecular weights, but also have distinguishable 

qualities. For instance, 2,4-DNT has higher melting and boiling points and a greater solubility in water 

than 2,6-DNT (HSDB 2012). 

DNTs are generally produced as a technical-grade mixture, which consists of approximately 76.5% 

2,4-DNT and 18.8% 2,6-DNT. The remaining ~5% consists of other isomers of DNT and minor 

contaminants such as TNT and the mononitrotoluenes (HSDB 2012). Unspecified forms of DNT (DNT 

not otherwise specified or NOS) can be characterized under the CAS Registry Number of 25321-14-6. 

Where information pertains to Tg-DNT or DNT NOS, it has been so noted. 
Table 4-1. Chemical Identity of Dinitrotoluenes 

Characteristic Informationa 

Chemical name 2,3-Dinitrotoluene 2,4-Dinitrotoluene 2,5-Dinitrotoluene 

Synonym(s) 1-Methyl­ 1-Methyl­ 2-Methyl2,3-dinitrobenzene; 2,4-dinitrobenzene; 1,4-dinitrobenzene; 

2,3-DNT 2,4-dinitrotoluol; 2,5-DNT 

2,4-DNT 

Registered trade name(s) No data No data No data 

Chemical formula C7H6N2O4 C7H6N2O4 C7H6N2O4 

Chemical structure 

NO2 NO2 NO2 

NO2 

O2N 

NO2 

Identification numbers: 

CAS registry 602-01-7 121-14-2 619-15-8 

NIOSH RTECS XT1400000 XT1575000 XT1750000 

EPA hazardous waste 

No data U105 No data 

OHM/TADS 7800118 

DOT/UN/NA/IMDG shipping IMO 6.1 IMO 6.1 IMO 6.1 

UN 1600 (molten) UN 1600 (molten) UN 1600 (molten) 

UN 2038 (solid or UN 2038 (solid or UN 2038 (solid or 

liquid) liquid) liquid) 

HSDB 5499 1144 5504 

NCI No data NCI-C01865 No data 

DINITROTOLUENES 182 

4. CHEMICAL AND PHYSICAL INFORMATION 
Table 4-1. Chemical Identity of Dinitrotoluenes 

Characteristic Informationa 

Chemical name 2,6-Dinitrotoluene 3,4-Dinitrotoluene 3,5-Dinitrotoluene 

Synonym(s) 1-Methyl­ 4-Methyl­ 1-methyl2,6-dinitrotoluene; 1,2-Dinitrobenzene; 3,5-dinitrobenzene; 

2,6-DNT 3,4-DNT 3,5-DNT 

Registered trade name(s) No data No data No data 

Chemical formula C7H6N2O4 C7H6N2O4 C7H6N2O4 

Chemical structure 

O2N NO2 

NO2 

O2N NO2 

NO2 

Identification numbers: 

CAS registry 606-20-2 610-39-9 618-85-9 

NIOSH RTECS XT1925000 XT2100000 XT2150000 

EPA hazardous waste 

U106 No data No data 

OHM/TADS 8300219 

DOT/UN/NA/IMDG shipping IMO 6.1 IMO 6.1 IMO 6.1 

UN 1600 (molten) UN 1600 (molten) UN 1600 (molten) 

UN 2038 (solid or UN 2038 (solid or UN 2038 (solid or 

liquid) liquid) liquid) 

HSDB 2931 5501 6463 

NCI No data No data No data 

 

DINITROTOLUENES 183 

4. CHEMICAL AND PHYSICAL INFORMATION 

aAll information obtained from HSDB 2012 

CAS = Chemical Abstracts Service; DOT/UN/NA/IMDG = Department of Transportation/United Nations/North 

America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency; 

HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NIOSH = National Institute for 

Occupational Safety and Health; OHM/TADS = Oil and Hazardous Materials/Technical Assistance Data System; 

RTECS = Registry of Toxic Effects of Chemical Substances 
DINITROTOLUENES 184 

4. CHEMICAL AND PHYSICAL INFORMATION 

Table 4-2. Physical and Chemical Properties of Dinitrotoluenes 

Property Informationa 

Chemical name 2,3-Dinitrotoluene 2,4-Dinitrotoluene 2,5-Dinitrotoluene 

Molecular weight 182.14 182.14 182.14 

Color Yellow Yellow No data 

Physical state Solid Solid Solid 

Melting point 59–61 °C 71 °C 52.5 °C 

Boiling point 284 °Cb 300 °C (slight 

decomposition) 

284 °Cb 

Density No data 1.3208 (71 °C) 1.282 (111 °C) 

Odor No data Slight No data 

Odor threshold: 

Water No data No data No data 

Air No data No data No data 

Solubility: 

Water 220 mg/L (25 °C, 

estimated)b 

270 mg/L (22 °C) 220 mg/L (25 °C, 

estimated)b 

Organic solvents No data Soluble in acetone, 

alcohol, benzene, 

ethanol, diethyl ether, 

pyridine, carbon 

disulfide 

Soluble in ethanol; very 

soluble in carbon 

disulfide 

Partition coefficients: 

Log Kow 2.18 (estimated)b 1.98 2.18 (estimated)b 

Log Koc No data 2.56c No data 

Vapor pressure at 20 °C 3.97x10-4 torr 

(estimated)d 

1.4x10-4 torr 3.97x10-4 torr 

(estimated)d 

Henry's law constant at 25 °C 

Autoignition temperature 9.26x10-8 atm-m3/mol 

(estimated)b 

5.4x10-8 atm-m3/mole 9.26x10-8 atm-m3/mol 

(estimated)b 

Flashpoint 

Flammability limits No data No data No data 

Conversion factors No data 404 °F 404 °F 

No data No data No data 

Explosive limits 1 ppm=7.40 mg/m3 

1 mg/m3=0.13 ppm 

1 ppm=7.40 mg/m3 

1 mg/m3=0.13 ppm 

1 ppm=7.40 mg/m3 

1 mg/m3=0.13 ppm 
DINITROTOLUENES 185 

4. CHEMICAL AND PHYSICAL INFORMATION 

Table 4-2. Physical and Chemical Properties of Dinitrotoluenes 

Property Informationa 

Chemical name 2,6-Dinitrotoluene 3,4-Dinitrotoluene 3,5-Dinitrotoluene 

Molecular weight 182.14 182.14 182.14 

Color Yellow to red Red Yellow to red 

Physical state Solid Solid Solid 

Melting point 66 °C 58.3 °C 93 °C 

Boiling point 285 °C 284 315 °Cb 

Density 1.2833 (111 °C) 1.2594 (111 °C) 1.2772 (111 °C) 

Odor Slight Slight No data 

Odor threshold: 

Water No data No data No data 

Air No data No data No data 

Solubility: 

Water 180 mg/L (20 °C) 100 mg/L (25 °C)f 145 mg/L (25 °C) 

Organic solvents Soluble in ethanol, 

chloroform 

Soluble in ethanol and 

carbon disulfide; 

slightly soluble in 

chloroform 

Soluble in benzene, 

ethyl ether, ethanol, 

chloroform, carbon 

disulfide 

Partition coefficients: 

Log Kow 2.10 2.08g 2.18 (estimated)b 

Log Koc No data No data No data 

Vapor pressure at 20 °C 5.67x10-4 torr 3.97x10-4 torr 

(estimated)d 

4.05x10-4 torrh 

Henry's law constant at 25 °C 

Autoignition temperature 7.47x10-7 atm-m3/mol 

(estimated)b 

9.26x10-8 atm-m3/mol 

(estimated)b 

9.26x10-8 atm-m3/mol 

(estimated)b 

Flashpoint 

Flammability limits No data No data No data 

Conversion factors 404 °F 404 °F No data 

No data No data No data 

Explosive limits 1 ppm=7.40 mg/m3 

1 mg/m3=0.13 ppm 

1 ppm=7.40 mg/m3 

1 mg/m3=0.13 ppm 

1 ppm=7.40 mg/m3 

1 mg/m3=0.13 ppm 

aAll information obtained from HSDB 2012 unless otherwise stated. 

bEPA 2011 

cU.S. Army 1980

dNeely and Blau 1985 

eAltschuh et al. 1999 

f

Chemicals Inspection Testing Institute 1992 

gNakagawa et al. 1992

hAIChE 1992 
DINITROTOLUENES 186 

4. CHEMICAL AND PHYSICAL INFORMATION 

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DINITROTOLUENES 187 

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

5.1 PRODUCTION 

Tables 5-1 and 5-2 list the facilities in each state that manufacture or process 2,4- and 2,6-DNT, 

respectively, and the intended use and range of maximum amounts of 2,4- and 2,6-DNT that are stored on 

site. The data listed in Tables 5-1 and 5-2 are derived from the Toxics Release Inventory (TRI13 2015). 

Only certain types of facilities were required to report; therefore, this is not an exhaustive list. In 2013, 

there were facilities located in nine states that produced, processed or used 2,4-DNT and facilities located 

in four states that produced, processed, or used 2,6-DNT. The number of individual facilities and the 

amount produced on site varied in each state. The other isomers of DNT are not contained in the TRI 

database. 

5.2 IMPORT/EXPORT 

No information was found concerning U.S. imports and exports of DNTs. 

According to the 2011 Directory of Chemical Producers, 2,4- and 2,6-DNT were domestically 

manufactured by three corporations including Air Products and Chemicals, Inc. (production site: 

Pasadena, Texas), the BASF Corporation (production site: Geismar, Louisiana) and Bayer Material 

Science, LLC (production site: Baytown, Texas) (SRI 2011). No information regarding the other 

isomers of DNT was included in the directory. While no production volumes were reported, data 

collected by the EPA Inventory Update Reporting (IUR) system indicated that <500,000 pounds of 

2,4-DNT were produced domestically in 2006 (EPA 2012a). The United Nations Screening Information 

Data Set (SIDS) Initial Assessment Report for Dinitrotoluene (mixed isomers) reported that the global 

production capacity of DNT is about 1.6 Mio t (1.6 million tons) annually (UNEP 2004). 

2,4- and 2,6-DNT are generally produced as a mixture called Tg-DNT, which contains approximately 

76.5% 2,4-DNT and 18.8% 2,6-DNT (with the remainder consisting of other isomers and minor 

contaminants such as TNT and mononitrotoluenes) (HSDB 2012). This mixture is commercially 

prepared in a two-step process. Toluene is nitrated with concentrated sulfuric and nitric acid resulting in 

the production of mononitrotoluene (U.S. Army 1987). Mononitrotoluene can be nitrated with mixed 

acids to yield dinitrotoluene (EPA 2008a). 
DINITROTOLUENES 188 

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

Table 5-1. Facilities that Produce, Process, or Use 2,4-Dinitrotoluene 

Minimum Maximum 

Number of amount on site amount on site 

Statea facilities in poundsb in poundsb Activities and usesc 

IA 1 100,000 999,999 8 

IL 1 1,000 9,999 8 

IN 3 0 999 12 

KY 1 100,000 999,999 12 

LA 1 0 99 14 

OH 1 1,000 9,999 12 

TX 3 1,000 9,999 8, 12 

UT 1 10,000 99,999 12 

VA 2 100,000 999,999 7 

aPost office state abbreviations used. bAmounts on site reported by facilities in each state. cActivities/Uses: 

1. Produce 6. Impurity 11. Chemical Processing Aid 

2. Import 7. Reactant 12. Manufacturing Aid 

3. Onsite use/processing 8. Formulation Component 13. Ancillary/Other Uses 

4. Sale/Distribution 9. Article Component 14. Process Impurity 

5. Byproduct 10. Repackaging 

Source: TRI13 2015 (Data are from 2013) 
DINITROTOLUENES 189 

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

Table 5-2. Facilities that Produce, Process, or Use 2,6-Dinitrotoluene 

Minimum Maximum 

Number of amount on site amount on site 

Statea facilities in poundsb in poundsb Activities and usesc 

IN 1 100 999 12 

KY 1 10,000 99,999 12 

OH 1 1,000 9,999 12 

TX 1 0 99 8 

aPost office state abbreviations used. bAmounts on site reported by facilities in each state. cActivities/Uses: 

1. Produce 6. Impurity 11. Chemical Processing Aid 

2. Import 7. Reactant 12. Manufacturing Aid 

3. Onsite use/processing 8. Formulation Component 13. Ancillary/Other Uses 

4. Sale/Distribution 9. Article Component 14. Process Impurity 

5. Byproduct 10. Repackaging 

Source: TRI13 2015 (Data are from 2013) 
 

 

 

 

 

 

 

DINITROTOLUENES 190 

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

5.3 USE 

The most commercially important use of DNTs is as a chemical intermediate in the production of TDI. 

TDI is a precursor to polyurethane polymers (HSDB 2012). Additionally, DNTs are used in the 

production of TNT, as a plasticizer in propellants, and as a waterproofing, plasticizing, and gelatinizing 

agent in explosives (HSDB 2012). DNTs are also used as intermediates in the production of dyes and as 

modifiers for smokeless powders in the munitions industry (HSDB 2012). 

5.4 DISPOSAL 

2,4-DNT and 2,6-DNT are listed as toxic substances under Section 313 of the Emergency Planning and 

Community Right to Know Act (EPCRA) under Title III of the Superfund Amendments and 

Reauthorization Act (SARA) (EPA 1995). Disposal of wastes containing DNTs is controlled by a 

number of federal regulations (see Chapter 8). 

Limited information is available regarding the appropriate disposal of DNTs. NIOSH recommends that 

small quantities be swept onto paper or other flammable material and incinerated in a suitable combustion 

chamber. Larger quantities should be reclaimed; if this is not practical, then they should be dissolved in 

fuel oil and atomized in a suitable combustion chamber (HSDB 2012). The ultimate disposal of DNTs 

can be achieved by controlled incineration in an incinerator unit equipped with an alkaline scrubber 

(HSDB 2012). DNTs have also been proposed as a potential candidate for rotary kiln incineration at 820– 

1,600 °C or fluidized bed incineration at 450–980 °C, with residence times of seconds for gases and 

liquids and longer for solids (HSDB 2012). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 191 

6. POTENTIAL FOR HUMAN EXPOSURE 

6.1 OVERVIEW 

2,4- and/or 2,6-DNT have been identified in at least 98 of the 1,699 hazardous waste sites that have been 

proposed for inclusion on the EPA National Priorities List (NPL) (HazDat 2007). Information on the 

other isomers was not available. The number of sites evaluated for DNTs is not known. The frequency 

of these sites can be seen in Figure 6-1. 

The available data provide a complex and incomplete view of the overall potential for human exposure to 

isomers of DNT. Little direct knowledge of the magnitude of environmental exposure pathways exists. 

Data regarding exposure of humans to DNTs have been obtained primarily from the workplace. 

DNTs have been found in waste water and groundwater in and around munitions sites (Jenkins et al. 

1986), and 4-nitrotoluene and dinitrobenzene, structural analogues of DNTs, are taken up by plants 

(McFarlane et al. 1987). However, predictions of environmental exposure pathways based on 

measurements of structural analogues of DNTs are severely limited by the complex abiotic reactions of 

DNTs in the environment and by the different pathways, rates, and products of biological reduction 

and/or oxidation of 2,4- and 2,6-DNT. 

The relatively low log octanol-water partition coefficients (log Kow) of the DNT isomers (1.98–2.18) 

suggest that DNTs released to the environment would not bioaccumulate. Measured bioconcentration 

factors also indicate that DNTs are not expected to bioaccumulate in fish and other aquatic species (Lang 

1997; NITE 2002). DNTs are not highly adsorbed to soil or sediment and may leach from the soil surface 

to groundwater. DNTs are degraded by oxidation, photolysis, and biotransformation in water or soil, 

resulting in a variety of degradation products. Volatilization is expected to occur slowly from water and 

soil surfaces and the rate of hydrolysis is negligible. 

Studies of occupational exposures to DNTs indicate that inhalation and dermal contact can result in 

absorption of DNTs into the body. The general population is not expected to be exposed to DNTs unless 

they reside near a source area, in which case, dermal contact and incidental ingestion pathways are the 

likely routes of exposure. 
DINITROTOLUENES 192

6. POTENTIAL FOR HUMAN EXPOSURE

Figure 6-1. Frequency of NPL Sites with 2,4- and 2,6-Dinitrotoluene

Contamination

Frequency 

of 

NPL Sites 

Derived from HazDat 

2007

1 

2 

3 

4 

5 

6-7
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 193 

6. POTENTIAL FOR HUMAN EXPOSURE 

6.2 RELEASES TO THE ENVIRONMENT 

The Toxics Release Inventory (TRI) data should be used with caution because only certain types of 

facilities are required to report (EPA 2005). This is not an exhaustive list. Manufacturing and processing 

facilities are required to report information to the TRI only if they employ 10 or more full-time 

employees; if their facility is included in Standard Industrial Classification (SIC) Codes 10 (except 1011, 

1081, and 1094), 12 (except 1241), 20–39, 4911 (limited to facilities that combust coal and/or oil for the 

purpose of generating electricity for distribution in commerce), 4931 (limited to facilities that combust 

coal and/or oil for the purpose of generating electricity for distribution in commerce), 4939 (limited to 

facilities that combust coal and/or oil for the purpose of generating electricity for distribution in 

commerce), 4953 (limited to facilities regulated under RCRA Subtitle C, 42 U.S.C. section 6921 et seq.), 

5169, 5171, and 7389 (limited S.C. section 6921 et seq.), 5169, 5171, and 7389 (limited to facilities 

primarily engaged in solvents recovery services on a contract or fee basis); and if their facility produces, 

imports, or processes ≥25,000 pounds of any TRI chemical or otherwise uses >10,000 pounds of a TRI 

chemical in a calendar year (EPA 2005). 

Section 112 of the Clean Air Act (CAA) lists 2,4-DNT as one of the original hazardous air pollutants 

(HAPs) known to cause, or suspected of causing, cancer or other serious human health effects or 

ecosystem damage (EPA 2000b). EPA's National Emission Inventory (NEI) database collects 

information about sources that emit criteria air pollutants and their precursors, and HAPs for the 

50 United States, Washington DC, Puerto Rico, and the U.S. Virgin Islands (prior to 1999, criteria 

pollutant emission estimates were maintained in the National Emission Trends [NET] database and HAP 

emission estimates were maintained in the National Toxics Inventory [NTI] database). The NEI database 

derives emission data from multiple sources including state and local environmental agencies; the TRI 

database; computer models for on-road and off-road emissions; and databases related to EPA's Maximum 

Achievable Control Technology (MACT) programs to reduce emissions of hazardous air pollutants. 

Using composite data from the NTI database from 1990 to 1993, it was estimated that the annual 

emissions of 2,4-DNT in the United States was approximately 3.5 tons per year during that time frame 

(EPA 2000b). Data downloaded from the 2005 NEI indicated that the total emission of 2,4-DNT was 

approximately 4 tons, with the biggest source arising from nonpoint source waste disposal. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 194 

6. POTENTIAL FOR HUMAN EXPOSURE 

6.2.1 Air 

Estimated releases of 7,621 pounds (~3.5 metric tons) of 2,4- and 2,6-DNT to the atmosphere from nine 

domestic manufacturing and processing facilities in 2013, accounted for about 99% of the estimated total 

environmental releases from facilities required to report to the TRI (TRI13 2015). These releases are 

summarized in Tables 6-1 and 6-2. The TRI database does not contain information regarding the other 

isomers of DNT. 

The Great Lakes Regional Air Toxic Emissions Inventory Project estimated that 1,894 pounds of 

2,4-DNT were emitted to the air of the Great Lakes watershed in 2002 (Great Lakes Commission 2006). 

The overwhelming majority of these emissions arose from local point and area sources such as production 

or use facilities. No data were available for the other isomers of DNT. 

Minute amounts of nitrotoluene are formed by the photochemical reaction of toluene, nitrogen oxides, and 

sunlight (Atkinson et al. 1980). Although DNTs could be formed subsequently, they would be subject to 

photolysis and would not be likely to accumulate enough to contribute significantly to human exposure. 

6.2.2 Water 

Estimated releases of 2 pounds (~0.001 metric tons) of 2,4- and 2,6-DNT to the water from nine domestic 

manufacturing and processing facilities in 2013, accounted for about 0.3% of the estimated total 

environmental releases from facilities required to report to the TRI (TRI13 2015). These releases are 

summarized in Tables 6-1 and 6-2. The TRI database does not contain information regarding the other 

isomers of DNT. 

The detection of DNTs in water has been reported (Feltes et al. 1990; Shackelford and Keith 1976; 

Staples et al. 1985). Both 2,4- and 2,6-DNT are recognized as major components in waste waters from 

TNT manufacturing facilities (Spanggord and Suta 1982; Spanggord et al. 1982a). DNTs occur in 

samples of TNT waste waters at concentrations of 0.04–48.6 mg/L (2,4-DNT) and 0.06–14.9 mg/L 

(2,6-DNT). The occurrence of DNTs in waste waters from other manufacturing uses (e.g., polyurethane 

forms) has not been reported. The frequency of detection of DNTs in surface waters, as indicated in the 

STORET database (Staples et al. 1985), is low. Slightly over 1% of the stations reported detectable 

quantities of DNTs, and the median of positive samples was <10 μg/L (0.01 mg/L). The presence of 

DNTs was not detected in samples of sediment or biota. 
DINITROTOLUENES 195 

6. POTENTIAL FOR HUMAN EXPOSURE 

Table 6-1. Releases to the Environment from Facilities that Produce, Process, or
 

Use 2,4-Dinitrotoluenea
 

Reported amounts released in pounds per yearb 

Total release 

Statec RFd Aire Waterf UI

g Landh Otheri On-sitej Off-sitek On- and off-site 

IA 1 500 0 0 0 0 500 0 500 

IL 1 0 0 0 0 0 0 0 0 

IN 3 2 0 0 0 0 2 0 2 

KY 1 5,500 0 0 0 0 5,500 0 5,500 

LA 1 0 0 0 0 0 0 0 0 

OH 1 2 0 0 21 0 2 21 23 

TX 3 14 0 0 0 0 14 0 14 

UT 1 2 0 0 0 0 2 0 2 

VA 2 0 2 0 0 1 2 1 3 

Total 14 6,020 2 0 21 1 6,022 22 6,044 

aThe TRI data should be used with caution since only certain types of facilities are required to report. This is not an 

exhaustive list. Data are rounded to nearest whole number.
 bData in TRI are maximum amounts released by each facility.
 cPost office state abbreviations are used.
 dNumber of reporting facilities.
 eThe sum of fugitive and point source releases are included in releases to air by a given facility.
 f

Surface water discharges, waste water treatment-(metals only), and publicly owned treatment works (POTWs) (metal
 

and metal compounds).
 gClass I wells, Class II-V wells, and underground injection.
 hResource Conservation and Recovery Act (RCRA) subtitle C landfills; other onsite landfills, land treatment, surface 

impoundments, other land disposal, other landfills.
 i

Storage only, solidification/stabilization (metals only), other off-site management, transfers to waste broker for
 

disposal, unknown.
 j

The sum of all releases of the chemical to air, land, water, and underground injection wells.
 kTotal amount of chemical transferred off-site, including to POTWs.
 

RF = reporting facilities; UI = underground injection 

Source: TRI13 2015 (Data are from 2013) 
 

DINITROTOLUENES 196 

6. POTENTIAL FOR HUMAN EXPOSURE 

Table 6-2. Releases to the Environment from Facilities that Produce, Process, or
 

Use 2,6-Dinitrotoluenea
 

Reported amounts released in pounds per yearb 

Total release 

Statec RFd Aire Waterf UI

g Landh Otheri On-sitej Off-sitek On- and off-site 

IN 1 1 0 0 0 0 1 0 1 

KY 1 1,600 0 0 0 0 1,600 0 1,600 

OH 1 0 0 0 21 0 0 21 21 

TX 1 0 0 0 0 0 0 0 0 

Total 4 1,601 0 0 21 0 1,601 21 1,622 

aThe TRI data should be used with caution since only certain types of facilities are required to report. This is not an 

exhaustive list. Data are rounded to nearest whole number.
 bData in TRI are maximum amounts released by each facility.
 cPost office state abbreviations are used.
 dNumber of reporting facilities.
 eThe sum of fugitive and point source releases are included in releases to air by a given facility.
 f

Surface water discharges, waste water treatment-(metals only), and publicly owned treatment works (POTWs) (metal
 

and metal compounds).
 gClass I wells, Class II-V wells, and underground injection.
 hResource Conservation and Recovery Act (RCRA) subtitle C landfills; other onsite landfills, land treatment, surface 

impoundments, other land disposal, other landfills.
 i

Storage only, solidification/stabilization (metals only), other off-site management, transfers to waste broker for
 

disposal, unknown
 j

The sum of all releases of the chemical to air, land, water, and underground injection wells.
 kTotal amount of chemical transferred off-site, including to POTWs.
 

RF = reporting facilities; UI = underground injection 

Source: TRI13 2015 (Data are from 2013) 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 197 

6. POTENTIAL FOR HUMAN EXPOSURE 

6.2.3 Soil 

Estimated releases of 42 pounds (~0.019 metric tons) of 2,4- and 2,6-DNT to soils from 6 domestic 

manufacturing and processing facilities in 2013, accounted for about 0.6% of the estimated total 

environmental releases from facilities required to report to the TRI (TRI13 2015). The TRI database does 

not contain information regarding the other isomers of DNT. There were no reported releases to 

underground injection wells. These releases are summarized in Tables 6-1 and 6-2. 

The extensive use of DNTs as intermediates in the synthesis of TDI and polyurethane foam is not a 

reported source of releases to soil. However, DNTs can be contaminants in the soil from explosives, 

propellants, etc. Residues of DNTs were observed to be deposited onto the surface of military live-fire 

training soil (Jenkins 2006). As a result, mean surface soil concentrations ranged from <0.001 to 

84 mg/kg for 2,4-DNT and from <0.001 to 4.6 mg/kg for 2,6-DNT (Jenkins 2006). 

6.3 ENVIRONMENTAL FATE 

6.3.1 Transport and Partitioning 

The water solubilities of DNTs are moderate (Callahan et al. 1979), and the octanol-water partition 

coefficients are low (EPA 1982c). As a result, there is a potential for DNTs to leach from soil into 

groundwater. 

The solubility of nitroaromatic compounds in seawater and pure water at different temperatures was 

examined by Luning Prak and O’Sullivan (2007) and Kholod et al. (2011). Luning Prak and O’Sullivan 

(2007) found that the solubility in both seawater and pure water was observed to increase with increasing 

temperatures (5–40 °C) and decrease with increasing salinity (0–100%) for 2,3-DNT and 2,6-DNT. 

Kholod et al. (2011) found that the Conductor-like Screening model for Real Solvents (COSMO-RS) was 

effective in estimating the solubility of 2,5-DNT. The same trends that were observed in the experimental 

values were also observed in the estimated values. These solubility models are useful in the 

characterization of the fate of nitro compounds in marine systems. 

U.S. Army (1980) determined partitioning of 2,4-DNT onto environmental media and found that the soil 

organic carbon partition coefficient (Koc), the octanol-water partition coefficient (log Kow), and the 

partition bioconcentration factor (Kb) were 364, 2, and 64, respectively. EPA (1982c) calculated 

sediment-water partitioning coefficients of 45 and 92 for 2,4- and 2,6-DNT, respectively. Depending on 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 198 

6. POTENTIAL FOR HUMAN EXPOSURE 

the nature of the sediment load, the total concentration of DNTs carried in the soil and water column 

could be high. The low Henry’s Law constants for the isomers of DNT (see Table 4-2) suggest that 

volatilization from water and moist soil surfaces will occur slowly. U.S. Army (1980) used 

experimentally determined volatilization rate constants of 2,4-DNT obtained under laboratory conditions, 

to extrapolate a volatilization rate constant for 2,4-DNT in an environmental aquatic water body with a 

depth of approximately 6.5 feet and ambient temperature of about 20 °C. Using this extrapolated rate 

constant, a volatilization half-life of >400 days is estimated for 2,4-DNT. 

Steady-state whole-fish bioconcentration factors (BCFs) of 2,4-DNT measured in carp during two 

different equilibrium periods were 9.15 and 4.15 (Lang et al. 1997). BCF values of DNT (mixed isomers 

CAS Registry Number 25321-14-6) measured in carp over a 6-week incubation period were 0.6–2.9 at a 

nominal concentration of 0.25 ppm and 3.2–21.2 at a nominal concentration of 0.025 ppm (NITE 2002). 

BCF values of <2.7 were also measured for 3,4-DNT in carp over a 6-week incubation period (NITE 

2002). These data suggest that bioaccumulation in fish and other aquatic organisms will be low. 

Direct measurement of plant uptake of DNTs has not been made, but plant uptake is predicted to occur 

based on its low octanol-water partition coefficient. Structural analogy with 1,3-dinitrobenzene and 

4-nitrotoluene (McFarlane et al. 1987) suggests that 2,4- and 2,6-DNT would be readily taken up by 

plants. Plant uptake of related nitroaromatic compounds such as 2,4,6-TNT and its byproduct, 4-amino2,6-DNT, has also been observed and is inversely proportional to soil organic carbon content (Pennington 

1988). The relative concentrations in the plants was root > stem > leaves > seed and food (U.S. Army 

1990). 

Uptake of 2,4-DNT was observed in a cell suspension culture of separate plantlets, Senecio jacobaea, 

Saponaria officinalis, and Phragmitis australis. The concentration of 2,4-DNT was reduced to 20 mg/L 

from 50 mg/L after 17 days by S. jacobaea (yellowweed). Concentrations of 2,4-DNT were rapidly 

decreased after 6 days by S. officinalis (soap wort). The reduction products, 4-amino-2-nitrotoluene and 

2-amino-4-nitrotoluene were observed after 2 days. No reduction products were observed after 

metabolism of 2,4-DNT with P. austrailis (reed) given that the uptake of 2,4-DNT by P. australis was 

much slower than in the other plantlets (Podlipna et al. 2015). 

Root uptake by plants can be inhibited by the increase of sorption to the soil. Sorption can reduce the 

bioavailability of organic compounds to target organisms. In a bioavailability and phytotoxicity study, 

the toxicity of 2,4-DNT to the aquatic duckweed plant was significantly reduced by sorption to potassium 
 

 

 

 

 

 

 

 

 

 

 

 

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smectite clay (Roberts 2007). In general, 2,4-DNT is expected to adsorb to suspended soil and sediment 

in water based on its Koc. Nitroaromatic compounds adsorb to the soil mainly by forming electron-donor 

acceptor complexes with clays (Haderlein 1996). In anoxic conditions, sorption of the tested aromatic 

compounds followed the order of DNTs>ANT>DATs, where monoamines (ANT) and diaminotoluenes 

(DAT) were the derivatives of DNTs (Yang 2008). 

The toxicity of the transformation products of 2,6-DNT was studied by Nipper et al. (2005) in spiked 

sandy, fine-grained marine sediments and in seawater. In environments with the spiked sediments, 

toxicity to the micro-algae, Ulva fasciata, decreased as 2,6-DNT was biotransformed to 2-amino6-nitrotoluene. Subsequent studies indicated that toxicity to the copepod Schizopera knabeni increased 

with biotransformation. Therefore, 2-amino-6-nitrotoluene was more toxic than 2,6-DNT to the copepod, 

and less toxic to the micro-algae. Likewise, in environments with the spiked seawater tests, 2-amino6-nitrotoluene was also more toxic than its parent compound to the copepod, but not to the micro-algae. 

These studies indicate that toxicity to the 2,6-DNT degradation products vary with the environment and 

species. 

The Pre-Biologic Screen (PBS) model for ecotoxicologic effects (Gillett 1983) estimates a score (heavy 

concern, concern, or no concern) for a compound determined by the octanol-water partition coefficient, 

the Henry's Law constant, and the half-life in the medium of interest. The score indicates the compound's 

potential for (a) bioaccumulation and multi-media/multispecies effects, (b) bioaccumulation and longterm effects, (c) persistence and interactions in the water column, including plant uptake and leaching, 

and (d) direct and indirect effects in the atmosphere (e.g., smog formation, plant fumigation, stratospheric 

modification). Both 2,4- and 2,6-DNT are of concern or heavy concern only for (c), persistence and 

interactions in the water column, depending on the value used for half-life. Since the degradation of 

DNTs is so dependent on environmental conditions and the presence of effective microorganisms, the 

protective view that DNTs are of heavy concern for persistence in water, plant uptake, and leaching to 

groundwater may be warranted. The lack of concern for bioaccumulation, multimedia/multispecies 

action, and atmospheric action also appears to be justified. 

6.3.2 Transformation and Degradation 

6.3.2.1 Air 

Based on their rapid photolysis in water, DNTs are presumed to be subject to oxidation of its methyl 

group, decarboxylation, ring oxidation, and/or nitroreduction in air and sunlight. DNTs are slowly 
 

 

 

 

 

 

 

 

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degraded in the atmosphere by reaction with photochemically produced hydroxyl radicals; the half-life in 

air is estimated to be approximately 50 days for the DNT isomers (EPA 2011). 

6.3.2.2 Water 

DNTs may be degraded in water through several mechanisms, including photolysis, microbial 

biodegradation, ozonation and chlorination, and oxidation by strong oxidants such as hydrogen peroxide, 

ozone, or oxone (Bausum et al. 1992; EPA 1979; Freedman et al. 1996; Ho 1986; Noguera and Freedman 

1996; U.S. Navy 1977). Ho (1986) studied photooxidation of 2,4-DNT in aqueous solution in the 

presence of hydrogen peroxide and suggested the following degradation pathway of 2,4-DNT: 2,4-DNT 

→ 1,3-dinitrobenzene → hydroxynitrobenzene derivatives → carboxylic acids → CO2, H2O, and HNO3. 

Oxidation of aqueous 2,4-DNT with hydrogen peroxide or ultraviolet (UV) irradiation alone was very 

slow, and elimination was not complete. Dillert et al. (1995) reported that degradations of DNTs and 

several other nitroaromatics were accelerated in irradiated TiO2 suspensions and that the degradation rates 

were dependent solution pH and light intensity. At given temperature, pH, and photo intensity, 

degradation rates were shown to decrease in order of 2-nitrotoluene > nitrobenzene > DNTs 

> 1,3-dinitrobenzene > TNT > trinitrobenzene, and the degradations followed first-order kinetics. The 

photocatalytic oxidation of 2,6-DNT in aqueous suspension of TiO2 produces ammonium and nitrate ions 

as the predominant species (Kumar and Davis 1997). 

The presence and potential toxicity of DNTs in waste water have spurred considerable study of the abiotic 

and biotic fate of 2,4- and 2,6-DNT. U.S. Army (1980) reported that the half-lives of DNTs in 

three sunlit natural waters were 3–10 hours, whereas the photolysis half-life in distilled water was 

43 hours. At different latitudes and sunlight conditions, they estimated that the photolysis half-life in 

waters would range from approximately 1.8 days (summer sunlight conditions) to 11.5 days (winter 

conditions). Simmons and Zepp (1986) found that dissolved or suspended humic substances greatly 

enhance (10–17 times) indirect photolysis of nitroaromatic compounds with a nitro group ortho to a 

methyl group. 

DNTs may be also degraded by ozonation and chlorination. Lee and Hunter (1985) reported that both 

ozone and chlorine produced <17% reduction of 2,6-DNT, whereas 2,4-DNT was more vulnerable, 

yielding about 35% reduction by chlorine and 60% reduction by ozone. Contact time did not appear to 

have any impact on the reduction rates. 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 201 

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Studies have shown that DNTs can be used as a sole carbon source for microorganisms commonly 

detected in natural waters. Using bay water obtained near an Army munitions facility as inoculum, >90% 

degradation of 2,4-DNT was observed after 6 days following a 2–3-day lag period (U.S. Army 1980). 

Under anaerobic conditions, the half-life of 2,6-DNT in non-acclimated sewage was found to be 28 days, 

with no loss of the compound under aerobic conditions during the same period (Hallas and Alexander 

1983). Liu et al. (1984) reported complete biotransformation of 2,4-DNT within 14 days under anaerobic 

conditions using a fresh sample of activated sludge diluted with distilled water as inoculum. The 

intermediates of biotransformation were identified as 2-amino-4-nitrotoluene, 4-amino-2-nitrotoluene, 

2-nitroso-4-nitrotoluene, and 4-nitroso-2-nitrotoluene. Parrish (1977) investigated 190 fungal species 

from 98 genera, but found only 5 capable of 2,4-DNT biotransformation. Valli et al. (1992) reported 

degradation of 2,4-DNT as the sole source of carbon and energy by the lignin-degrading fungus 

Phanerochaete chrysosporium under aerobic conditions, resulting in stoichiometric release of nitrate. 

Several additional studies have shown biodegradation of DNTs from microorganisms isolated from areas 

that are frequently exposed to DNTs and other structurally similar compounds such as TNT (Bausum et 

al. 1992; Freedman et al. 1996). Bausum et al. (1992) found complete degradation of 20 ppm 2,4-DNT 

and 20 ppm 2,6-DNT in water samples taken downstream a short distance from the Radford Army 

Ammunition Plant in Radford, Virginia. A lag time was noted prior to the breakdown for both of the two 

compounds with 2,4-DNT exhibiting the shorter lag time. Microbial enrichment cultures were developed 

from the collected water samples by exposing the cultures to increasing concentrations of 2,4- and 

2,6-DNT. Degradation and visible turbidity in the suspension medium were noted up to a level of 

130 ppm. In a separate but related study, degraded DNTs were shown to be converted to CO2 with 

2,4-DNT conversion occurring at a greater rate than that of 2,6-DNT; concentrations ranged from 0.004 to 

10.0 ppm (Bausum et al. 1992). The rate of mineralization to CO2 was concentration dependent and 

increased with increasing concentration. 

In the Bradley et al. (1994) study, a culture of microorganisms taken from aquifer sediments at an 

explosives-contaminated site was observed to be capable of transforming 2,4- and 2,6-DNT. Breakdown 

products from 2,4-DNT degradation included 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene. 

Carbon dioxide was released during the degradation process. Aminonitrotoluene isomers were also 

detected as breakdown products of a solution of 2,4-DNT and ethanol (Freedman et al. 1996). The 

Freedman et al. (1996) study exposed an inoculum from a wastewater treatment plant at an ammunition 

plant to a solution of 2,4-DNT and ethanol and a solution of 2,4-DNT and ether. The concentration of 

2,4-DNT at each application was 0.55 mM and the concentrations of ethanol and ether were 600 and 
 

 

 

 

 

 

 

 

 

 

 

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142 mg/L, respectively. Ethanol and ether were chosen because they are often found in munitions 

manufacturing waste-water streams along with 2,4-DNT. As stated above, aminonitrotoluene isomers 

were detected as products of the solution containing 2,4-DNT and ethanol. The degradation of ethanol 

was believed to be driving a partial reduction of 2,4-DNT before the oxidation of 2,4-DNT took place. In 

contrast, ether at the applied concentration slowed the rate of 2,4-DNT degradation. Low chemical 

oxygen demand during the studies suggests that DNTs were mineralized to a significant degree. 

Spanggord et al. (1991) studied the degradation pattern of 2,4-DNT using, water samples obtained from 

the Waconda Bay located near a TNT manufacturing facility (Volunteer Army Ammunition Plant) in 

Chattanooga, Tennessee. Samples were enriched with 2,4-DNT and incubated for 5 days. The 

Pseudomonas sp. strain aerobically degraded 2,4-DNT as a sole source of carbon, and the nitro groups 

were oxidatively removed without prior reduction to the amines. Since this Pseudomonas sp. strain used 

2,4-DNT as a carbon source, it may be useful for the removal of other nitrotoluenes from contaminated 

soils or from industrial waste streams. Likewise, it may also be useful since the oxidative removal of the 

nitro groups bypasses the accumulation of toxic amino derivatives. 

The co-metabolism of 2,4-DNT with ethanol, methanol, and acetic acid has been studied under anaerobic 

conditions (Cheng et al. 1995). High concentrations of ethanol accelerated the reductive transformation 

of 2,4-DNT by supporting the growth of the anaerobic bacteria. The rate of 2,4-DNT biotransformation 

was much higher with the addition of ethanol than with the addition of methanol or acetic acid in 

anaerobic conditions (Cheng et al. 1995). In a culture using a continuous flow laboratory fermentor under 

anaerobic conditions with both 2,4-DNT and ethanol as substrates, 2,4-DNT was completely transformed 

to 2,4-diaminotoluene (Cheng et al. 1996). During the biotransformation, two intermediates were formed: 

2-amino-4-nitrotoluene and 4-amino-2-nitrotoluene. 

2-Amino-4-nitrotoluene and 4-amino-2-nitrotoluene were also formed in the transformation of 2,4-DNT 

to 2,4-aminotoluene by the marine microbial culture, Shweanella marisflava EP1. S. marisflavi EP1 acts 

to reduce 2,4-DNT during respiration under anaerobic conditions by using lactate as an electron donor 

(Huang et al. 2015). 

The products formed from anoxic biotransformation of 2,4-DNT by two denitrifying enrichment cultures 

with ethanol provided as a primary substrate were characterized in one study (Noguera and Freedman 

1997). One culture was developed with inoculum acclimated to 2,4-DNT, the other with activated sludge 

that was not routinely exposed to nitroaromatic compounds. The acclimated culture consumed 2,4-DNT 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 203 

6. POTENTIAL FOR HUMAN EXPOSURE 

twice as fast as the unacclimated culture, with reduction of 2,4-DNT to aminonitrile toluenes as the initial 

pathway. The principal metabolites identified in the acclimated culture were 6-nitroindazole, 

2-nitrotoluene, and 4-nitrotoluene, as well as products from acetylation at the para position (4-acetamide2-nitrotoluene and 4-acetamidetoluene). Reduction of aminonitrotoluenes to 2,4-diaminotoluene also 

occurred, and its subsequent disappearance resulted in accumulation of significant amount of 

nonfilterable material in both cultures. The soluble metabolites formed from the unacclimated culture 

were more hydrophilic. Initial characterization of the highly hydrophilic metabolites indicated 

approximately equal amounts of negatively-charged and neutral compounds. 

Biotransformation of DNT by a Pseudomonas aeruginosa strain, which was isolated from the waste water 

of the Radford Army Ammunition Plant (RAAP) located in Radford, Virginia was observed under both 

aerobic and anoxic conditions (Noguera and Freedman 1996). The biotransformation was mainly 

reductive under both of these conditions and was reflective of the cometabolic transformations that can 

occur in the presence of easily degradable organic matter such as ethanol. P. aeruginosa reduced both 

nitro groups of DNTs resulting in the primary breakdown products of 4-amino-2-nitrotoluene and 

2-amino-4-nitrotoluene, with small amounts of 2,4-diaminotoluene also formed. Several DNT 

metabolites from acetylation of the arylaminos were also identified, including 4-acetamide-2-nitrotoluene, 

2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene. 

Clostridium acetobutylicum has received interest in anaerobic TNT bioremediation systems due to their 

ability to rapidly reduce aryl nitro groups. Results indicated that dihydroxylaminotoluenes are the 

predominant primary intermediates in the transformation of the DNT isomers by C. acetobutylicum and 

that further metabolism of the dihydroxylaminotoluenes in cell cultures and extracts resulted in the 

formation of arylamines through hydroxylamine reduction (Hughes 1999). 

6.3.2.3 Sediment and Soil 

Microorganisms indigenous to surface soils collected at a munitions-contaminated site were reported to 

transform 2,4- and 2,6-DNT to amino-nitro intermediates within 70 days (Bradley et al. 1994). Another 

study showed that composting can decrease the concentrations of explosives, such as TNT, in 

contaminated soil, but neither 2,4- nor 2,6-DNT was detected in the compost (Griest et al. 1993). A study 

of soil sample handling times indicated that lower temperatures retard the breakdown of 2,4-DNT (Grant 

et al. 1995). 2,4-DNT was observed to be more stable than TNT in contaminated soils (Grant et al. 1995). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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DNTs were not readily biodegradable using a standardized Organisation for Economic Co-operation and 

Development (OECD) test. 3,4-DNT and mixed isomers of DNT (CAS Registry Number 25321-14-6) 

achieved 0% of their theoretical biochemical oxygen demand (BOD) over a 14-day incubation period 

using an activated sludge inoculum and the modified MITI (OECD 301 C) test (NITE 2002). The DT50 of 

2,4-DNT in an organic soil (82% sand, 13% silt, 5.1% clay, 2.3% organic carbon) was reported as 7 days 

and the DT90 was 191 days (UNEP 2004); however, other studies cited in this report indicated limited 

degradation of DNT under environmental conditions. In a test system maintained under anaerobic 

conditions, performed according to EPA-Guideline No. 796.3140, 0% biodegradation was observed 

within 56 days (UNEP 2004). 

In a study conducted by Nishino et al. (1999), the mineralization of 2,4- and 2,6-DNT was examined in 

soil slurries. DNT-degrading bacteria cultures commonly used in contaminated water systems were 

examined to see if they were able to degrade DNTs in contaminated soils. It was found that 

microorganisms indigenous to the soils did not convert DNTs to aminonitrotoluenes during the short 

incubation times required to mineralize DNTs. DNT-degrading bacteria, however, removed over 99% of 

the initial DNTs in the soil after 2–3 days of incubation. Disappearance of DNTs was accompanied by 

14CO2 release and stoichiometric appearance of nitrite. 

In another study, soil from a former ammunition plant was anaerobically treated in a laboratory slurry 

reactor. 2,4-DNT was completely reduced to undetectable levels in the reactor. The contaminated soil 

was also tested in a larger technical scale where the soil slurry was treated anaerobically and subsequently 

aerobically to complete the bioremediation process. An overall reduction of > 99% of the contaminants 

was observed, and ecotoxicological tests showed that the toxicity of the soil could not be detected after 

the anaerobic/aerobic process (Lenke et al. 1998). 

The biotransformation of 2,4- and 2,6-DNT by the indigenous microflora contained in marine sediment 

from a shipwreck site near Halifax Harbour was studied by Yang et al. (2008). Incubation of 2,4- and 

2,6-DNT in anaerobic sediment-water slurries at 10 °C led to the disappearance of both 2,4- and 2,6-DNT 

and their reduction to their monoamine derivatives in 10 days. The derivatives were progressively 

reduced even further to 2,4- and 2,6-diaminotoluenes in a series of 50 days for 2,4-DNT and 35 days for 

2,6-DNT. 

The biodegradation of sorbed 2,4-DNT by the Burkholderia sp. strain was examined in a clay-rich 

aggregated porous medium by Ortega-Calvo et al. (1999). Burkholderia sp. exhibited sensitivity to the 
 

 

 

 

 

 

 

 

 

 

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6. POTENTIAL FOR HUMAN EXPOSURE 

cold and was unable to complete the degradation process at low temperatures (Monti 2005). Therefore, 

P. fluorescens was genetically modified (by intergeneric transformation of the DNT genes from the 

Burkholderia sp. strain to the P. fluorescens strain) to use 2,4-DNT as the sole nitrogen source to 

completely degrade the compound (Monti 2005). P. fluorescens was shown to be capable of degrading 

2,4-DNT in temperatures as low as 10 °C and significantly decreased the toxic effects of 2,4-DNT on 

specific plants. 

The correlation between the mutagenicity and biodegradability of TNT and its analogs, including 2,4- and 

2,6-DNT, was reported in Maeda et al. (2007). The umu test using luminescent bacteria was employed to 

assess the mutagenicity and biodegradability of the nitroaromatic compounds. The Pseudomonas sp. 

strain TM15 bacteria isolated from TNT contaminated soils in Japan was found to biotransform the 

aromatic compounds harboring three nitro groups efficiently, although 2,4- and 2,6-DNT had low 

degradation rates. It was demonstrated that the mutagenicity, induced by the nitroaromatic compounds, 

increased with biodegradability. 

In the presence of ultraviolet (UV) exposure, photo-transformation of 2,6-DNT began immediately after 

simulated solar radiation (SSR) in sandy and fine-grained sediment, with 89% being photo-transferred 

after 24 hours, and none remaining for photo-transfer after 72 hours (Nipper et al. 2004). 

6.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT 

Reliable evaluation of the potential for human exposure to DNTs depends in part on the reliability of 

supporting analytical data from environmental samples and biological specimens. Concentrations of 

DNTs in unpolluted atmospheres and in pristine surface waters are often so low as to be near the limits of 

current analytical methods. In reviewing data on DNT levels monitored or estimated in the environment, 

it should also be noted that the amount of chemical identified analytically is not necessarily equivalent to 

the amount that is bioavailable. The analytical methods available for monitoring DNTs in a variety of 

environmental media are detailed in Chapter 7. 

6.4.1 Air 

Concentrations of DNTs in the ambient air have been reported to be low (0.03–0.1 mg/m3

) or 

undetectable (Woollen et al. 1985). Occupationally, concentrations of 2,4-DNT found in the stationary 

air and the personal air of the mechanical plant operating room, rest rooms, and office ranged from 

<0.1 to 20 μg/m3 (<1x10-4–0.020 mg/m3

) (Letzel et al. 2003). NIOSH (1980) measured breathing zone air 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 206 

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concentrations of Tg-DNT that ranged from not detected (below detection limits) to 23 μg/m3 

(0.023 mg/m3

) (TWA). Tg-DNT concentrations in area air samples ranged from not detected to 

420 μg/m3 (0.42 mg/m3

) (TWA). NIOSH (1982) reported that area air samples in a manufacturing 

facility contained TWA concentrations of Tg-DNT that ranged from not detected to 890 μg/m3 

(0.89 mg/m3

). Levine et al. (1985b) also measured personal air samples of 2,4-DNT at 10–440 μg/m3 

(0.01–0.44 mg/m3

) and 2,4- and 2,6-DNT at 50–590 μg/m3 (0.05–0.590 mg/m3) in the workplace. 

6.4.2 Water 

DNTs are rarely detected in public drinking water supplies. 2,4-DNT and 2,6-DNT are priority 

pollutants, and amendments to the Safe Drinking Water Act require that they be monitored for by large 

community water systems (CWSs) and non-transient non-community water systems (NTNCWSs) and a 

statistically representative sample of qualifying small CWSs and NTNCWSs (EPA 2008a, 2008b). 

Contaminant occurrence data collected under the First Unregulated Contaminant Monitoring Regulation 

(UCMR 1) showed only a single detection of DNTs. Since the health reference level (0.05 μg/L or 

5x10-5 mg/L) for each isomer was below the minimum reporting level of 2 μg/L (0.002 mg/L), the data 

were reported as only as detections exceeding this level. Among the small public water systems 

(797 tested), there were no detections of 2,4-DNT in 3,251 samples taken, and there was only 1 detection 

out of 30,513 samples obtained from large systems throughout the United States (3,076 tested). This 

single detection of 333 μg/L (0.333 mg/L) was in a surface water sample obtained from an entry point 

source at a large public water system in the State of Tennessee (EPA 2008b). 2,6-DNT was not detected 

in any of the 33,765 samples (both large and small systems) for which it was tested. A total of 

3,873 public water systems were tested for 2,6-DNT, of which 1,970 relied on ground water sources and 

1,903 relied on surface water sources (EPA 2008b). 

DNTs have been detected in surface water and groundwater near source locations such as munitions sites. 

Concentrations of 2,4- and 2,6-DNT obtained from a small brook and the Losse River, Germany adjacent 

to a former ammunition plant were 0.5–13.0 and 0.1–7.6 μg/L (5x10-4–0.013 and 1x10-4–0.0076 mg/L), 

respectively (Feltes et al. 1990). Two ponds located on a closed munition site in Germany had 

concentrations of 2,4-DNT ranging from 0.8 to 1.2 μg/L (8x10-4–0.0012 mg/L) and concentrations of 

2,6-DNT ranging from 0.07 to 0.3 μg/L (7x10-5–3x10-4 mg/L) (Feltes et al. 1990). Monitoring studies at 

three polluted locations of the Elbe River in Germany found concentrations of 2,4-DNT of 0.1–1.3 μg/L 

(1x10-4–0.0013 mg/L), while concentrations of 2,6-DNT were 0.08–0.5 μg/L (8x10-5–5x10-4 mg/L) 

(Feltes et al. 1990). Sohr et al. (1995) reported 2,4- and 2,6-DNT concentrations of 0.7 and 3.1 μg/L 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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(7x10-4 and 0.0031 mg/L), respectively, at contaminated warfare sites in Germany. Other lesscontaminated sites contained 28 ng/L (2.8x10-6 mg/L) 2,4-DNT and 19 ng/L 2,6-DNT (1.95x10-5 mg/L) 

(Sohr et al. 1995). In the seawater of Dokai Bay in Japan, 2,4- and 2,6-DNT were measured at levels of 

not detected (ND)–206 and ND–14.8 μg/L (ND–0.206 and ND–0.0148 mg/L), respectively, in an area 

receiving effluent from an industrial plant (Hashimoto et al. 1982). 2,3-DNT was also detected, but at 

much lower levels (ND–0.412 μg/L) (ND–4.12x10-4 mg/L). DNT levels as high as 10,000 μg/L 

(10 mg/L) were reported in potable groundwater at the Joliet Army Ammunition Plant located in Will 

County, Illinois (EPA 2008a). The dates that these levels were obtained are not known. Levels were also 

reported as 3,200 (3.2 mg/L) and 2,700 mg/L (2.7 mg/L) in 1989 (Agency for Toxic Substances and 

Disease Registry 2009); however, current levels of DNTs may be significantly lower. 2,3-, 2,5-, 3,4-, and 

3,5-DNT isomers were identified in both monitoring wells and a few private water supply wells near the 

Badger Army Ammunition Plant site in Wisconsin (EPA 2008a). 

6.4.3 Sediment and Soil 

Hoke et al. (1993) reported that only low concentrations of 2,4- and 2,6-DNT were detected in sediment 

of the Great Calumet River-Indian Harbor. Concentrations of 2,4-DNT in sediment pore water ranged 

from the detection limit of 0.01 to 0.07 μg/L (1x10-5–7x10-5 mg/L). 2,4-DNT concentrations in sediment 

pore water ranged from 0.1 to 1.7 μg/L (1x10-4–0.0017 mg/L). 2,6-DNT was not detected in sediment 

samples (limit of detection [LOD]=0.01 μg/L or 1x10-5 mg/L) and subsequently was not analyzed for in 

sediment pore water. 2,4-DNT and 2,6-DNT were identified in bed sediment collected from 20 major 

river basins in the United States for the 1992–1995 sampling period (Lopes and Furlong 2001). The 

maximum detected concentrations were reported as 170 μg/kg for 2,4-DNT (detected in 0.6% of 519 sites 

sampled) and 93 μg/kg (detected in 1% of 518 sites sampled) for 2,6-DNT. 

Concentrations ranging from <0.1 mg/kg (detection limit) to 117 mg/kg of 2,4-DNT were found at the 

Joliet Army Ammunition Plant, in Joliet, Illinois, an NPL site. 2,6-DNT was detected on this site at 

concentrations ranging from <0.1 to 8 mg/kg (Simini et al. 1995). The concentrations of 2,4- and 

2,6-DNT were reported as 19 and 1.38 g/kg, respectively, in soil samples obtained at the Volunteer Army 

Ammunition Plant located in Chattanooga, Tennessee and levels of 2,4- and 2,6-DNT were reported as 

8.9 and 0.48 g/kg, respectively, in soil samples obtained at the Badger Army Ammunition Plant located in 

Wisconsin (UNEP 2004). The concentration of 2,4-DNT in soil samples at the Gyttorp facility in 

Sweden, which was used for explosives manufacturing from 1864 to 1995 was 4 g/kg (UNEP 2004). The 

concentrations of 2,4- and 2,6-DNT measured in soil samples obtained from the decommissioned TNT 
 

 

 

 

 

 

 

 

 

 

 

 

 

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manufacturing facility in Hessisch Lichtenau Germany were reported as 3.6 and 2.5 g/kg, respectively 

(UNEP 2004). 

Residues of DNTs were detected on the surface of military live-fire training soil (Jenkins 2006). Mean 

surface soil concentrations ranged from <0.001 to 84 mg/kg for 2,4-DNT and from <0.001 to 4.6 mg/kg 

for 2,6-DNT at 23 military firing ranges in the United States and Canada (Jenkins 2006). 

6.4.4 Other Environmental Media 

Neither 2,4- nor 2,6-DNT were detected in samples of fish obtained from Lake Michigan tributaries and 

Grand Traverse Bay (Camanzo et al. 1987). DNTs were monitored for, but were not detected in, fish 

from Great Lakes harbors and tributaries in Ohio and Wisconsin (DeVault 1985). 

6.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE 

Sources of exposure to DNTs for the general population include processing facilities that manufacture or 

process DNTs, as well as hazardous waste sites that release this chemical. Populations residing near 

ammunition or explosive manufacturing facilities may be exposed to contaminated groundwater or soil. 

Exposure pathways to DNTs include dermal contact and incidental ingestion pathways. Since DNTs are 

not frequently detected in air, inhalation exposure to the general population is expected to be low. 

Occupational exposure to DNTs may occur from its use in the manufacture of toluene diisocyanate, in the 

production of explosives, in the manufacture of azo dye intermediates, and in organic synthesis in the 

preparation of toluidines and dyes (IARC 1996). Exposure may also occur at facilities that store or 

dispose the substance. Occupational exposure will involve inhalation, dermal contact, and incidental 

ingestion, with inhalation the most likely exposure pathway. 

Studies on occupational exposure to DNTs are limited. Levine et al. (1985b) evaluated the 7-hour TWA 

personal exposure of workers to Tg-DNT and measured urinary metabolites of DNTs at a DNT 

manufacturing plant. Breathing zone exposure level of production unit operators to both 2,4- and 

2,6-DNT averaged 0.26 mg/m3 (2.6x10-4 mg/L). Air exposure concentrations of loaders, who load storage 

tanks, collect samples, and perform cleaning tasks, averaged 0.32 mg/m3 (3.2x10-4 mg/L). Exposure of 

maintenance mechanics averaged 0.12 mg/m3 (1.2x10-4 mg/L), and the exposure of acid-stripper operators 

was 0.06 mg/m3 (6x10-5 mg/L). The highest personal air monitoring concentrations and levels of urinary 

metabolites were found to be for loaders, followed by process operators. The levels of urinary 

metabolites of DNTs in loaders and operators exceeded those that would have resulted from the inhaled 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 209 

6. POTENTIAL FOR HUMAN EXPOSURE 

concentrations, although the workers wore gloves for operations that might have led to dermal exposure. 

Woollen et al. (1985) carried out biological monitoring studies of 28 workers at an explosives factory. 

The 2,4-DNT metabolite, 2,4-dinitrobenzoic acid was not detected or was present at very low levels in 

urine samples prior to the start of work; however, it was detected in all post-shift urine samples. The 

weakly mean post-shift urine concentration of 2,4-dinitrobenzoic acid was 17 mg/L. Concentrations of 

2,4-DNT in air samples of the plant ranged from 0.02 to 2.68 mg/m3 (2.0x10-5–0.00268 mg/L). It was 

concluded that air concentrations could not solely account for the observed excretion levels of the 

metabolite, 2,4-dinitrobenzoic acid, indicating probable dermal uptake or inadvertent ingestion. 

A cross-sectional study with 82 employees who dismantled military waste at the mechanical plant in 

Saxony, Germany was undertaken by Letzel et al. (2003). The maximum concentrations of 2,4-DNT in 

the ambient air was 20 μg/m3 (0.02 mg/m3

). The maximum concentrations in the urine of workers 

regularly exposed to the ammunition were 2.1 μg/L (0.0021 mg/L) for 2,4-DNT, 95 μg/L (0.095 mg/L) 

for 2,4-dinitrobenzoic acid, and 3.6 μg/L (0.0036 mg/L) for 2,6-DNT. 

In another study, Levine et al. (1986a) reported increases of heart disease among workers at two 

ammunition plants; one of which is located in Joliet, Illinois and the other located in Radford, Virginia. 

DNTs were manufactured and purified at the plant in Joliet, Illinois and were used in single-base 

propellant formulations at the plant in Radford, Virginia. 

OSHA established an 8-hour TWA Permissible Exposure Limit (PEL) for DNTs (mixed isomers) as 

1.5 mg/m3

, with skin designation to indicate the potential significant contribution to the overall exposure 

by the cutaneous route (OSHA 2013). The American Conference of Governmental Industrial Hygienist 

(ACGIH)’s Threshold Limit Value (TLV) for DNTs (mixed isomers) is 0.2 mg/m3 (2x10-4 mg/L), with 

skin notation (ACGIH 2014). TLV is the time-weighted average concentration for a conventional 8-hour 

workday and a 40-hour workweek to which it is believed that nearly all workers may be repeatedly 

exposed without adverse effect. The National Institute of Occupational Safety and Health (NIOSH) 

determined the Recommended Exposure Limit (REL) for DNTs (mixed isomers) as 1.5 mg/m3 

(0.0015 mg/L), with skin designation (NIOSH 2015). 

6.6 EXPOSURES OF CHILDREN 

This section focuses on exposures from conception to maturity at 18 years in humans. Differences from 

adults in susceptibility to hazardous substances are discussed in Section 3.7, Children’s Susceptibility. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 210 

6. POTENTIAL FOR HUMAN EXPOSURE 

Children are not small adults. A child’s exposure may differ from an adult’s exposure in many ways. 

Children drink more fluids, eat more food, breathe more air per kilogram of body weight, and have a 

larger skin surface in proportion to their body volume. A child’s diet often differs from that of adults. 

The developing human’s source of nutrition changes with age: from placental nourishment to breast milk 

or formula to the diet of older children who eat more of certain types of foods than adults. A child’s 

behavior and lifestyle also influence exposure. Children crawl on the floor, put things in their mouths, 

sometimes eat inappropriate things (such as dirt or paint chips), and spend more time outdoors. Children 

also are closer to the ground, and they do not use the judgment of adults to avoid hazards (NRC 1993). 

No studies are available that monitor the level of exposure of children to DNTs. No measurements have 

been made of DNTs or their metabolite levels in amniotic fluid, meconium, cord blood, or neonatal blood 

to test for prenatal exposure, nor have measurements been made of DNTs or metabolite levels in breast 

milk. However, because of the low octanol-water partition coefficients of DNTs and excretion in the 

urine, they are not expected to accumulate in maternal tissues. 

Although DNTs can degrade in the environment, they have been detected at high levels at artillery sites or 

ammunition producing facilities. Therefore, children playing in soil contaminated with DNTs have the 

potential to be more exposed than adults, both because of this behavior and because of their larger skin 

surface area in proportion to their body weight for dermal absorption. Also, children drinking well water 

contaminated with DNTs might be exposed to more of the chemical than adults would be due to the fact 

that children drink more fluids per kilogram of body weight than adults. Significant dietary exposure is 

unlikely as DNTs are not expected to accumulate in animal tissues. However, ingestion of vegetables and 

crops grown in DNT-contaminated areas could be a source of exposure. 

There were no studies that examine potential exposure of children from their parents work clothes, skin, 

hair, tools, or other objects removed from the workplace. No information is available concerning 

exposure from consumer products because DNTs are used mainly for military and industrial purposes. 

Although DNTs are genotoxic in in vivo test systems, they have been found to be negative in dominant 

lethal mutations (U.S. Army 1979) and spermatocyte DNA repair (Working and Butterworth 1984). 

There is no evidence that exposure of parental germ cells to the active forms of DNTs could plausibly 

occur since DNTs do not accumulate in tissue. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 211 

6. POTENTIAL FOR HUMAN EXPOSURE 

6.7 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES 

In addition to individuals who are occupationally exposed to DNTs (see Section 6.5), there may be groups 

within the general population that have potentially high exposures (higher than background levels) to 

DNTs. These populations include individuals living in proximity to sites where DNTs were produced or 

sites where DNTs were disposed, and individuals living near one of the NPL hazardous waste sites where 

isomers of DNT have been detected in some environmental media (HazDat 2007). 

Based on the available information, it appears that the highly-exposed populations would be workers 

exposed in manufacturing facilities. 

Members of the general population are likely to be exposed only if they are near a local source of 

contamination, such as an industrial discharge or an abandoned waste site. There is also the possibility of 

exposure to DNTs from the gradual turnover of former military bases to local communities for public use. 

DNTs do not appear to be widespread in the environment, and they were not frequently detected at 

hazardous waste sites. 

6.8 ADEQUACY OF THE DATABASE 

Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the 

Administrator of EPA and agencies and programs of the Public Health Service) to assess whether 

adequate information on the health effects of DNTs is available. Where adequate information is not 

available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of research 

designed to determine the health effects (and techniques for developing methods to determine such health 

effects) of DNTs. 

The following categories of possible data needs have been identified by a joint team of scientists from 

ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would 

reduce the uncertainties of human health assessment. This definition should not be interpreted to mean 

that all data needs discussed in this section must be filled. In the future, the identified data needs will be 

evaluated and prioritized, and a substance-specific research agenda will be proposed. 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 212 

6. POTENTIAL FOR HUMAN EXPOSURE 

6.8.1 Identification of Data Needs 

Physical and Chemical Properties. Information regarding the physical and chemical properties of 

a chemical is essential for estimating the partitioning of the chemical in the environment. Information on 

the physical and chemical properties of DNTs is presented in Chapter 4 and the data appear to be 

adequate (HSDB 2012). The isomers of DNT have many similar traits, including identical molecular 

weights, but 2,4-DNT has higher melting and boiling points and a greater solubility in water than 

2,6-DNT (HSDB 2012). DNTs are generally produced as a technical-grade mixture comprised of 95% 

2,4- and 2,6-DNT and 5% other substances. 

Production, Import/Export, Use, Release, and Disposal. According to the Emergency 

Planning and Community Right-to-Know Act of 1986, 42 U.S.C. Section 11023, industries are required 

to submit substance release and off-site transfer information to the EPA. The TRI, which contains this 

information for 2013, became available in October 2014. This database is updated yearly and should 

provide a list of industrial production facilities and emissions. 

USITC statistics on synthetic organic chemical production (USITC 1987) do not describe DNT 

production. Uses of DNTs appear to be well characterized (HSDB 2012). The most commercially 

important use of DNT is as a chemical intermediate in the production of toluene diisocyanate, a precursor 

to polyurethane polymers (HSDB 2012). DNTs are recognized as potentially hazardous chemicals and 

are subject to a variety of regulations (see Chapter 8), but disposal practices and restrictions are not 

adequately documented. 

Environmental Fate. The low octanol-water partition coefficients of the DNT isomers predict that 

DNTs released to the environment would not bioaccumulate and would be weakly bound to soil organic 

matter. Bioconcentration data also supports the notion that 2,4-DNT is not bioaccumulative in fish (NITE 

2002; Lang 1997). The relatively low volatility and high solubility of DNTs indicate that they will tend 

to remain in water for long periods of time unless acted upon by light, oxygen, or biota, creating the 

potential for transportation to groundwater or surface water (Jenkins et al. 1986). DNTs have been found 

in waste water and groundwater in and around sites where munitions have been historically used (Jenkins 

et al. 1986; Spanggord and Suta 1982; UNEP 2004). The occurrence of DNTs in waste water from other 

manufacturing uses such as polyurethane forms has not been reported. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 213 

6. POTENTIAL FOR HUMAN EXPOSURE 

Given the importance of information about the behavior of DNTs in the water column, and the extensive 

range of available information relative to that topic (EPA 1982c; Gillett 1983; Hashimoto et al. 1982, 

1984; Jenkins et al. 1986; U.S. Army 1980), the absence of substantive information about DNT releases 

to, or fate in, soils and air is less troublesome than it might be for many chemicals. Data on the 

persistence of DNTs in the vadose zone (the unsaturated zone lying between the ground level and the top 

of the groundwater) and groundwater are needed, as well as measured rates of plant uptake and 

metabolism. Because of the structurally specific nature of biotransformations, more information on the 

fate of DNT metabolites would be welcome. 

Bioavailability from Environmental Media. No information is currently available that describes 

the bioavailability of DNTs in food. Data on bioavailability of soil/sediment residues would be helpful. 

Neither 2,4- nor 2,6-DNT were detected in samples of fish obtained from Lake Michigan tributaries and 

Grand Traverse Bay (Camanzo et al. 1987). DNTs were not detected in fish from Great Lakes harbors 

and tributaries in Ohio and Wisconsin (DeVault 1985). 

Food Chain Bioaccumulation. Limited information indicates that DNTs are not widely distributed 

in the environment. Residues of DNTs have a low frequency of detection in water samples. These data 

indicate that bioaccumulation may not be an area of concern (Callahan et al. 1979; EPA 1982c). The log 

Kow values for 2,4- and 2,6-DNT are 1.98 and 2.10, respectively (Callahan et al. 1979; EPA 1982c), 

indicating that bioaccumulation is not likely to occur. BCFs also indicate that DNTs are not expected to 

bioaccumulate in fish (NITE 2002; Lang 1997). Degradation of DNTs forms a variety of products. 

Additional information would help to confirm or refute indications of low potential for bioaccumulation 

of DNTs in foods. 

Exposure Levels in Environmental Media. Reliable monitoring data for the levels of DNTs in 

contaminated media at hazardous waste sites are needed so that the information obtained on levels of 

DNTs in the environment can be used in combination with the known body burden of DNTs to assess the 

potential risk of adverse health effects in populations living in the vicinity of hazardous waste sites. 

The sparse information base suggests that widespread contamination by DNTs has not occurred (Staples 

et al. 1985). DNTs were detected at very low frequencies in drinking water (EPA 2008b). Analyses of 

wastewaters indicate that local contamination may occur (Feltes et al. 1990; Shackelford and Keith 1976; 

Spanggord and Suta 1982; Spanggord et al. 1982a). DNTs were also present in the sediment and soil near 

source areas (Hoke 1993; Simini 1995; Jenkins 2006). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 214 

6. POTENTIAL FOR HUMAN EXPOSURE 

Exposure Levels in Humans. No studies of exposure of the general population were found, and the 

occupational studies (Levine et al. 1985b; Woollen et al. 1985) are inadequate to ascertain "background" 

or nonoccupational exposure. Based on available information, the highly-exposed populations are those 

workers exposed in manufacturing facilities. Members of the general population are likely to be exposed 

only in that they are near a local source of contamination. Toxicokinetic data on occupationally- and 

environmentally-exposed humans will be helpful. Measurements of DNTs and their metabolite levels in 

blood and urine will be useful to provide an estimate of internal dose of exposure. 

This information is necessary for assessing the need to conduct health studies on these populations. 

Exposures of Children. No exposure or body burden studies have been conducted on children; 

consequently, it is not known if children differ from adults in their weight-adjusted intake of DNTs, or if 

unique exposure pathways for children exist. Since DNTs are not a widespread environmental 

contaminant, there are only two likely potential sources of exposure for children. Children living near a 

DNT-contaminated site might be exposed if DNTs have moved offsite in contaminated environmental 

media. If such a situation were identified, further site-specific studies of children’s exposure could be 

conducted. Children whose parents work in manufacturing facilities that produce or use DNTs and are 

occupationally exposed to significant quantities of DNTs might potentially be exposed to DNTs 

transported home on their parents’ work clothes, skin, hair, tools, or other objects removed from the 

workplace. If such a significant occupational exposure setting were identified, they might be the subject 

of a take-home exposure study. 

Child health data needs relating to susceptibility are discussed in Section 3.12.2, Identification of Data 

Needs: Children’s Susceptibility. 

Exposure Registries. No exposure registries for 2,4- and 2,6-DNT were located. This substance is 

not currently one of the compounds for which a sub-registry has been established in the National 

Exposure Registry. The substance will be considered in the future when chemical selection is made for 

sub-registries to be established. The information that is amassed in the National Exposure Registry 

facilitates the epidemiological research needed to assess adverse health outcomes that may be related to 

exposure to this substance. 
 

 

 

 

 

 

DINITROTOLUENES 215 

6. POTENTIAL FOR HUMAN EXPOSURE 

The development of an exposure registry would provide valuable data on exposure levels and frequency. 

In addition to providing information on exposure levels and duration, a registry would be useful in 

identifying sources of exposure such as hazardous waste sites and manufacturing and use facilities. 

Knowledge about exposure levels and sources would be valuable in developing strategies to control 

unnecessary sources and these exposures. The ability to correlate sources and exposure levels with health 

effects would be useful in identifying disease conditions that may result from exposure to the chemical. 

6.8.2 Ongoing Studies 

No ongoing studies sponsored by NIH or EPA were identified for DNTs. 
DINITROTOLUENES 216 

6. POTENTIAL FOR HUMAN EXPOSURE 

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DINITROTOLUENES 217 

7. ANALYTICAL METHODS 

The purpose of this chapter is to describe the analytical methods that are available for detecting, 

measuring, and/or monitoring DNTs, their metabolites, and other biomarkers of exposure and effect to 

DNTs. The intent is not to provide an exhaustive list of analytical methods. Rather, the intention is to 

identify well-established methods that are used as the standard methods of analysis. Many of the 

analytical methods used for environmental samples are the methods approved by federal agencies and 

organizations such as EPA and the National Institute for Occupational Safety and Health (NIOSH). Other 

methods presented in this chapter are those that are approved by groups such as the Association of 

Official Analytical Chemists (AOAC) and the American Public Health Association (APHA). 

Additionally, analytical methods are included that modify previously used methods to obtain lower 

detection limits and/or to improve accuracy and precision. 

7.1 BIOLOGICAL MATERIALS 

The need to determine DNTs in biological materials could arise from occupational exposure in the 

manufacture and processing of 2,4,6-TNT and from exposure to waste water and waste disposal sites 

associated with TNT manufacture. It has been noted (Jenkins et al. 1986) that "one of the Army's most 

serious water pollution problems is the disposal of wash waters used to clean equipment and interior 

surfaces at munitions manufacturing and demilitarization facilities." The same reference mentions the 

generation of large quantities of waste water from these facilities. 

Although there are numerous occupational monitoring studies, a limited number of methods regarding the 

determination of DNTs in biological samples are available in the literature. DNTs have been determined 

in ocean floor fauna using thin layer chromatography (TLC) (U.S. Navy 1972). Procedures have been 

described for the examination of swabs for traces of explosives, including 2,6-DNT using highperformance liquid chromatography (HPLC) with electrochemical detection at a pendent drop electrode 

(Lloyd 1983a). These techniques can be applied to biological materials such as skin surfaces exposed to 

explosives. DNTs and their metabolites were determined in blood and urine by gas chromatography (GC) 

techniques (Turner et al. 1985; Woollen et al. 1985) and in urine by TLC (Woollen et al. 1985). 

Qualitative determination of DNTs and their metabolites can also be performed after reduction to primary 

arylamines and subsequent coupling of diazo compounds to produce a colored complex, which absorbs 

light at 550 nm (Smith et al. 1995). 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 218 

7. ANALYTICAL METHODS 

Dichloromethane is the solvent of choice for extracting DNTs from water samples (EPA 1982a) and from 

wastes (EPA 1986a). Reversed-phase high-performance liquid chromatography (RP-HPLC) is attractive 

for the determination of DNTs in waste water because it enables direct analysis of aqueous samples 

(Jenkins et al. 1986). A medium similar to the mobile phase used in this HPLC separation, i.e., 50/38/12 

(v/v/v) water/methanol/acetonitrile, should be suitable for extracting DNTs from low-lipid biological 

samples and for subsequent HPLC determination after sample cleanup. 

In a study conducted by Honeychurch et al. (2003), screen-printed carbon electrodes (SPCEs) were used 

as disposable sensors for the measurement of 2,6-DNT using a stripping voltammetric method. 

Voltammograms showed the appearance of two reduction peaks that corresponded to the formation of the 

hydroxylamine derivatives from the reduction of the nitro groups and one oxidation peak possibly from 

the formation of the nitrosamines. The detection limit was 161 ng/mL (0.161 mg/L) and the sensors were 

evaluated by determining concentrations of 2,6-DNT in spiked and unspiked potable water, saliva, and 

dust wipes. Percent recoveries for spiked samples were 92.8–113% for potable water, 41.0–52.0% for 

saliva, and 67.6–79.1% for dust wipes. 2,6-DNT was not detected in unspiked samples. This new study 

indicates that SPCEs could possibly be used in the analytical detection of DNTs in occupational settings. 

Methods for the determination of the DNTs in biological samples are given in Table 7-1. 

7.2 ENVIRONMENTAL SAMPLES 

The basic method for collecting DNTs from the ambient atmosphere is adsorption on a solid phase, such 

as granular adsorbents (silica gel), filters, and impingers, followed by removal with solvents such as 

chloroform. Bubbler collectors can also be used for direct collection of analyte in a non-volatile solvent 

such as ethylene glycol. Other instrumentation for the detection of 2,4-DNT and other explosives will 

accept both air and surface particulate samples (Nacson et al. 1994). The instruments consist of capillary 

GC columns terminating in an electron capture detector (ECD); the detection limit for 2,4-DNT is 20 ppt 

(2x10-5 ppm) (Nacson et al. 1994). Also available is a portable version that is useful for a wide variety of 

applications, such as security checks, mail or passports, or in high-risk facilities (Nacson et al. 1994). 
 

 

 

 

DINITROTOLUENES 219 

7. ANALYTICAL METHODS 

Table 7-1. Analytical Methods for Determining Dinitrotoluenes in Biological
 

Materials
 

Analytical Sample Percent 

Sample matrix Preparation method method detection limit recovery Reference 

Urine containing Hydrolysis of metabolites, GC/MS 0.1 mg/L NR Turner et al. 

DNTs and extraction, derivatization 1985 

metabolites 

Urine containing Zinc-catalyzed reduction of UV/VIS 100 ng/mL (0.1 NR Smith et al. 

DNTs and DNT with hydrochloric acid to mg/L) 1995 

metabolites primary arylamines; diazotize 

and couple with N-(1-naphthyl)ethylene diamine to 

produce a colored complex 

with characteristic adsorption 

at 550 nm 

Urine containing Extraction with ethyl acetate GC 0.1 mg/L NR Woollen et 

metabolites al. 1985 

Urine Extraction with ethyl acetate TLC 0.1 mg/La NR Woollen et 

al. 1985 

Plant tissue Extract with sonication; Reverse­ 0.086B0.109 98.2 U.S. Army 

cleanup on flonsil; alumina phase LC mg/L 1998 

(LC); inject UV/VIS 

Skin Swab with ethanol HPLC/ED 5.6 ng/mLa 97 Lloyd 1983a 

(0.0056 mg/L) (2,6-DNT); 

93 

(2,4-DNT) 

Blood Extraction with toluene GC 0.00001 mg/mLa Woollen et 

(0.01 mg/L) al. 1985 

Ocean floor NR TLC NR U.S. Navy 

fauna 1972 

Skin Swab with ethanol HPLC/ED 5.6 ng/mLa 97 Lloyd 1983a 

(0.0056 mg/L) (2,6-DNT); 

93 

(2,4-DNT) 

aLowest detected concentration. 

DNT = dinitrotoluene; ECD = electron capture detection; ED = electrochemical detection; GC = gas chromatography; 

HPLC = high-performance liquid chromatography; LC = liquid chromatography; MS = mass spectrometry; NR = not 

reported; TLC = thin layer chromatography; UV/VIS = ultra-violet/visible spectroscopy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 220 

7. ANALYTICAL METHODS 

Sylvia et al. (2000) developed a Surface-Enhanced Raman Spectroscopy (SERS) to potentially detect 2,4-DNT 

vapors above TNT-based landmines. This method of detection exhibited reproducible results, sensitive detection 

levels of ≤5 ppb (0.005 ppm) 2,4-DNT, and spectra that demonstrated high signal-to-noise ratios collected in 

30 seconds. While only preliminary blind testing was done, vapor-phase SERS is a promising 

instrumental method for the detection of explosive vapors. 

Albert and Walt (2000) developed a novel cross-reactive optical microsensor for high-speed detection of 

low-level explosives and explosive-like vapors. This porous silica-based sensor, was used to detect 

DNTs in water vapor at a level of 23 ppb in clean dry air. 

Likewise, a field developed instrument with artificial cross-reactive optical sensors was employed for the 

detection of 2,4-DNT vapors in spiked soil and aqueous and ground samples. Two types of fluorescence 

based vapor sensors were developed: one sensor was semiselective for the nitroaromatic compounds 

(NAC), while the other was non-specific and cross reactive. The fluorescent sensors were exposed to 

multiple solvents and were validated/calibrated by making measurements of known and estimated 

concentrations of 2,4-DNT. The system has demonstrated the ability to detect 120 ppb 2,4-DNT vapor in 

blind humidified samples and has been exclusively used in field tests (Albert et al. 2001). 

DNTs are most commonly extracted with dichloromethane from water samples (EPA 1982a) and from 

wastes (EPA 1986a). A continuous countercurrent liquid-liquid extraction method is useful in extracting 

DNTs from surface water samples (Deroux et al. 1996). The advantage of this method is that it is capable 

of extractions from large sample volumes and unfiltered natural water samples (Deroux et al. 1996). A 

sonic extraction-liquid chromatographic method has been used for detection of 2,4-DNT in soils (Bauer et 

al. 1990; Griest et al. 1993). A simple screening method has been developed for the detection of 2,4-DNT 

in field soil samples that utilizes the spectrophotometer for identification by colorometrics after an initial 

reaction of the extract with potassium hydroxide and sodium sulfite (Jenkins and Walsh 1992). 

A biosensor has been developed by Smirnova et al. (2004) for the detection of DNTs in soil and 

groundwater. The genetic system from the Burkholderia sp. strain DNT was examined and used to 

develop the prototype cell-based biosensor. The central element of the biosensor was found to be two 

crystal structures of the transcriptional regulator DntR with acetate and thiocyanate occupying the 

inductor-binding cavity. Analysis of this biosensor was done to model how 2,4-DNT might bind to DntR 

and to ultimately make conclusions on how the specificity of the biosensor might be enhanced. 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 221 

7. ANALYTICAL METHODS 

The analysis of DNTs is normally done by GC with a variety of detectors, including flame ionization 

detector (FID), electron capture detection (ECD), Hall electrolytic conductivity detector (HECD), 

thermionic specific detector (TSD), fourier transform infrared (FT-IR), thermal energy analyzer (TEA), or 

mass spectrometry (MS). It has been noted (EPA 1986b) that 2,4-DNT is "subject to erratic (gas) 

chromatographic behavior." When mass spectrometry is used to analyze water samples for DNT, electron 

impact (EI) is preferentially used because many structure-specific fragments will be formed, which can be 

used for identification of isomers (Feltes et al. 1990). To improve the accuracy of mass spectroscopic 

techniques in the identification of pollutants in aqueous and solid matrices, EPA has developed the 

method of isotope dilution (EPA 1989). Isotope dilution employs stable, isotopically labeled analogs of 

both 2,4- and 2,6-DNT to be used as internal standards in GC/MS analysis (EPA 1989). Negative-ion 

chemical ionization has been shown to have a higher sensitivity and selectivity than EI, however, and 

should be used when determining traces of nitroaromatic compounds in complex aqueous mixtures 

(Feltes et al. 1990). 

The quenching of the photoluminescence of porous silicone films in a flowing air stream once the films 

were exposed to DNTs was monitored and the quantity of DNTs was detected in a study by Content et al. 

(2000). This detection was achieved by catalytic oxidation of DNTs. The detection limit of 2 ppb was 

observed for 2,4-DNT. 

TLC and high-performance thin layer chromatography (HPTLC) have also been used to identify and 

quantify 2,4- and 2,6-DNT in soil and water samples from contaminated waste sites (Griest et al. 1993; 

Sohr et al. 1995; Steuckart et al. 1994). GC analysis is difficult because of the large amounts of humic 

acids present which cause overlap of matrix signals without cleanup; therefore, HPTLC can be a more 

advantageous method (Steuckart et al. 1994). Cleanup is not necessary with HPTLC, except in the 

analysis of soil samples (Steuckart et al. 1994). 

A sensitive method for the analysis of DNTs in drinking water has been developed using wide-bore fused 

silica capillary column GC with an ECD (Hable et al. 1991). The detection limits of this method are 

0.04 μg/L (4x10-5 mg/L) for 2,4-DNT and 0.003 μg/L (3x10-6 mg/L) for 2,6-DNT; these detection limits 

are sensitive enough to meet the suggested requirements for EPA health advisories and water quality 

criteria. 

Also, HPLC with UV-detection for nitroaromatic compounds and amperometric detection for 

aminoaromatic and phenolic compounds were used by Spiegel and Welsch (1997) for the monitoring of 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 222 

7. ANALYTICAL METHODS 

the degradation products of explosives in groundwater. These methods of detection were reported to be 

extremely sensitive. 2,4-DNT and 2,6-DNT were observed to be nearly completely metabolized as seen 

by their decrease in the chromatograms. Spiegel et al. (2005) found that HPLC-EC was more effective in 

detecting trace compounds, whereas HPLC-UV was found to be effective in detecting chemicals in highly 

contaminated groundwater. Detection limits ranged from 1 to 40 pg/L for HPLC-EC. 

For the determination of 2,4-DNT in munitions manufacture waste water, RP-HPLC was chosen by 

Jenkins et al. (1986) because it enables direct analysis of samples in aqueous solution without prior 

extraction, attains adequate detection limits without preconcentration, and avoids problems with analyte 

thermal instability. The detection limit for 2,4-DNT was 10 μg/L (0.01 mg/L) with a standard deviation 

of 3.4 μg/L (0.034 mg/L) for concentrations up to 250 μg/L (0.25 mg/L). A convenient method for 

analysis of 2,4- and 2,6-DNT in contaminated soils used high performance liquid chromatography with 

minimal sample preparation (Preslan et al. 1993). Likewise, DNTs were also measured in field soil 

samples by using a method that included the fortification of DNTs in the soil, recovery of DNTs by 

pressurized fluid extraction, and measurement of DNTs by gas and liquid chromatography-MS (Campbell 

et al. 2003). Compared with the former EPA method SW-846 8330, this method provided a quicker time 

frame needed to complete an extraction (15 minutes as opposed to 18 hours) and MS confirmation of the 

analytes. The limit of quantitation for 2,4-DNT was 0.05 μg/g. 

Methods for the determination of DNTs in environmental samples are summarized in Table 7-2. 

7.3 ADEQUACY OF THE DATABASE 

Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the 

Administrator of EPA and agencies and programs of the Public Health Service) to assess whether 

adequate information on the health effects of DNTs is available. Where adequate information is not 

available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of research 

designed to determine the health effects (and techniques for developing methods to determine such health 

effects) of DNTs. 

The following categories of possible data needs have been identified by a joint team of scientists from 

ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if 
 

 

 

 

 

DINITROTOLUENES 223 

7. ANALYTICAL METHODS 

Table 7-2. Analytical Methods for Determining Dinitrotoluenes in Environmental
 

Samples
 

Sample Analytical Sample Percent 

matrix Preparation method method detection limit recovery Reference 

Air 

Air 

Air 

Air 

Water 

Water 

Water 

Water 

Water 

Water 

Water 

Silica gel, desorb with 

chloroform 

Dispersed on glass 

beads, glass tube, and 

Flowmeter/mixer; 

exposure to dry synthetic 

air 

Collect sample and insert 

into analyzer 

Collect sample and insert 

into analyzer 

Extraction with 

dichloromethane 

Extraction with 

dichloromethane 

Extraction with 

dichloromethane; add 

methanol 

Adjust pH of spiking 

solution to >11 with 

NaOH; extraction with 

dichloromethane; add 

anhydrous sodium 

sulfate; filter; rotary 

evaporate 

Counter current liquidliquid extraction method 

Adjust to pH 12 and 

pump into column; 

remove organic phase; 

add to retirnate and 

adjust to pH 2 before 

pumping into column 

Spike H2O sample with 

standards; conduct SPE; 

elute and dry under 

nitrogen 

GC 

FT-IR/MCT 

detector, 

NMR 

GC/ECD 

Fluorescent 

sensing film 

GC/ECD or 

TEA 

GC/MS 

(electron 

impactBfull 

scan) 

GC/MS 

Liquid-liquid 

extraction 

with GC/MS 

GC/MS 

(EI and 

PICI) 

GC/MS 

TLC 

(254 nm) 

0.1 mg/m3 

(1x10-4 mg/L) 

2 ppb 

(0.002 ppm) 

(2,4-DNT) 

20 ppt 

(2x10-5 ppm) 

(2,4-DNT) 

NR 

ECD: 

3.8x10-14 g/s 

(2,4-DNT); 

TEA: 

1.77x10-11 g/s 

(2,4-DNT) 

47 pg (2,6-DNT) 

5 ppb 

(0.005 ppm) 

0.8 μg/L 

(8x10-4 mg/L)(2, 

4-DNT); 1.4 μg/L 

(1.4x10-4 mg/L) 

(2,6-DNT) 

2 ng/L 

(2x10-6 mg/L) 

2 ng/L 

(2x10-6 mg/L) 

20 ng (scanner) 

40 ng (visually) 

70–84 

96 

(2,4-DNT); 

100 

(2,6-DNT) 

89 

89 

diol-119­

115.3 

RP-18­

100.6-102 

Hunt et al. 1980 

Content et al. 2000 

Nacson et al. 1994 

Xin et al. 2011 

Feltes et al. 1990 

Feltes et al. 1990 

Yinon 1996 

Yook et al. 1994 

Deroux et al. 1996 

Deroux et al. 1996 

Kessel and Hauck 

1996 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 224 

7. ANALYTICAL METHODS 

Table 7-2. Analytical Methods for Determining Dinitrotoluenes in Environmental
 

Samples
 

Sample Analytical Sample Percent 

matrix Preparation method method detection limit recovery Reference 

Drinking Extraction with 0.5 mL 

water toluene; rotate 

30 minutes at 15 RPM 

Groundwater Extraction with 

and surface dichloromethane; acidify 

water water sample with HCl 

and extract with isobutylmethyl ketone; 

concentrate in rotary 

evaporator; dissolve 

residues in 

dichloromethane 

Groundwater	 Extraction with 

dichloromethane 

Groundwater	 Expose three samples to 

different amounts of 

sunlight; inject sample, 

loop 

Groundwater	 Salting-Out Extraction 

(SOE) 

Waste water Diluted directly with 

(for 2,4-DNT) methanol and acetonitrile 

Waste water	 Extraction with 

dichloromethane 

Waste water	 Extraction with dichloromethane, exchange to 

hexane 

Waste water	 Extraction with 

dichloromethane; 

EPA Method 8090 

Non-water Extraction with 

miscible dichloromethane; 

waste EPA Method 8090 

GC/ECD 

TLC 

HPTLCAMD 

HPLC/UV 

(245 nm) 

HPLC/EC 

HPLC/UV 

GC/IDMS 

GC/ECD 

GC/MS 

GC/ECD 

0.003 μg/L 

(3x10-6 mg/L) 

(2,6-DNT); 

0.04 μg/L 

(4x10-5 mg/L) 

(2,4-DNT) 

10–20 ng/spot 

20 ng (2,4- and 

2,6-DNT) 

0.01–0.1 μg/L 

(1x10-5– 

1x10-4 mg/L) 

1–40 pg/L 

4.6 μg/L 

(0.0046 mg/L) 

10 μg/L 

(0.01 mg/L) 

NR 

5.7 μg/L 

(0.0057 mg/L) 

(2,4-DNT); 

1.9 μg/L 

(0.0019 mg/L) 

(2,6-DNT) 

2,000 μg/L 

(2 mg/L) 

(2,4-DNT); 

1,000 μg/L 

(1 mg/L) 

(2,6-DNT) 

93–103 

(2,6-DNT); 

93–96 

(2,4-DNT) 

– 

– 

NR 

10 

(2,4-DNT); 

17 

(2,6-DNT) 

Hable et al. 1991 

Sohr et al. 1995 

Steukart et al. 1994 

Spiegel and Welsch 

1997 

Spiegel et al. 2005 

Jenkins et al. 1986 

EPA 1980 

EPA 1982a 

EPA 1986a 

EPA 1986a 
 

 

 

 

 

 

DINITROTOLUENES 225 

7. ANALYTICAL METHODS 

Table 7-2. Analytical Methods for Determining Dinitrotoluenes in Environmental
 

Samples
 

Sample Analytical Sample Percent 

matrix Preparation method method detection limit recovery Reference 

Biosludge Extraction with sulfuric 

acid and 

dichloromethane 

Soil Extraction with 

acetonitrile in ultrasonic 

bath; flocculate 

supernatant with CaCl2; 

filter 

Soil Dilution directly with 

methanol 

Soil Extraction with acetone; 

filter supernatant; react 

with potassium hydroxide 

and sodium sulfite 

Soil Grind soil; extraction with 

acetone in ultrasonic 

bath; centrifuge; add 

5 mL toluene and remove 

acetone; dry toluene 

extract over anhydrous 

sodium sulfate 

Soil Fortification, extraction 

Soil, Extraction 

sediment, 

solid waste 

Soil, Extraction with 

sediment, dichloromethane 

solid waste 

Soil, Extraction 

sediment, 

solid waste 

Soil, water, Extraction with dichloroand methane; addition of 

municipal isotopically labeled 

sludges analog 

Soil/compost	 Acid leaching followed by 

sonic extraction 

Soil/compost	 Extraction with 

acetonitrile; combine with 

CaCl2; derivatize with 

TFAA, then 

deactivate with H2O 

GC/TEA 

SE/LC 

HPLC/PDA 

Spectrophotometry 

HPTLC/ 

AMD 

HPLC-DAD, 

LC/MS, 

GC/MS 

GC/MS 

GC/FT-IR 

GC/FT-IR 

GC/MS 

HPLC 

HPLC 

0.05 mg/L 

40–80 pg/μL 

(0.04– 

0.08 mg/L) 

2 μg/g (2,4-DNT) 

20 ng (2,4- and 

2,6-DNT) 

0.05 μg/g 

660 μg/kg 

10 μg/L 

(0.01 mg/L) 

10 μg/L 

(0.01 mg/L) 

10 μg/mL 

(10 mg/L) 

0.055– 

0.248 mg/Lc 

– 

84 

95–97 

NR 

NR 

NR 

NR 

Phillips et al. 1983 

Bauer et al. 1990 

Emmrich et al. 1993 

Jenkins and Walsh 

1992 

Steuckart et al. 1994 

Campbell et al. 2003 

EPA 1986b 

EPA 1986c 

Gurka et al. 1987 

EPA 1989 

Griest et al. 1993 

Preslan et al. 1993 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 226 

7. ANALYTICAL METHODS 

Table 7-2. Analytical Methods for Determining Dinitrotoluenes in Environmental
 

Samples
 

Sample Analytical Sample Percent 

matrix Preparation method method detection limit recovery Reference 

Materials Swab with ethanol HPLC/ED 5.6 μg/L 97 Lloyd 1983b 

exposed to (0.056 mg/L) (2,6-DNT); 

DNT (bomb 93 

debris) (2,4-DNT) 

Materials Swab surface and insert GC/ECD 20 ppt Nacson et al. 1994 

exposed to into outlet port (2x10-5 mg/L) 

DNT (2,4-DNT) 

Phenolic and Inject sample, loop- SFC oxidative-250 pg – Wallenborg et al. 

nitroaromatic separate using super­ (230 nm) reductive-100 pg 1997 

compounds critical CO2 as mobile 

phase 

Explosives Extract in acetonitrile and MECC 0.55–0.74 mg/L – Oehrle 1996 

dilute in pH 7 buffer; (214 nm) 

inject hydrostatistically 

and detect by UV 

aAnalyses for both 2,4-DNT and 2,6-DNT unless otherwise noted. bMinimum detection to ECD.
 cVaried over course of experiment.
 

AMD = automated multiple development; CaCl2 = calcium chloride; CO2 = carbon dioxide; DAD = diode array
 

detection; DNT = dinitrotoluene; ECD = electron-capture detection; ED = electrochemical detection; EI = electron
 

ionization; FT-IR = fourier transform infrared; GC = gas chromatography; HCl = hydrochloric acid; HPLC = highperformance liquid chromatography; HPTLC = high-performance thin layer chromatography; IDMS = isotope dilution 

mass spectrometry; LC = liquid chromatography; MCT = mercury cadmium telluride; MECC = micellar electrokinetic
 

capillary chromatography; MS = mass spectrometry; NaOH = sodium hydroxide; NMR = nuclear magnetic
 

resonance; NR = not reported; PDA = photodiode array detection; PICI = positive ion chemical ionization;
 

RPM = revolutions per minute; SE = solid extraction; SFC = super critical fluid chromatography; SFE = supercritical
 

fluid extraction; SPE = solid-phase extraction; TDM = thermal desorption modulator interface; TEA = thermal energy
 

analysis; TFAA = trifluoroacetic anhydride; TLC = thin layer chromatography; UV = ultraviolet absorption
 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 227 

7. ANALYTICAL METHODS 

met would reduce the uncertainties of human health assessment. This definition should not be interpreted 

to mean that all data needs discussed in this section must be filled. In the future, the identified data needs 

will be evaluated and prioritized, and a substance-specific research agenda will be proposed. 

7.3.1 Identification of Data Needs 

Methods for Determining Biomarkers of Exposure and Effect. The available methods for the 

determination of DNTs and their metabolites in biological samples are inadequate. Although one method 

exists for determination of DNTs and their metabolites in urine (Smith et al. 1995; Turner et al. 1985; 

Woolen et al. 1985), blood (Woolen et al. 1985), and skin (Lloyd 1983a; Nacson et al. 1994), there is a 

need for modern validated standard methods of analysis for such data in plant and animal tissues and 

exudates. Methods do exist for water and waste water (EPA 1982a, 1982b) and for solid wastes (EPA 

1986a, 1986b, 1986c). A method that employs the use of SPCEs has the potential to be useful for 

examining biological samples such as potable water, saliva, and dust wipes (Honeychurch et al. 2003). 

The need also exists for good methods to determine DNT biomarkers in biological materials. The 

determination of this compound in plant and animal tissues and exudates would be useful to help 

determine exposure. 

7.3.2 Ongoing Studies 

No ongoing studies regarding analytical methods sponsored by NIH or EPA were identified for DNTs. 

Methods for Determining Parent Compounds and Degradation Products in Environmental 

Media. DNTs can be analyzed in water, air, and waste samples with reasonable selectivity and 

sensitivity (EPA 1986a, 1986b, 1986c, 1989; Gurka et al. 1987; Nacson et al. 1994; Yinon 1996; Yook et 

al. 1994). Therefore, there is a reasonable database in this area. 

There exists an ongoing effort to develop a "Master Analytical Scheme" for organic compounds in water 

(Michael et al. 1988). The overall goal is to detect and measure quantitatively organic compounds at 

0.1 μg/L in drinking water, l μg/L in surface waters, and 10 μg/L in effluent waters. Analytes include 

numerous semivolatile compounds and some compounds that are only "semi-soluble" in water, as well as 

volatile compounds (boiling point <150 °C). It may be anticipated that improved methods for the 

determination of semivolatile DNT isomers in environmental samples may be developed as part of this 

effort. 
 

DINITROTOLUENES 228 

7. ANALYTICAL METHODS 

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DINITROTOLUENES 229 

8. REGULATIONS, ADVISORIES, AND GUIDELINES
 

MRLs are substance specific estimates, which are intended to serve as screening levels, are used by 

ATSDR health assessors and other responders to identify contaminants and potential health effects that 

may be of concern at hazardous waste sites. 

As summarized in Table 8-1, ATSDR has derived individual acute-duration oral MRLs for 6 DNT 

isomers (2,3-DNT, 2,4 DNT, 2,5-DNT, 2,6-DNT, 3,4-DNT and 3,5-DNT), intermediate-duration oral 

MRLs for 2,4-DNT and 2,6-DNT, and a chronic-duration oral MRL for 2,4-DNT. 

The International Agency for Research on Cancer (IARC) classifies 2,4- and 2,6-DNT as Group 2B 

carcinogens (possibly carcinogenic to humans) and 3,5-DNT as a Group 3 carcinogen (not classifiable as 

to its carcinogenicity to humans) (IARC 2015). The U.S. EPA classifies the 2,4-/2,6-DNT mixture as B2 

(probable human carcinogen) (IRIS 2003). The American Conference of Governmental Industrial 

Hygienists (ACGIH) has classified the 2,4-/2,6-DNT mixture as an A3 carcinogen (confirmed animal 

carcinogen with unknown relevance to humans) (ACGIH 2014). 

OSHA requires employers of workers who are occupationally exposed to the 2,4-/2,6-DNT mixture to 

institute engineering controls and work practices to reduce and maintain employee exposure at or below 

permissible exposure limits (PEL) of 1.5 mg/m3 (OSHA 2013). 

The EPA (IRIS 2002) has derived an oral reference dose (RfD) of 0.002 mg/kg/day for 2,4-DNT based on 

a NOAEL of 0.2 mg/kg/day for neurotoxicity, Heinz bodies, and biliary tract hyperplasia in Beagle dogs 

in a 2-year feeding study (Ellis et al. 1985). The EPA (IRIS 2003) also derived an oral slope factor (q1*) 

of 0.068 (mg/kg/day)-1 based on multiple tumor types in female rats (U.S. Army 1979) for the 

2,4-/2,6-DNT mixture. 

Tg-DNT and 3,4-DNT are on the Comprehensive Environmental Response, Compensation, and Liability 

Act (CERCLA) list of hazardous substances and reportable quantities and have been assigned a reportable 

quantity (RQ) limit of 10 pounds combined (EPA 2014d). 2,4-DNT, and 2,6-DNT also appear on the 

CERCLA list of chemicals and have been assigned RQ limits of 10 and 100 pounds, respectively (EPA 

2014d). 
 

 

 

DINITROTOLUENES 230 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

The 2,4-/2,6-DNT mixture is designated as a hazardous substances under Section 311 of the Clean Water 

Act; any discharge of these chemicals over a specified threshold level (10 pounds) into navigable waters 

is subject to reporting requirements (EPA 2013d). 

The international and national regulations, advisories, and guidelines regarding DNTs in air, water, and 

other media are summarized in Table 8-2. 
 

 

 

DINITROTOLUENES 231 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

Table 8-1. Oral Minimal Risk Levels (MRLs) for Dinitrotoluenes 

Isomer 2,3-DNT 2,4-DNT 2,5-DNT 2,6-DNT 3,4-DNT 3,5-DNT 

Acute-duration 

MRL 

(mg/kg/day) 0.09 

Critical effect Kidney and 

hematopoietic 

effects in rats 

Point of 275 mg/kg/day 

departure (LOAEL) 

Uncertainty 3,000 (10L, 

factora 10A, 10H, 3D) 

Intermediate-duration 

MRL
 

(mg/kg/day)
 

Critical effect
 

Point of 

departure
 

Uncertainty
 

factora
 

Chronic-duration 

MRL
 

(mg/kg/day)
 

Critical effect
 

Point of 

departure
 

Uncertainty
 

factora
 

0.05 0.007 

Neurotoxicity in Splenic lesions 

dogs in rats 

5 mg/kg/day 2.05 mg/kg/day 

(NOAEL) (BMDL10) 

100 (10A, 10H)	 300 (10A, 10H, 

3D) 

0.007 

Hematological 

effects in dogs 

0.67 mg/kg/day 

(BMDL1SD) 

100 (10A, 10H) 

0.001 

Hematological 

effects in dogs 

0.12 mg/kg/day 

(BMDL1SD) 

100 (10A, 10H) 

0.09	 0.03 

Hematological Hematopoietic 

effects in dogs effects in rats 

9.31 mg/kg/day 8.05 mg/kg/day 

(BMDL1SD) (BMDL10) 

100 (10A, 10H)	 300 (10A, 10H, 

3D) 

0.004 

Splenic lesions 

in dogs 

4 mg/kg/day 

(LOAEL) 

1000 (10L, 10A, 

10H) 

0.03 

Testicular 

effects in rats 

10 mg/kg/day 

(NOAEL) 

300 (10A, 10H, 

3D) 

aUncertainty factors are comprised of uncertainty associated with use of a LOAEL (L), extrapolation from animals to 

humans (A), human variability (H), and/or database limitations (D). 

BMDL10 = lower 95% confidence limit on the benchmark dose (BMD10) associated with a response rate that differs 

from the control response rate by 10%; BMDL1SD = lower 95% confidence limit on the benchmark dose (BMD1SD) 

associated with a change of 1 standard deviation from the controls; DNT = dinitrotoluene; LOAEL = lowest-observedadverse-effect-level; NOAEL = no-observed-adverse-effect level 
DINITROTOLUENES 232 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

Table 8-2. Regulations, Advisories, and Guidelines Applicable to Dinitrotoluenes 

Agency Description Information Reference 

INTERNATIONAL 

Guidelines: 

IARC Carcinogenicity classification IARC 2015 

2,4-DNT Group 2Ba 

2,6-DNT Group 2Ba 

3,5-DNT Group 3b 

WHO Air quality guidelines No data WHO 2010 

Drinking water quality guidelines No data WHO 2011 

NATIONAL 

Regulations and 

Guidelines: 

a. Air 

ACGIH TLV (8-hour TWA) ACGIH 2014 

DNTc,d 0.2 mg/m3 

AIHA ERPGs No data AIHA 2014 

DOE PAC-1e DOE 2012a 

DNTc, 2,4-, 2,6-, and 3,4-DNT 0.6 mg/m3 

PAC-2e 

DNTc 1.1 mg/m3 

2,4-DNT 4.9 mg/m3 

2,6-DNT 12 mg/m3 

3,4-DNT 33 mg/m3 

PAC-3e 

DNTc, 2,4-, 2,6-, and 3,4-DNT 200 mg/m3 

EPA AEGLs No data EPA 2014a 

Hazardous air pollutant EPA 2013a 

2,4-DNT Yes 42 USC 7412 

NAAQS No data EPA 2014c 

NIOSH REL (10-hour TWA) NIOSH 2015 

DNTc,f,g 1.5 mg/m3 

IDLH 

DNTc,f,g 50 mg/m3 

OSHA PEL (8-hour TWA) for general industry OSHA 2013 

DNTc,g 1.5 mg/m3 29 CFR 1910.1000, 

Table Z-1 

PEL (8-hour TWA) for construction 

DNTc,g 1.5 mg/m3 

OSHA2014a 

29 CFR 1926.55, 

Appendix A 

PEL (8-hour TWA) for shipyards 

DNTc,g 1.5 mg/m3 

OSHA 2014b 

29 CFR 1915.1000 
 

 

DINITROTOLUENES 233 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

Table 8-2. Regulations, Advisories, and Guidelines Applicable to Dinitrotoluenes 

Agency Description Information Reference 

NATIONAL(cont.) 

b. Water 

EPA Designated as hazardous substances in EPA 2013b 

accordance with Section 311(b)(2)(A) of 40 CFR 116.4 

the Clean Water Act 

DNTc, 2,4-, 2,6-, and 3,4-DNT Yes 

Drinking water standards and health EPA 2012b 

advisories 

1-day health advisory for a 10-kg 

child 

2,4-DNT 1 mg/L 

2,6-DNT 0.4 mg/L 

10-day health advisory for a 10-kg 

child 

2,4-DNT 1 mg/L 

2,6-DNT 0.04 mg/L 

DWEL 

2,4-DNT 0.1 mg/L 

2,6-DNT 0.04 mg/L 

10-4 Cancer risk 

2,4-DNT 0.005 mg/L 

2,6-DNT 0.005 mg/L 

National primary drinking water standards No data EPA 2009 

National recommended water quality 

criteria: human health for the 

consumption of 

EPA 2013c 

2,4-DNT 

Water plus organism 0.11 μg/Lh,i 

Organism only 3.4 μg/Lh,i 

Reportable quantities of hazardous 

substances designated pursuant to 

Section 311 of the Clean Water Act 

EPA 2013d 

40 CFR 117.3 

DNT 10 pounds 

c. Food 

FDA EAFUSj No data FDA 2013 

d. Other 

ACGIH Carcinogenicity classification ACGIH 2014 

DNT A3c,k 

EPA Carcinogenicity classification 

2,4-/2,6-DNT mixture; no CASRN B2l 

IRIS 2003 

Oral slope factor 

2,4-/2,6-DNT mixture; no CASRN 0.68 (mg/kg/day)-1 
 

DINITROTOLUENES 234 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

Table 8-2. Regulations, Advisories, and Guidelines Applicable to Dinitrotoluenes 

Agency Description Information Reference 

NATIONAL(cont.)
 

EPA (cont.)
 RfC 

2,4-DNT 

RfD 

2,4-DNT 

Identification and listing of hazardous 

waster 

2,4-DNT 

2,6-DNT 

Inert pesticide ingredients in pesticide 

products 

Superfund, emergency planning, and 

community right-to-know 

Designated CERCLA hazardous 

substance and reportable quantity 

DNTc and 3,4-DNT 

2,4-DNT 

2,6-DNT 

Effective date of toxic chemical 

release reporting 

DNT
 

2,4-DNT
 

2,6-DNT
 

Extremely hazardous substances 

and its threshold planning quantity 

TSCA chemical lists and reporting 

periods 

2,4-DNT 

Effective date 

Reporting date 

TSCA health and safety data reporting 

2,4-DNT
 

Effective date
 

Reporting date
 

IRIS 2002 

No data 

0.002 mg/kg/day 

EPA 2013e 

40 CFR 261, 

U105 Appendix VIII 

U106 

No data EPA 2014c 

EPA 2014d 

40 CFR 302.4 

10 poundsm 

10 poundsn 

100 poundso 

EPA 2014e 

40 CFR 372.65 

01/01/1990 

01/01/1987 

01/01/1987 

No data EPA 2014f 

40 CFR 355, 

Appendix A 

EPA 2014f 

40 CFR 712.30 

03/11/1994 

05/10/1994 

EPA 2014g 

40 CFR 716.120 

03/11/1994 

06/30/1998 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 235 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

Table 8-2. Regulations, Advisories, and Guidelines Applicable to Dinitrotoluenes 

Agency Description Information Reference 

NATIONAL(cont.) 

NTP Carcinogenicity classification No data NTP 2014 

aGroup 2B: possibly carcinogenic to humans. bGroup 3: not classifiable as to its carcinogenicity to humans
 cTg-DNT is composed primarily of the 2,4- and 2,6-DNT isomers and the other four isomers (2,3-, 2,5-, 3,4-, and 

3,5-DNT) make up only 5% of Tg-DNT. dSkin: refers to the potential significant contribution to the overall exposure by the cutaneous route.
 eDefinitions of PAC terminology are available from U.S. Department of Energy (DOE 2012b).
 f

NIOSH potential occupational carcinogen.
 gSkin designation. hThis criterion is based on carcinogenicity of 10-6 risk.
 i

EPA has updated its national recommended water quality criteria for human health. The comment period for the 

draft water quality criteria ended August 13, 2014. A final update has not been issued yet, but the proposed criteria 

for 2,4-DNT is 0.0378 μg/L for water + organism and 0.711 μg/L for organism only (EPA 2014b).
 j

The EAFUS list of substances contains ingredients added directly to food that FDA has either approved as food 

additives or listed or affirmed as GRAS.
 kA3: confirmed animal carcinogen with unknown relevance to humans.
 l

Group B2: probable human carcinogen.
 mDesignated CERCLA hazardous substance pursuant to Section 311(b)(2) and Section 307(a) of the Clean Water
 

Act.
 nDesignated CERCLA hazardous substance pursuant to Section 311(b)(2) and Section 307(a) of the Clean Water
 

Act, Section 112 of the Clean Air Act, and Section 3001 of RCRA.
 oDesignated CERCLA hazardous substance pursuant to Section 311(b)(2) and Section 307(a) of the Clean Water
 

Act, and Section 3001 of RCRA.
 

ACGIH = American Conference of Governmental Industrial Hygienists; AEGL = acute exposure guideline levels;
 

AIHA = American Industrial Hygiene Association; CERCLA = Comprehensive Environmental Response,
 

Compensation, and Liability Act; CFR = Code of Federal Regulations; DNT = dinitrotoluene; DOE = Department of
 

Energy; DWEL = drinking water equivalent level; EAFUS = Everything Added to Food in the United States;
 

EPA = Environmental Protection Agency; ERPG = emergency response planning guidelines; FDA = Food and Drug 

Administration; GRAS = Generally Recognized As Safe; IARC = International Agency for Research on Cancer;
 

IDLH = immediately dangerous to life or health; IRIS = Integrated Risk Information System; NAAQS = National
 

Ambient Air Quality Standards; NIOSH = National Institute for Occupational Safety and Health; NTP = National
 

Toxicology Program; OSHA = Occupational Safety and Health Administration; PAC = Protective Action Criteria;
 

PEL = permissible exposure limit; RCRA = Resource Conservation and Recovery Act; REL = recommended 

exposure limit; RfC = inhalation reference concentration; RfD = oral reference dose; TLV = threshold limit values;
 

TSCA = Toxic Substances Control Act; TWA = time-weighted average; WHO = World Health Organization
 
DINITROTOLUENES 236 

8. REGULATIONS, ADVISORIES, AND GUIDELINES 

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DINITROTOLUENES 237 

9. REFERENCES
 

Abernethy DJ, Couch DB. 1982. Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese hamster 

ovary cells. Mutat Res 103:53-59. 

ACGIH. 2014. Dinitrotoluene. In: TLVs and BEIs based on the documentation of the threshold limit 

values for chemical substances and physical agents and biological exposure indices. Cincinnati, OH: 

American Conference of Governmental Industrial Hygienists, 28, 72-77. 

Adlercreutz H. 1995. Phytoestrogens: Epidemiology and a possible role in cancer protection. Environ 

Health Perspect Suppl 103(7):103-112. 

Agency for Toxic Substances and Disease Registry. 1989. Decision guide for identifying substancespecific data needs related to toxicological profiles; Notice. Agency for Toxic Substances and Disease 

Registry, Division of Toxicology. Fed Regist 54(174):37618-37634. 

Agency for Toxic Substances and Disease Registry. 2009. Public health assessments and health 

consultations. Joliet Army Ammunition Plant (Manufacturing Area) and Joliet Army Ammunition Plant 

(Lap Area), Joliet, Will County, Illinois. http://www.atsdr.cdc.gov/HAC/pha/pha.asp?docid=558&pg=1. 

April 15, 2015. 

Agency for Toxic Substances and Disease Registry. 2013. ATSDR case studies in environmental 

medicine. Nitrate/nitrite toxicity. Atlanta, GA: Agency for Toxic Substances and Disease Registry. 

U.S. Department of Health and Human Services. 

http://www.atsdr.cdc.gov/csem/nitrate_2013/docs/nitrite.pdf. May 20, 2014. 

AIChE. 1992. 3,5-Dinitrotoluene. C7H6N204. In: Physical and thermodynamic properties of pure 

chemicals. American Institute of Chemical Engineers, Design Institute for Physical Property Data. 

Philadelphia, PA: Taylor and Francis. 

AIHA. 2014. Current ERPG Values (2014). Fairfax, VA: American Industrial Hygiene Association. 

https://www.aiha.org/getinvolved/AIHAGuidelineFoundation/EmergencyResponsePlanningGuidelines/Documents/2014%20ERP 

G%20Values.pdf. March 4, 2015. 

Albert KJ, Walt DR. 2000. High-speed fluorescence detection of explosives-like vapors. Anal Chem 

72(9):1947-1955. 

Altman PL, Dittmer DS. 1974. Biological handbooks: Biology data book. Vol. III. 2nd ed. Bethesda, 

MD: Federation of American Societies for Experimental Biology, 1987-2008, 2041. 

Altschuh J, Brüggemann R, Santi H, et al. 1999. Henry's law constants for a diverse set of organic 

chemicals: Experimental determination and comparison of estimation methods. Chemosphere 

39(11):1871-1887. 

* Not cited in text 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 238 

9. REFERENCES 

Andersen ME, Krishnan K. 1994. Relating in vitro to in vivo exposures with physiologically based tissue 

dosimetry and tissue response models. In: Salem H, ed. Animal test alternatives: Refinement, 

reduction, replacement. New York, NY: Marcel Dekker, Inc., 9-25. 

Andersen ME, Clewell HJ, Gargas ML, et al. 1987. Physiologically based pharmacokinetics and the risk 

assessment process for methylene chloride. Toxicol Appl Pharmacol 87(2):185-205. 

Ashby J, Burlinson B, Lefevre PA, et al. 1985. Non genotoxicity of 2,4,6-trinitrotoluene (TNT) to the 

mouse bone marrow and the rat liver: Implications for its carcinogenicity. Arch Toxicol 58:14-19. 

Atkinson R, Carter WPL, Darnall KR, et al. 1980. A smog chamber and modeling study of the gas phase 

nitrogen oxides air photo-oxidation of toluene and the cresols. Int J Chem Kinet 12:779-836. 

Banerjee HN, Verma M, Hou LH, et al. 1999. Cytotoxicity of TNT and its metabolites. Yale J Biol Med 

72(1):1-4. 

Barnes DG, Dourson M. 1988. Reference dose (RfD): Description and use in health risk assessments. 

Regul Toxicol Pharmacol 8(4):471-486. 

Bauer CF, Koza SM, Jenkins TF. 1990. Liquid chromatographic method for determination of explosives 

residues in soil: Collaborative study. J Assoc Off Anal Chem 73:541-552. 

Bausum HT, Mitchell WR, Major MA. 1992. Biodegradation of 2,4- and 2,6-dinitrotoluene by 

freshwater microorganisms. J Environ Sci Health A27:663-695. 

Bentur Y, Keyes DC. 2004. Explosives. In: Dart RC, ed. Medical Toxicology. 3rd ed. Philadelphia, 

PA: Lippincott Williams & Wilkins, 1258-1279. 

Berger GS, ed. 1994. Epidemiology of endometriosis. In: Endometriosis: Advanced management and 

surgical techniques. New York, NY: Springer-Verlag, 3-7. 

Bloch E, Gondos B, Gatz M, et al. 1988. Reproductive toxicity of 2,4-dinitrotoluene in the rat. Toxicol 

Appl Pharmacol 94:466-472. 

Bond JA, Medinsky MA, Dent JG, et al. 1981. Sex dependent metabolism and biliary excretion of 

carbon-14 labeled dinitrotoluene in isolated perfused rat livers. J Pharmacol Exp Ther 219:598-603. 

Bond JA, Rickert DE. 1981. Metabolism of 2,4-dinitrotoluene by freshly isolated Fischer-344 rat 

primary hepatocytes. Drug Metab Dispos 9:10-14. 

Bradley PM, Chapelle FH, Landmeyer JE et al. 1994. Microbial transformation of nitroaromatics in 

surface soils and aquifer materials. Appl Environ Microbiol 60:2170-2175. 

Bruning T, Chronz C, Thier R, et al. 1999. Occurrence of urinary tract tumors in miners highly exposed 

to dinitrotoluene. J Occup Environ Med 41(3):144-149. 

Brüning T, Thier R, Mann H. et al. 2001. Pathological excretion patterns of urinary proteins in miners 

highly exposed to dinitrotoluene. J Occup Environ Med 43(7):610-615. 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 239 

9. REFERENCES 

Callahan MA, Slimak MW, Gabel N, et al. 1979. Water-related environmental fate of 129 priority 

pollutants. Volume II. Washington, DC: Monitoring and Data Support Division (WH-553), U.S. 

Environmental Protection Agency. EPA440479029b. PB80204381, 81-1 to 82-8. 

Camanzo J, Rice CP, Jude DG, et al. 1987. Organic priority pollutants in nearshore fish from 14 Lake 

Michigan (USA) tributaries and embayments, 1983. J Great Lakes Res 13:296-309. 

Campbell S, Ogoshi R, Uehara G, et al. 2003. Trace analysis of explosives in soil: Pressurized fluid 

extraction and gas and liquid chromatography-mass spectrometry. J Chromatogr Sci 41(6):284-288. 

Chadwick RW, George SE, Chang J, et al. 1990. Comparative gastrointestinal enzyme activity and 

activation of the promutagen 2,6-dinitrotoluene in male CD-1 mice and male Fischer 344 rats. Cancer 

Lett 52:13-19. 

Chadwick RW, George SE, Kohan MJ, et al. 1993. Potentiation of 2,6-dinitrotoluene genotoxicity in 

Fischer-344 rats by pretreatment with Aroclor 1254. Toxicology 80:153-171. 

Chapman DE, Michener SR, Powis G. 1993. In vitro metabolism of [3

H]2,6-dinitrotoluene by human 

and rat liver. Toxicol in Vitro 7(3):213-220. 

Cheng J, Kanjo Y, Suidan MT, et al. 1996. Anaerobic biotransformation of 2,4-dinitrotoluene with 

ethanol as primary substrate: Mutual effect of the substrates on their biotransformation. Water Research 

30:307-314. 

Cheng J, Suldan MT, Venosa AD. 1995. Anaerobic biotransformation of 2,4-dinitrotoluene with 

different primary substrates. Proceedings - Water and Environmental Federation Annual Conference 68th, 

99-109. 

Chiu CW, Lee LH, Wang CY, et al. 1978. Mutagenicity of some commercially available nitro 

compounds for Salmonella typhimurium. Mutat Res 58:11-22. 

Clewell HJ, Andersen ME. 1985. Risk assessment extrapolations and physiological modeling. Toxicol 

Ind Health 1(4):111-131. 

CMA. 1991. Initial submission from Chemical Manufacturers Association to U.S. EPA submitting 

information on 2,6-dinitrotoluene acute (6-hour) inhalation toxicity study in rats with attachments. 

Chemical Manufacturers Association. NTIS OTS0533663. 

Content S, Trogler WC, Sailor MJ. 2000. Detection of nitrobenzene, DNT, and TNT vapors by 

quenching of porous silicon photoluminescence. Chemistry 6(12):2205-2213. 

Costa LG, Aschner M, Vitalone A, et al. 2004. Developmental neuropathology of environmental agents. 

Annu Rev Pharmacol Toxicol 44: 87-110. 

Couch DB, Allen PF, Abernethy DJ. 1981. The mutagenicity of dinitrotoluenes in Salmonella 

typhimurium. Mutat Res 90:373-383. 

Currance PL, Clements B, Bronstein AC. 2007. Dinitrophenol and related compounds. In: Emergency 

care for hazardous materials exposure. St. Louis, MO: Mosby Jems, 335-338. 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 240 

9. REFERENCES 

Dellarco VL, Prival MJ. 1989. Mutagenicity of nitro compounds in Salmonella typhimurium in the 

presence of flavin mononucleotide in a preincubation assay. Environ Mol Mutagen 13:116-127. 

Deng Y, Meyer SA, Guan X, et al. 2011. Analysis of common and specific mechanisms of liver function 

affected by nitrotoluene compounds. PLoS One 6(2):e14662. 

Deroux JM, Gonzalez C, Le Cloirec P, et al. 1996. Analysis of extractable organic compounds in water 

by gas chromatography mass spectrometry: Applications to surface water. Talanta 43:365-380. 

De Vault DS. 1985. Contaminants in fish from Great Lakes harbors and tributary mouths. Arch Environ 

Contam Toxicol 14:587-594. 

Dillert R, Brandt M, Fornefett I, et al. 1995. Photocatalytic degradation of trinitrotoluene and other 

nitroaromatic compounds. Chemosphere 30:2333-2341. 

DOE. 2012a. Table 3: PACs by CASRN (pdf). PAC Rev 27 Tables - PAC data and chemical properties 

presented in pdf and excel tables. Protective Action Criteria (PAC) with AEGLs, ERPGs, & TEELs: 

Rev. 27 for chemicals of concern - March 2012. Oak Ridge, TN: U.S. Department of Energy. 

http://energy.gov/ehss/protective-action-criteria-pac-aegls-erpgs-teels-rev-27-chemicals-concern-march2012. March 4, 2015. 

DOE. 2012b. Protective action criteria (PAC): Chemicals with AEGLs, ERPGs, & TEELs. Definition 

of PACs (AEGLs, ERPGs or TEELs). Protective Action Criteria (PAC) with AEGLs, ERPGs, & TEELs: 

Rev. 27 for Chemicals of Concern - March 2012. Oak Ridge, TN: U.S. Department of Energy. 

http://energy.gov/ehss/protective-action-criteria-pac-aegls-erpgs-teels-rev-27-chemicals-concern-march2012. March 24, 2015. 

Dunkel VC, Zeiger E, Brusik D, et al. 1985. Reproducibility of microbial mutagenicity assays: II. 

Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ 

Mutagen 7:1-248. 

Einistö P, Watanabe M, Ishidate M Jr., et al. 1991. Mutagenicity of 30 chemicals in Salmonella 

typhimurium strains possessing different nitroreductase or O-acetyltransferase activities. Mutat Res 

259:95-102. 

Ek CJ, Dziegielewska KM, Habgood MD, et al. 2012. Barriers in the developing brain and 

neurotoxicology. Neurotoxicology 33(3):586-604. 

Ellis HV, Hong CB, Lee CC, et al. 1985. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. 

Part I. Beagle dog. J Am Coll Toxicol 4:233-242. 

Emmrich M, Kaiser M, Rueden H, et al. 1993. Determination of RDX, 2,4,6-trinitrotoluene and other 

nitroaromatic compounds by high-performance liquid chromatography with photodiode-array detection. J 

Chromatogr 645:89-94. 

Engelhardt G, Schwind KR, Mussler B. 2004. The testing of chemicals in the Syrian hamster embryo 

(SHE) cell transformation assay for assessment of carcinogenic potential. Toxicol in Vitro 18(2):213­

218. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 241 

9. REFERENCES 

EPA. 1979. Evaluation of the ultraviolet-ozone and ultraviolet-oxidant treatment of pink water. 

Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and Development, Industrial 

Environmental Research Laboratory. EPA600279129. PB300763. 

EPA. 1980. Semivolatile organic compounds by isotope dilution GC-IDMS-method 1625. U.S. 

Environmental Protection Agency. 

EPA. 1982a. Nitroaromatics and isophorone-method 609. Methods for organic chemical analysis of 

municipal and industrial wastewater. Cincinnati, OH: U.S. Environmental Protection Agency. 

Environmental Monitoring and Support Laboratory, 609-1 to 609-8. 

EPA. 1982b. Base/neutrals and acids-method 625. Methods for organic chemical analysis of municipal 

and industrial wastewater. Cincinnati, OH: U.S. Environmental Protection Agency. Environmental 

Monitoring and Support Laboratory, 625-1 to 625-19. 

EPA. 1982c. Aquatic fate process data for organic priority pollutants. U.S. Environmental Protection 

Agency. EPA440481014. PB87169090. 

EPA. 1986a. Nitroaromatics and cyclic ketones-method 8090. In: Test methods for evaluating solid 

wastes, SW-846. 3rd ed. Washington, DC: U.S. Environmental Protection Agency, Office of Solid 

Waste and Emergency Response, 8090-1 to 8090-15. 

EPA. 1986b. Gas chromatography/mass spectrometry for semivolatile organics: Capillary column 

technique-method 8270. In: Test methods for evaluating solid wastes, SW-846. 3rd ed. Washington, 

DC: U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response, 8270-1 to 

8270-32. 

EPA. 1986c. Capillary column analysis of semivolatile organic compounds by gas 

chromatography/Fourier transform infrared (GC/FT-IR) spectrometry-method 8410. In: Test methods 

for evaluating solid wastes, SW-846. 3rd ed. Washington, DC: U.S. Environmental Protection Agency, 

Office of Solid Waste and Emergency Response, 8410-1 to 8410-17. 

EPA. 1989. Method 1624: Volatile organic compounds by isotope dilution GCMS; Method 1625: 

Semivolatile organic compounds by isotope dilution GCMS. U.S. Environmental Protection Agency, 

Office of Water Regulations and Standards, Industrial Technology Division. EPA440189023. 

EPA. 1990. Interim methods for development of inhalation reference concentrations. Washington, DC: 

U.S. Environmental Protection Agency, Office of Health and Environmental Assessment, Office of 

Research and Development, Environmental Criteria and Assessment Office. EPA600890066A. 

EPA. 1992. Health advisory for 2,4- and 2,6-dinitrotoluene (DNT). Washington, DC: U.S. 

Environmental Protection Agency, Office of Water, Office of Science and Technology, Health and 

Ecological Criteria Division. PB92189315. 

EPA. 1995. Health effects assessment summary tables. Washington, DC: U.S. Environmental 

Protection Agency, Office of Research and Development, Office of Emergency and Remedial Response. 

EPA540R95036. PB95921199. 

EPA. 1996. Drinking water regulations and health advisories. Washington, DC: U.S. Environmental 

Protection Agency. EPA822R96001. 
 

 

 

 

 

DINITROTOLUENES 242 

9. REFERENCES 

EPA. 1997. Special report on environmental endocrine disruption: An effects assessment and analysis. 

Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. EPA630R96012. 

EPA. 2000a. Benchmark dose technical guidance document. Washington, DC: U.S. Environmental 

Protection Agency, Risk Assessment Forum. EPA630R00001. 

EPA. 2000b. National air pollutant emission trends, 1900-1998. Research Triangle Park, NC: U.S. 

Environmental Protection Agency, Office of Air Quality Planning and Standards. EPA454R00002. 

http://www.epa.gov/ttn/chief/trends/trends98/trends98.pdf. May 08, 2012. 

EPA. 2005. Toxic chemical release inventory reporting forms and instructions: Revised 2004 version. 

Section 313 of the Emergency Planning and Community Right-to-Know Act (Title III of the Superfund 

Amendments and Reauthorization Act of 1986). U.S. Environmental Protection Agency, Office of 

Environmental Information. EPA260B05001. 

EPA. 2008a. Drinking water health advisory for 2,4-dinitrotoluene and 2,6-dinitrotoluene. Washington, 

DC: U.S. Environmental Protection Agency. EPA822R08010. 

http://nepis.epa.gov/Exe/ZyPURL.cgi?Do ckey=P100165L.txt. May 02, 2012. 

EPA. 2008b. The analysis of occurrence data from the first unregulated contaminant monitoring 

regulation (UCMR 1) in support of regulatory determinations for the second drinking water contaminant 

candidate list (CCL 2). U.S. Environmental Protection Agency. EPA815R08013. 

http://www.epa.gov/ogwdw/ccl/pdfs/reg_determine2/report_ccl2-reg2_ucmr1_occurrencereport.pdf. 

May 02, 2012. 

EPA. 2009. National primary drinking water regulations. Washington, DC: U.S. Environmental 

Protection Agency, Office of Ground Water and Drinking Water. EPA816F090004. 

http://water.epa.gov/drink/contaminants/upload/mcl-2.pdf. March 4, 2015. 

EPA. 2011. Dinitrotoluenes. EPI Suite results for CAS 602-01-7, 619-15-8, 606-20-2, 618-85-9, 610­

39-9. Download EPI SuiteTM v4.10. U.S. Environmental Protection Agency. 

http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm. May 08, 2012. 

EPA. 2012a. CAS 121-14-2. Non-confidential 2006 IUR records by chemical, including manufacturing, 

processing and use information. U.S. Environmental Protection Agency. 

http://cfpub.epa.gov/iursearch/index.cfm?err=t#chemical. May 03, 2012. 

EPA. 2012b. 2012 Edition of the drinking water standards and health advisories. Washington, DC: U.S. 

Environmental Protection Agency, Office of Water. EPA822S12001. 

http://water.epa.gov/action/advisories/drinking/upload/dwstandards2012.pdf. March 4, 2015. 

EPA. 2012c. National ambient air quality standards (NAAQS). Washington, DC: U.S. Environmental 

Protection Agency, Office of Air and Radiation. http://www.epa.gov/air/criteria.html. January 08, 2014. 

EPA. 2013a. Title 42 - The public health and welfare. Chapter 85 - Air pollution prevention and control. 

Subchapter I - programs and activities. Part A - Air quality and emission limitations. Hazardous air 

pollutants. United States Code 42 USC 7412. http://www.gpo.gov/fdsys/pkg/USCODE-2013­

title42/pdf/USCODE-2013-title42-chap85-subchapI-partA-sec7412.pdf. April 9, 2015. 
 

 

 

 

 

DINITROTOLUENES 243 

9. REFERENCES 

EPA. 2013b. Designation of hazardous substances. U.S. Environmental Protection Agency. Code of 

Federal Regulations 40 CFR 116.4. http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol22/pdf/CFR2014-title40-vol22-sec116-4.pdf. March 4, 2015. 

EPA. 2013c. National recommended water quality criteria. Washington, DC: U.S. Environmental 

Protection Agency, Office of Water, Office of Science and Technology. 

http://water.epa.gov/scitech/swguidance/standards/criteria/current/index.cfm. March 4, 2015. 

EPA. 2013d. Determination of reportable quantities for hazardous substances. Subpart A - General 

provisions. U.S. Environmental Protection Agency. Code of Federal Regulations 40 CFR 117.3. 

http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol22/pdf/CFR-2014-title40-vol22-sec117-3.pdf. 

March 4, 2015. 

EPA. 2013e. Appendix VIII to Part 261-Hazardous constituents. U.S. Environmental Protection 

Agency. Code of Federal Regulations 40 CFR 261, Appendix VIII. http://www.gpo.gov/fdsys/pkg/CFR2013-title40-vol27/pdf/CFR-2013-title40-vol27-part261-appVIII.pdf. September 10, 2014. 

EPA. 2013f. Appendix A to Part 355—The list of extremely hazardous substances and their threshold 

planning quantities. U.S. Environmental Protection Agency. Code of Federal Regulations 40 CFR 355. 

http://www.gpo.gov/fdsys/pkg/CFR-2013-title40-vol29/pdf/CFR-2013-title40-vol29-part355-appA.pdf. 

September 10, 2014. 

EPA. 2014a. Final AEGLs (162). Washington, DC: U.S. Environmental Protection Agency, Office of 

Pollution Prevention and Toxics. 

http://www.epa.gov/oppt/aegl/pubs/compiled_aegls_update_03oct2014.pdf. March 4, 2015. 

EPA. 2014b. Draft: Updated national recommended water quality criteria - human health. Washington, 

DC: U.S. Environmental Protection Agency, Office of Water, Office of Science and Technology. 

http://water.epa.gov/scitech/swguidance/standards/criteria/current/hhdraft.cfm. April 9, 2015. 

EPA. 2014c. InertFinder. U.S. Environmental Protection Agency. 

http://iaspub.epa.gov/apex/pesticides/f?p=101:1:. March 31, 2015. 

EPA. 2014d. Designation of hazardous substances. U.S. Environmental Protection Agency. Code of 

Federal Regulations 40 CFR 302.4. http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol28/pdf/CFR2014-title40-vol28-sec302-4.pdf. March 4, 2015. 

EPA. 2014e. Chemicals and chemical categories to which this part applies. Subpart D - Specific toxic 

chemical listings. U.S. Environmental Protection Agency. Code of Federal Regulations 40 CFR 372.65. 

http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol28/pdf/CFR-2014-title40-vol28-sec372-65.pdf. 

March 4, 2015. 

EPA. 2014f. Chemical lists and reporting periods. Subpart B - Manufacturers reporting - preliminary 

assessment information. U.S. Environmental Protection Agency. Code of Federal Regulations 40 CFR 

712.30. http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol31/pdf/CFR-2014-title40-vol31-sec712­

30.pdf. April 9, 2015. 

EPA. 2014g. Substances and listed mixtures to which this subpart applies. Subpart B - Specific 

chemical listings. U.S. Environmental Protection Agency. Code of Federal Regulations 40 CFR 

716.120. http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol31/pdf/CFR-2014-title40-vol31-sec716­

120.pdf. April 9, 2015. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 244 

9. REFERENCES 

FDA. 2013. Everything added to food in the United States (EAFUS). Washington, DC: U.S. Food and 

Drug Administration. http://www.accessdata.fda.gov/scripts/fcn/fcnnavigation.cfm?rpt=eafuslisting. 

April 9, 2015. 

Feltes J, Levsen K, Volmer D, et al. 1990. Gas chromatographic and mass spectrometric determination 

of nitroaromatics in water. J Chromatogr 518:21-40. 

Fomon SJ. 1966. Body composition of the infant: Part I: The male reference infant. In: Falkner F, ed. 

Human development. Philadelphia, PA: WB Saunders, 239-246. 

Fomon SJ, Haschke F, Ziegler EE, et al. 1982. Body composition of reference children from birth to age 

10 years. Am J Clin Nutr 35(Suppl 5):1169-1175. 

Ford LS. 1981. Eye irritation test of 1-methyl-2,4-dinitrotoluene in rabbits with cover letter outlining 

current study of tetrahydrofuran dated 05/10/94. Submitted to the U.S. Environmental Protection Agency 

under TSCA Section 8D. OTS0557136. 

Freedman DL, Shanley RS, Scholze RJ. 1996. Aerobic biodegradation of 2,4-dinitrotoluene, 

aminonitrotoluene isomers, and 2,4-diaminotoluene. J Haz Mater 49:1-14. 

George SE, Allison JC, Brooks LR, et al. 1998. Modulation of 2,6-dinitrotoluene genotoxicity by 

alachlor treatment of Fischer 344 rats. Environ Mol Mutagen 31(3):274-281. 

George SE, Chadwick RW, Chang JJ, et al. 1992. 2,4,5-Trichlorophenoxyacetic acid influence on 

2,6-dinitrotoluene-induced urine genotoxicity in Fischer 344 rats: Effect on gastrointestinal microflora 

and enzyme activity. Fundam Appl Toxicol 18:240-246. 

George SE, Kohan MJ, Warren SH. 1996. Hepatic DNA adducts and production of mutagenic urine in 

2,6-dinitrotoluene-treated B6C3F1 male mice. Cancer Lett 102:107-111. 

Gillett JW. 1983. A comprehensive pre-biologic screen for ecotoxicologic effects. Environ Toxicol 

Chem 2:463-476. 

Giwercman A, Carlsen E, Keiding N, et al. 1993. Evidence for increasing incidence of abnormalities of 

the human testis: A review. Environ Health Perspect Suppl 101(2):65-71. 

Goldsworthy TL, Hamm Jr. TE, Rickert DE et al. 1986. The effect of diet on 2,6-dinitrotoluene 

hepatocarcinogenesis. Carcinogenesis 7:1909-1915. 

Grant CL, Jenkins TF, Myers KF, et al. 1995. Holding-time estimates for soils containing explosive 

residues: Comparison of fortification vs. field contamination. Environ Toxicol Chem 14:1865-1874. 

Great Lakes Commission. 2006. 2002 Inventory of toxic air emissions for the Great Lakes Region. 

http://glc.org/air/inventory/2002/2002report_Full.pdf. May 03, 2012. 

Griest WH, Stewart AJ, Tyndall RL, et al. 1993. Chemical and toxicological testing of composted 

explosives-contaminated soil. Environ Toxicol Chem 12:1105-1116. 

Gruener N. 1976. Ontogenetic development of NADH-dependent methemoglobin reductase in 

erythrocytes of man and rat. J Toxicol Environ Health 1:787-791. 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 245 

9. REFERENCES 

Guest D, Schnell SR, Rickert DE, et al. 1982. Metabolism of 2,4-dinitrotoluene by intestinal 

microorganisms from rat, mouse and man. Toxicol Appl Pharmacol 64:160-168. 

Gurka DF, Titus R, Griffiths PR, et al. 1987. Evaluation of an improved single-beam gas 

chromatography/Fourier transform infrared interface for environmental analysis. Anal Chem 

59:2362-2369. 

Guzelian PS, Henry CJ, Olin SS, eds. 1992. Similarities and differences between children and adults: 

Implications for risk assessment. Washington, DC: International Life Sciences Institute Press. 

Hable M, Stern C, Asowata C, et al. 1991. The determination of nitroaromatics and nitroamines in 

ground and drinking water by wide-bore capillary gas chromatography. J Chromatogr Sci 29:131-135. 

Haderlein SB, Weissmahr KW, Schwarzenbach RP. 1996. Specific adsorption of nitroaromatic 

explosives and pesticides to clay minerals. Environ Sci Technol 30(2):612-622. 

Hallas LE, Alexander M. 1983. Microbial transformation of nitro aromatic compounds in sewage 

effluent. Appl Environ Microbiol 45:1234-1241. 

Hamill PVV, Steinberger E, Levine RJ, et al. 1982. The epidemiologic assessment of male reproductive 

hazard from occupational exposure to TDA and dinitrotoluene. J Occup Med 24:985-993. 

Harth V, Bolt HM, Bruning T. 2005. Cancer of the urinary bladder in highly exposed workers in the 

production of dinitrotoluenes: A case report. Int Arch Occup Environ Health 78(8):677-680. 

Hashimoto A, Sakino H, Kojima T, et al. 1982. Sources and behavior of dinitrotoluene isomers in sea 

water. Water Res 16:891-898. 

Hashimoto Y, Tokura K, Kishi H, et al. 1984. Prediction of sea-water solubility of aromatic compounds. 

Chemosphere 13:881-888. 

HazDat. 2007. 2,4- and 2,6-DNT. HazDat Database: ATSDR’s Hazardous Substance Release and 

Health Effects Database. Atlanta, GA: Agency for Toxic Substances and Disease Registry. 

http://www.atsdr.cdc.gov/hazdat.html. 

Hazleton Laboratories. 1977. A thirty-day toxicology study in Fischer-344 rats given dinitrotoluene, 

technical grade. Full report. Submitted to Chemical Industry Institute of Toxicology, Research Triangle 

Park, NC. Submitted to the U.S. Environmental Protection Agency under TSCA Section 8D. Document 

No. 878212040. OTS0205947. 

Hazleton Laboratories. 1982. 104-week chronic study in rats. Dinitrotoluene. Final report Volume I of 

II. Submitted to Chemical Industry Institute of Toxicology, Research Triangle Park, NC. Submitted to 

the U.S. Environmental Protection Agency under TSCA Section 8D. Document No. 878212041. 

OTS0205947. 

Ho PC. 1986. Photooxidation of 2,4-dinitrotoluene in aqueous solution in the presence of hydrogen 

peroxide. Environ Sci Technol 20:260-267. 

Hodgson JR, Kowalski MA, Glennon JP, et al. 1976. Mutagenicity studies on 2,4-dinitrotoluene 

[Abstract]. Mutat Res 38:387. 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 246 

9. REFERENCES 

Hoel DG, Davis DL, Miller AB, et al. 1992. Trends in cancer mortality in 15 industrialized countries, 

1969-1986. J Natl Cancer Inst 84(5):313-320. 

Hoke RA, Giesy JP, Zabik M, et al. 1993. Toxicity of sediments and sediment pore waters from the 

Grand Calumet River-Indiana Harbor are of concern. Ecotoxicol Environ Saf 26:86-112. 

Holen I, Mikalsen SO, Sanner T. 1990. Effects of dinitrotoluenes on morphological cell transformation 

and intercellular communication in Syrian hamster embryo cells. J Toxicol Environ Health 29:89-98. 

Honeychurch KC, Hart JP, Pritchard PR, et al. 2003. Development of an electrochemical assay for 2,6­

dinitrotoluene, based on a screen-printed carbon electrode, and its potential application in bioanalysis, 

occupational and public health. Biosens Bioelectron 19(4):305-312. 

Hong CB, Ellis JV, Lee CC, et al. 1985. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. 

Part III. CD-l mice. J Am Coll Toxicol 4:257-269. 

HSDB. 2012. Dinitrotoluenes. Hazardous Substances Data Bank. National Library of Medicine. 

http://toxnet.nlm.nih.gov. May 08, 2012. 

Huang J, Ning G, Li F, et al. 2015. Biotransformation of 2,4-dinitrotoluene by obligate marine 

Shewanella marisflavi EP1 under anaerobic conditions. Bioresour Technol 180:200-206. 

10.1016/j.biortech.2014.12.108. 

Huang Q, Wang L, Han S. 1995. The genotoxicity of substituted nitrobenzenes and the quantitative 

structure-activity relationship studies. Chemosphere 30:915-923. 

Huang QG, Kong LR, Liu YB et al. 1996. Relationships between molecular structure and chromosomal 

aberrations in in vitro human lymphocytes induced by substituted nitrobenzenes. Bull Environ Contam 

Toxicol 57:349-353. 

Hughes JB, Wang C, Zhang C. 1999. Anaerobic biotransformation of 2,4-dinitrotoluene and 

2,6-dinitrotoluene by Clostridium acetobutylicum: A pathway through dihydroxylamino intermediates. 

Environ Sci Technol 33(7):1065-1070. 

Hunt RJ, Neubauer NR, Picone RF. 1980. An improved procedure for sampling and analysis of 

dinitrotoluene vapor concentrations in workplace air. Am Ind Hyg Assoc J 41:592-594. 

IARC. 1996. 2,4- Dinitrotoluene and 2,6-dinitrotoluene. IARC monographs on the evaluation of 

carcinogenic risks to humans. Vol. 65: Lyon, France: World Health Organization, International Agency 

for Research on Cancer. 

IARC. 2015. Agents classified by the IARC monographs. Volumes 1–112. Lyon, France: International 

Agency for Research on Cancer. 

http://monographs.iarc.fr/ENG/Classification/ClassificationsCASOrder.pdf. March 31, 2015. 

IRIS. 2002. 2,4-Dinitrotoluene (CASRN 121-14-2). Integrated Risk Information System. Washington, 

DC: U.S. Environmental Protection Agency. http://www.epa.gov/iris/subst/0524.htm. April 9, 2015. 
 

 

 

 

 

 

 

 

DINITROTOLUENES 247 

9. REFERENCES 

IRIS. 2003. 2,4-/2,6-Dinitrotoluene mixture; no CASRN. Integrated Risk Information System. 

Washington, DC: U.S. Environmental Protection Agency. http://www.epa.gov/iris/subst/0397.htm. 

April 9, 2015. 

Jenkins TF, Walsh ME. 1992. Development of field screening methods for TNT, 2,4-DNT and RDX in 

soil. Talanta 39:419-428. 

Jenkins TF, Hewitt AD, Grant CL, et al. 2006. Identity and distribution of residues of energetic 

compounds at army live-fire training ranges. Chemosphere 63:1280-1290. 

Jenkins TF, Leggett DC, Grant CL, et al. 1986. Reversed-phase high-performance liquid 

chromatographic determination of nitroorganics in munitions wastewater. Anal Chem 58:170-175. 

Jokinen MP, Clarkson TB, Prichard RW. 1985. Animal models in atherosclerosis research. Exp Mol 

Pathol 42:1-28. 

Jones CR, Liu YY, Sepai O, et al. 2005a. Hemoglobin adducts in workers exposed to nitrotoluenes. 

Carcinogenesis (Oxford) 26(1):133-143. 

Jones CR, Sepai O, Liu YY, et al. 2005b. Urinary metabolites of workers exposed to nitrotoluenes. 

Biomarkers 10(1):10-28. 

Jones-Price C, Marks TA, Ledoux TA, et al. 1982. Teratological and postnatal evaluation of 

dinitrotoluene in Fischer-344 rats. Final report. E.I. Dupont de Nemours & Co. Submitted to the U.S. 

Environmental Protection Agency under TSCA Section 8D. EPA86940000730S. OTS0557140. 

Kawai A, Goto S, Matsumoto Y, et al. 1987. Mutagenicity of aliphatic and aromatic nitro compounds, 

industrial materials, and related compounds. Jpn J Ind Health 29(1):34-54. 

Kearns GL, Abdel-Rahman SM, Alander SW, et al. 2003. Developmental pharmacology--drug 

disposition, action, and therapy in infants and children. N Engl J Med 349(12):1157-1167. 

Kedderis GL, Dyroff MC, Rickert DE. 1984. Hepatic macromolecular binding of the hepatocarcinogen 

2,6-DNT and its 2,4-isomer in vivo; modulation by the sulfotransferase inhibitors pentachlorophenol and 

2,6-dichloro-4-nitrophenol. Carcinogenesis 5:1199-1204. 

Kessel S, Hauck HE. 1996. Qualitative and quantitative determination of 2,4,6-TNT, hexogen, octogen, 

aminonitro- and nitrocompounds in ammunition wastes with modified TLC and HPTLC precoated layers. 

Chromatographia 43:401-404. 

Kholod YA, Gryn'ova G, Gorb L, et al. 2011. Evaluation of the dependence of aqueous solubility of 

nitro compounds on temperature and salinity: A COSMO-RS simulation. Chemosphere 83:287-294. 

Komori M, Nishio K, Kitada M, et al. 1990. Fetus-specific expression of a form of cytochrome P-450 in 

human livers. Biochemistry 29(18):4430-4433. 

Kozuka H, Mori M, Katayama K, et al. 1978. Studies on the metabolism and toxicity of dinitrotoluenesmetabolism of dinitrotoluenes by Rhodotorula glutinis and rat liver homogenate. Jpn J Toxicol Environ 

Health (Eisei Kagaku) 24(5):252-259. 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 248 

9. REFERENCES 

Kozuka H, Mori M, Nause Y. 1979. Studies on the metabolism and toxicity of dinitrotoluenes. 

Toxicological study of 2,4-dinitrotoluene (2,4-DNT) in rats in long-term feeding. J Toxicol Sci 

4:221-228. 

Krishnan K, Andersen ME. 1994. Physiologically based pharmacokinetic modeling in toxicology. In: 

Hayes AW, ed. Principles and methods of toxicology. 3rd ed. New York, NY: Raven Press, Ltd., 149­

188. 

Krishnan K, Andersen ME, Clewell HJ, et al. 1994. Physiologically based pharmacokinetic modeling of 

chemical mixtures. In: Yang RSH, ed. Toxicology of chemical mixtures: Case studies, mechanisms, 

and novel approaches. San Diego, CA: Academic Press, 399-437. 

Kumar S, Davis AP. 1997. Heterogeneous photocatalytic oxidation of nitrotoluenes. Water Environ Res 

69:1238-1245. 

La DK, Froines JR. 1992. Comparison of DNA adduct formation between 2,4- and 2,6-dinitrotoluene by 

32P-postlabelling analysis. Arch Toxicol 66:633-640. 

La DK, Froines JR. 1993. Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and 

its noncarcinogenic analog 2,6-diaminotoluene. Mutat Res 301:79-85. 

Lane RW, Simon GS, Dougherty RW, et al. 1985. Reproductive toxicity and lack of dominant lethal 

effects of 2,4-dinitrotoluene in the male rat. Drug Chem Toxicol 8:265-280. 

Lang PZ, Wang Y, Chen DB, et al. 1997. Bioconcentration, elimination and metabolism of 

2,4-dinitrotoluene in carps (Cyprinus Carpio L.). Chemosphere 35(8):1799-1815. 

Lee CC, Hong CB, Ellis HV, et al. 1985. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. 

Part II. CD rats. J Am Coll Toxicol 4:243-256. 

Lee YS, Hunter JV. 1985. Effect of ozonation and chlorination of Environmental Protection Agency 

priority pollutant. In: Jolley RL, Bull RJ, Davis WP, et al., eds. Water chlorination: Chemistry, 

environmental impact and health effects. Vol. 5. Chelsea, MI: Lewis Publishers, Inc. 1515-1526. 

Leeder JS, Kearns GL. 1997. Pharmacogenetics in pediatrics: Implications for practice. Pediatr Clin 

North Am 44(1):55-77. 

Leikin JB, Paloucek FP. 2008. Poisoning and toxicology handbook. 4th ed. Boca Raton, FL: CRC 

Press, 830. 

Lenke H, Warrelmann J, Daun G, et al. 1998. Biological treatment of TNT-contaminated soil. 

2. Biologically induced immobilization of the contaminants and full-scale application. Environ Sci 

Technol 32:1964-1971. 

Lent EM, Crouse LC, Quinn MJ, Jr., et al. 2012a. Comparison of the repeated dose toxicity of isomers 

of dinitrotoluene. Int J Toxicol 31(2):143-157. 

Lent EM, Crouse LC, Quinn MJ, Jr., et al. 2012b. Assessment of the in vivo genotoxicity of isomers of 

dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays. Mutat Res 742(1­

2):54-60. 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 249 

9. REFERENCES 

Leonard TB, Adams T, Popp JA. 1986. Dinitrotoluene isomer-specific enhancement of the expression of 

diethylnitrosamine-initiated hepatocyte foci. Carcinogenesis 7:1797-1803. 

Leonard TB, Graichen ME, Popp JA. 1987. Dinitrotoluene isomer-specific hepatocarcinogenesis in 

F344 rats. JNCI 79:1313-1319. 

Leonard TB, Lyght O, Popp JA. 1983. Dinitrotoluene structure-dependent initiation of hepatocytes in 

vivo. Carcinogenesis 4:1059-1061. 

Letzel S, Goen T, Bader M, et al. 2003. Exposure to nitroaromatic explosives and health effects during 

disposal of military waste. Occup Environ Med 60(7):483-488. 

Leung HW. 1993. Physiologically-based pharmacokinetic modelling. In: Ballentyne B, Marrs T, 

Turner P, eds. General and applied toxicology. Vol. 1. New York, NY: Stockton Press, 153-164. 

Levine RJ, Andjelkovich DA, Kersteter SL, et al. 1986a. Heart disease in workers exposed to 

dinitrotoluene. J Occup Med 28:811-816. 

Levine RJ, Andjelkovich DA, Kersteter SL, et al. 1986b. Mortality of munitions workers exposed to 

dinitrotoluene. Final Report. Research Triangle Park, NC: Chemical Industry Institute of Toxicology. 

Government Accession No. ADA 167600. 

Levine RJ, Corso RDD, Blunden PB. 1985a. Fertility of workers exposed to dinitrotoluene and TDA at 

three chemical plants. In: Rickert DE, ed. Toxicity of nitroaromatic compounds. Chemical Industry 

Institute of Toxicology Series. Washington, DC: Hemisphere Publishing Corp., 243-254. 

Levine RJ, Turner MJ, Crume YS, et al. 1985b. Assessing exposure to dinitrotoluene using a biological 

monitor. J Occup Med 279:627-638. 

Liu D, Thomson K, Anderson AC. 1984. Identification of nitroso compounds from biotransformation of 

2,4-dinitrotoluene. Appl Environ Microbiol 47:1295-1298. 

Livingston AL. 1978. Forage plant estrogens. J Toxicol Environ Health 4(2-3):301-324. 

Lloyd JBF. 1983a. Clean-up procedures for the examination of swabs for explosive traces by highperformance liquid chromatography with electrochemical detection at a pendent drop electrode. J 

Chromatogr 261:391-406. 

Lloyd JBF. 1983b. High-performance liquid chromatography of organic explosives components with 

electrochemical detection at a pendant mercury drop electrode. J Chromatogr 257:227-236. 

Long LM, Rickert DE. 1982. Metabolism and excretion of 2,6-dinitro-[14C]toluene in vivo and in 

isolated perfused rat livers. Drug Metab Dispos 10:455-458. 

Lopes TJ, Furlong ET. 2001. Occurrence and potential adverse effects of semivolatile organic 

compounds in streambed sediment, United States, 1992-1995. Environ Toxicol Chem 20(4):727-737. 

Loveday KS, Lugo MH, Resnick MA, et al. 1989. Chromosome aberration and sister chromatid 

exchange tests in Chinese hamster ovary cells in vitro: II. Results with 20 chemicals. Environ Mol 

Mutagen 13:60-94. 
 

 

 

 

 

 

 

DINITROTOLUENES 250 

9. REFERENCES 

Luning Prak DJ, O'Sullivan DW. 2007. Solubility of 4-nitrotoluene, 2,6-dinitrotoluene, 

2,3-dinitrotoluene, and 1,3,5-trinitrobenzene in pure water and seawater. J Chem Eng Data 52:2446­

2450. 

Maeda T, Nakamura R, Kadokami K, et al. 2007. Relationship between mutagenicity and reactivity or 

biodegradability for nitroaromatic compounds. Environ Toxicol Chem 26(2):237-241. 

Maksimov YY. 1968. Vapor pressures of aromatic nitrocompounds at various temperatures. Russian J 

Phys Chem 42:1550-1552. 

Mayr U, Butsch A, Schneider S. 1992. Validation of two in vitro test systems for estrogenic activities 

with zearalenone, phytoestrogens and cereal extracts. Toxicology 74(2-3):135-149. 

McFarlane C, Nolt C, Wickliff C, et al. 1987. The uptake, distribution and metabolism of four organic 

chemicals by soybean plants and barley roots. Environ Toxicol Chem 6:847-856. 

McGee LC, McCausland A, Plume CA, et al. 1942. Metabolic disturbances in workers exposed to 

dinitrotoluene. Am J Digest Dis 9:329-331. 

McGee LC, Reed HL, Nereim TJ, et al. 1947. Metabolic disturbances in workers exposed to 

dinitrotoluene during World War II. Gastroenterology 8:293-295. 

McGown EL, Knudsen JJ, Makovec GT, et al. 1983. Fourteen-day feeding study of 2,4-dinitrotoluene in 

male and female rats. U.S. Army Medical Research and Development Command, Division of Research 

Support, Letterman Army Institute of Research. ADA126069. 

Medinsky MA, Dent JG. 1983. Biliary excretion and enterohepatic circulation of 2,4-dinitrotoluene 

metabolites in Fischer-344 Rats. Toxicol Appl Pharmacol 68:359-366. 

Michael LC, Pellizari ED, Wiseman RW. 1988. Development and evaluation of a procedure for 

determining volatile organics in water. Environ Sci Technol 22:565-570. 

Mirsalis JC, Butterworth BE. 1982. Induction of unscheduled DNA synthesis in rat hepatocytes 

following in vivo treatment with dinitrotoluene. Carcinogenesis 3:241-245. 

Mirsalis JC, Tyson CK, Steinmetz KL, et al. 1989. Measurement of unscheduled DNA synthesis and 

S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds. Environ 

Mol Mutagen 14:155-164. 

Monti MR, Smania AM, Fabro G, et al. 2005. Engineering pseudomonas fluorescens for biodegradation 

of 2,4-dinitrotoluene. Appl Environ Microbiol 71(12):8864-8872. 

Mori M, Kudo Y, Nunozawa T, et al. 1985. Intestinal metabolism of 2,4-dinitrotoluene in rats. Chem 

Pharm Bull 33:327-332. 

Mori MA, Miyahara T, Hasegawa Y, et al. 1984. Metabolism of dinitrotoluene isomers by Escherichia 

coli isolated from human intestine. Chem Pharm Bull (Tokyo) 32(10):4070-4075. 

Mori M, Miyahara T, Taniguchi K, et al. 1982. Mutagenicity of 2,4-dinitrotoluene and its metabolites in 

Salmonella typhimurium. Toxicol Lett 13:1-5. 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 251 

9. REFERENCES 

Mori MA, Sayama M, Shoji M et al. 1997. Biliary excretion and microfloral transformation of major 

conjugated metabolites of 2,4-dinitrotoluene and 2,6-dinitrotoluene in the male Wistar rat. Xenobiotica 

27:1225-1236. 

Mori MA, Shoji M, Dohrin M, et al. 1996. Further studies on the urinary metabolites of 

2,4-dinitrotoluene and 2,6-dinitrotoluene in the male Wistar rat. Xenobiotica 26:79-88. 

Mori M, Shoji M, Sayama M, et al. 2000. Secondary metabolism of dinitrobenzyl glucuronide related to 

production of genotoxic compounds of dinitrotoluene in male Wistar rat. J Health Sci 46(5):329-335. 

Morselli PL, Franco-Morselli R, Bossi L. 1980. Clinical pharmacokinetics in newborns and infants: 

Age-related differences and therapeutic implications. Clin Pharmacokin 5(6):485-527. 

Nacson S, Legrady O, Siu T, et al. 1994. Improved and novel approaches for the detection of explosives. 

Proc SPIE Int Soc Opt Eng 2276:69-78. 

NAS/NRC. 1989. Report of the oversight committee. In: Biologic markers in reproductive toxicology. 

Washington, DC: National Academy of Sciences, National Research Council, National Academy Press, 

15-35. 

NCI. 1978. Bioassay of 2,4-dinitrotoluene for possible carcinogenicity. CAS No. 121-14-2. 

Washington, DC: National Cancer Institute, U.S. Department of Health, Education, and Welfare, Public 

Health Service, National Institutes of Health. NCI-CG-TR-54. 

Neuwoehner J, Schofer A, Erlenkaemper B, et al. 2007. Toxicological characterization of 

2,4,6-trinitrotoluene, its transformation products, and two nitramine explosives. Environ Toxicol Chem 

26(6):1090-1099. 

NIOSH. 1980. Health hazard evaluation determination. Report no. HE 79-113-728. Olin Chemical Co., 

Brandenberg, KY. Hazard Evaluations and Technical Assistance Branch. National Institute of 

Occupational Safety and Health. OTS0204878, Section 8E. 

NIOSH. 1982. Health Hazard Evaluation Report, No. HETA-81-295-1155, Olin (formerly Allied) 

Chemical Co., Moundsville, WV. Cincinnati, OH: National Institute of Occupational Safety and Health, 

Hazard Evaluations and Technical Assistance Branch. OTS0528962, Section 4. PB84150465. 

NIOSH. 1997. NIOSH pocket guide to chemical hazards. Cincinnati, OH: U.S. Department of Health 

and Human Services, Public Health Service, Centers for Disease Control, National Institute for 

Occupational Safety and Health. NIOSH publication No. 94-116. 

NIOSH. 2015. Dinitrotoluene. NIOSH pocket guide to chemical hazards. Atlanta, GA: National 

Institute for Occupational Safety and Health, Centers for Disease Control and Prevention. 

http://www.cdc.gov/niosh/npg/npgd0235.html. April 1, 2015. 

Nipper M, Carr RS, Biedenbach JM, et al. 2005. Fate and effects of picric acid and 2,6-DNT in marine 

environments: Toxicity of degradation products. Mar Pollut Bull 50(11):1205-1217. 

Nipper M, Qian Y, Carr RS, et al. 2004. Degradation of picric acid and 2,6-DNT in marine sediments 

and waters: The role of microbial activity and ultra-violet exposure. Chemosphere 56(6):519-530. 
 

 

 

 

 

 

 

 

DINITROTOLUENES 252 

9. REFERENCES 

Nishino SF, Spain JC, Lenke H, et al. 1999. Mineralization of 2,4- and 2,6-dinitrotoluene in soil slurries. 

Environ Sci Technol 33(7):1060-1064. 

NITE. 2002. Dinitrotoluene. National Institute of Technology and Evaluation. 

http://www.safe.nite.go.jp/data/hazkizon/pk_e_kizon_data_result.home_data. May 02, 2012. 

Noguera DR, Freedman DL. 1996. Reduction and acetylation of 2,4-dinitrotoluene by a Pseudomonas 

aeruginosa strain. Applied Environ Microbiol 62:2257-2263. 

Noguera DR, Freedman DL. 1997. Characterization of products from the biotransformation of 2,4­

dinitrotoluene by denitrifying enrichment cultures. Water Environ Res 69:260-268. 

NRC. 1993. Pesticides in the diets of infants and children. National Research Council, Washington DC: 

National Academy Press. 

NTP. 2014. Report on Carcinogens. Thirteenth edition. Research Triangle Park, NC: U.S. Department 

of Health and Human Services, Public Health Service, National Toxicology Program. 

http://ntp.niehs.nih.gov/pubhealth/roc/roc13/. April 9, 2015. 

Oehrle SA. 1996. Analysis of nitramine and nitroaromatic explosives by capillary electrophoresis. J 

Chromatogr A 745:233-237. 

Ortega-Calvo JJ, Fesch C, Harms H. 1999. Biodegradation of sorbed 2,4-dinitrotoluene in a clay-rich, 

aggregated porous medium. Environ Sci Technol 33(21):3737-3742. 

OSHA. 2010. 2,4-Dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT). Method No. 44. Sampling and 

analytical methods. Occupational Safety and Health Administration. 

http://www.osha.gov/dts/sltc/methods/organic/org044/org044.html. May 03, 3012. 

OSHA. 2013. Subpart Z - Toxic and hazardous substances. Air contaminants. Occupational Safety and 

Health Standards. Code of Federal Regulations 29 CFR 1910.1000. http://www.gpo.gov/fdsys/pkg/CFR2014-title29-vol6/pdf/CFR-2014-title29-vol6-sec1910-1000.pdf. March 4, 2015. 

OSHA. 2014a. Gases, vapors, fumes, dusts, and mists. Appendix A to 1926.55-1970. American 

Conference Of Governmental Industrial Hygienists’ threshold limit values of airborne contaminants for 

construction. Subpart D-Occupational health and environmental controls. Occupational Safety and 

Health Standards. Code of Federal Regulations 29 CFR 1926.55. http://www.gpo.gov/fdsys/pkg/CFR2014-title29-vol8/pdf/CFR-2014-title29-vol8-sec1926-55.pdf. March 4, 2015. 

OSHA. 2014b. Subpart Z - Toxic and hazardous substances. Air contaminants. Table Z - Shipyards. 

Occupational Safety and Health Standards. Code of Federal Regulations 29 CFR 1915.1000. 

http://www.gpo.gov/fdsys/pkg/CFR-2013-title29-vol7/pdf/CFR-2013-title29-vol7-sec1915-1000.pdf. 

March 4, 2015. 

Owen GM, Brozek J. 1966. Influence of age, sex and nutrition on body composition during childhood 

and adolescence. In: Falkner F, ed. Human development. Philadelphia, PA: WB Saunders, 222-238. 

Ozturk K, Durusoy M. 1999. The detection and comparison of the genotoxic effects of some nitro 

aromatic compounds by the umu and SOS chromotest systems. Toxicol Lett 108(1):63-68. 
 

 

 

 

 

DINITROTOLUENES 253 

9. REFERENCES 

Padda RS, Wang C, Hughes JB, et al. 2003. Mutagenicity of nitroaromatic degradation compounds. 

Environ Toxicol Chem 22(10):2293-2297. 

Parrish FW. 1977. Fungal transformation of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene. Appl Environ 

Microbiol 34:232-233. 

Pearson JG, Glennon JP, Barkley JJ, et al. 1979. An approach to the toxicological evaluation of a 

complex industrial wastewater. Annual Symposium on Aquatic Toxicology 2:284-301. 

Pennington JC. 1988. Plant uptake of 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrotoluene, and 

2-amino-4,6-dinitrotoluene using 14C-labeled and unlabeled compounds. Army Engineer Waterways 

Experiment Station, Vicksburg, MS. Report No. WES/TR/EL-88-20. ADA203690. 

Perkins RG. 1919. A study of the munitions intoxications in France. US Pub Health Rep 34:2335-2374. 

Phillips JH, Coraor RJ, Prescott SR. 1983. Determination of nitroaromatics in biosludges with a gas 

chromatograph/thermal energy analyzer. Anal Chem 55:889-892. 

Podlipna R, Pospisilova B, Vanek T. 2015. Biodegradation of 2,4-dinitrotoluene by different plant 

species. Ecotoxicol Environ Saf 112:54-59. 

Popp JA, Leonard TB. 1982. The use of in vivo hepatic initiation-promotion systems in understanding 

the hepatocarcinogenesis of technical grade dinitrotoluene. Toxicol Pathol 10(2):190-196. 

Preslan JE, Hatrel BB, Emerson M, et al. 1993. An improved method for analysis of 2,4,6-trinitrotoluene 

and its metabolites from compost and contaminated soils. J Hazard Mater 33:329-337. 

*Ramos K, McMahon K, Alipui C, et al. 1991a. Modulation of aortic smooth muscle cell proliferation 

by dinitrotoluene. Adv Exp Med Biol 283(Biological Reactive Intermediates 4):805-808. 

*Ramos KS, McMahon KK, Alipui C, et al. 1991b. Modulation of DNA synthesis in aortic smooth 

muscle cells by dinitrotoluenes. Cell Biol Toxicol 7:111-128. 

Reader SC, Foster P. 1990. The in vitro effects of four isomers of dinitrotoluene on rat Sertoli and 

Sertoli-germ cell cocultures: Germ cell detachment and lactate and pyruvate production. Toxicol Appl 

Pharmacol 106:287-294. 

Reifenrath WG, Kammen HO, Palmer WG, et al. 2002. Percutaneous absorption of explosives and 

related compounds: An empirical model of bioavailability of organic nitro compounds from soil. 

Toxicol Appl Pharmacol 182(2):160-168. 

Rickert DE, Long RM. 1980. Tissue distribution of 2,4-dinitrotoluene and its metabolites in male and 

female Fischer-344 rats. Toxicol Appl Pharmacol 56:286-293. 

Rickert DE, Long RM. 1981. Metabolism and excretion of 2,4-dinitrotoluene in male and female 

Fischer-344 rats after different doses. Drug Metab Dispos 9:226-232. 

Rickert DE, Butterworth BE, Popp JA. 1984. Dinitrotoluene: Acute toxicity, oncogenicity, genotoxicity, 

and metabolism. CRC Crit Rev Toxicol 13:217-234. 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES 254 

9. REFERENCES 

Rickert DE, Long RM, Krakowka S, et al. 1981. Metabolism and excretion of 2,4-(14C)dinitrotoluene in 

conventional and axenic Fischer-344 rats. Toxicol Appl Pharmacol 59:574-579. 

Rickert DE, Schnell SR, Long RM. 1983. Hepatic macromolecular covalent binding and intestinal 

disposition of 2,4-(14C)dinitrotoluene. J Toxicol Environ Health 11: 555-568. 

Roberts MG, Rugh CL, Li H, et al. 2007. Reducing bioavailability and phytotoxicity of 

2,4-dinitrotoluene by sorption on K-smectite clay. Environ Toxicol Chem 26(2):358-360. 

Rothfuss A, O'Donovan M, De Boeck M, et al. 2010. Collaborative study on fifteen compounds in the 

rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies. Mutat Res 702(1):40-69. 

Sabbioni G, Jones CR, Sepai O, et al. 2006. Biomarkers of exposure, effect, and susceptibility in 

workers exposed to nitrotoluenes. Cancer Epidemiol Biomarkers Prev 15(3):559-566. 

Saunders NR, Ek CJ, Habgood M, et al. 2008. Barriers in the brain: A renaissance? Trends Neurosci 

31(6):279-286. 

Saunders NR, Liddelow SA, Dziegielewska KM. 2012. Barrier mechanisms in the developing brain. 

Front Pharmacol 3(46):1-18. 

Sayama M, Mori MA, Maruyama Y, et al. 1993. Intestinal transformation of 2,6-dinitrotoluene in male 

Wistar rats. Xenobiotica 23:123-131. 

Sayama M, Mori M, Shirokawa T, et al. 1989. Mutagenicity of 2,6-dinitrotoluene and its metabolites, 

and their related compounds in Salmonella typhimurium. Mutat Res 226:181-184. 

Sayama M, Mori M, Shoji M, et al. 1998. Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their 

reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing 

Ames test strains. Mutat Res 420(1-3):27-32. 

Scheuplein R, Charnley G, Dourson M. 2002. Differential sensitivity of children and adults to chemical 

toxicity. I. Biological basis. Regul Toxicol Pharmacol 35(3):429-447. 

Schut HAJ, Loeb TR, Grimes LA, et al. 1983. Distribution, elimination, and test for carcinogenicity of 

2,6-dinitrotoluene after intraperitoneal and oral administration to strain A mice. J Toxicol Environ Health 

12:659-670. 

Seidler A, Bruning T, Taeger D, et al. 2014a. Cancer incidence among workers occupationally exposed 

to dinitrotoluene in the copper mining industry. Int Arch Occup Environ Health 87(2):117-124. 

Seidler A, Harth V, Taeger D, et al. 2014b. Dinitrotoluene exposure in the copper mining industry and 

renal cancer: A case-cohort study. Occup Environ Med 71(4):259-265. 

Seifert SA. 2004. Nitrates and nitrites. In: Dart RC, ed. Medical toxicology. 3rd ed. Philadelphia, PA: 

Lippincott Williams & Williams, 1174-1180. 

Shackelford WM, Keith LH. 1976. Frequency of organic compounds identified in water. Athens, GA: 

U.S. Environmental Protection Agency, Environmental Research Laboratory. EPA600476062. 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 255 

9. REFERENCES 

Short RD, Lee CC. 1980. Effect of some nitrotoluenes on the biotransformation of xenobiotics in rats. 

Experimentia (Basel) 36:100-101. 

Simini M, Wentsel RS, Checkai RT, et al. 1995. Evaluation of soil toxicity at Joliet Army Munition 

Plant. Environ Toxicol Chem 14:623-630. 

Simmon VF, Kauhanen K, Tardiff RG. 1977. Mutagenic activity of chemicals identified in drinking 

water. In: Scott S, Bridges BA, Sohels FH, eds. Progress in genetic toxicology, 249-258. 

Simmons MS, Zepp RG. 1986. Influence of humic substances on photolysis of nitroaromatic compounds 

in aqueous systems. Water Res 20:899-904. 

Smirnova IA, Dian C, Leonard GA, et al. 2004. Development of a bacterial biosensor for nitrotoluenes: 

The crystal structure of the transcriptional regulator DntR. J Mol Biol 340(3):405-418. 

Smith EF II, Smith HJ, Kuchar EJ. 1995. Monitoring of dinitrotoluene and its metabolites in urine by 

spectrophotometry of their coupled aryldiazonium salts. Am Ind Hyg Assoc J 56:1175-1179. 

Smith RP. 1996. Toxic responses of the blood. In: CD Klaassen, MJ Wonsiewicz, LA Sheinis, eds. 

Casarett and Doull's toxicology, the basic science of poisons, 5th ed. New York, NY: Macmillan 

Publishing Company, 335-354. 

Soares ER, Lock LF. 1980. Lack of indication of mutagenic effects of dinitrotoluenes and 

diaminotoluenes in mice. Environ Mutagen 2:111-124. 

Sohr J, Janes W, Bongartz A. 1995. TLC analysis of nitro compounds in residual warfare site 

contamination. Analysis Magazine 23:M25-M26. 

Sorenson DR, Brabec M. 2003. The response of adult rat Sertoli cells, immortalized by a temperaturesensitive mutant of SV40, to 1,2-dinitrobenzene, 1,3-dinitrobenzene, 2,4-dinitrotoluene, 3,4­

dinitrotoluene, and cadmium. Cell Biol Toxicol 19(2):107-119. 

Spanggord RJ, Suta BE. 1982. Effluent analysis of wastewater generated in the manufacture of 

2,4,6-trinitrotoluene 2. Determination of a representative discharge of ether extractable components. 

Environ Sci Technol 16:233-236. 

Spanggord RJ, Gibson BV, Keck RG, et al. 1982a. Effluent analysis of wastewater generated in the 

manufacture of 2,4,6-trinitrotoluene 1. Characterization study. Environ Sci Technol 16:229-232. 

Spanggord RJ, Mortelmans KE, Griffin AF, et al. 1982b. Mutagenicity in Salmonella typhimurium and 

structure-activity relationships of wastewater components emanating from the manufacture of 

trinitrotoluene. Environ Mutagen 4:163-179. 

Spanggord RJ, Myers CH, LeValley SE, et al. 1990. Structure-activity relationship for the intrinsic 

hepatotoxicity of dinitrotoluenes. Chem Res Toxicol 3:551-558. 

Spanggord RJ, Spain JC, Nishino SF, et al. 1991. Biodegradation of 2,4-dinitrotoluene by a 

Pseudomonas sp. Appl Environ Microbiol 57(11):3200-3205. 

Spiegel K, Welsch T. 1997. Monitoring degradation processes of explosives by HPLC analysis with 

UV- and amperometric detection. Fresenius J Anal Chem 357:333-337. 
 

 

 

 

 

 

DINITROTOLUENES 256 

9. REFERENCES 

Spiegel K, Headley JV, Peru KM, et al. 2005. Residues of explosives in groundwater leached from soils 

at a military site in eastern Germany. Commun Soil Sci Plant Anal 36:133-153. 

SRI. 2011. Dinitrotoluenes. 2011 Directory of chemical producers United States. SRI Consulting, 532­

533. 

Staples CA, Werner AF, Hoogheem TJ. 1985. Assessment of priority pollutant concentrations in the 

United States using STORET database. Environ Toxicol Chem 4:131-142. 

Stayner LT, Dannenberg AL, Bloom T, et al. 1993. Excess hepatobiliary cancer mortality among 

munitions workers exposed to dinitrotoluene. J Occup Med 35:291-296. 

Steuckart C, Berger-Preiss E, Levsen K. 1994. Determination of explosives and their biodegradation 

products in contaminated soil and water from former ammunition plants by automated multiple 

development high-performance thin-layer chromatography. Anal Chem 66:2570-2577. 

Stoner GD, Greisiger EA, Schut AJ, et al. 1984. A comparison of the lung adenoma response in strain 

A/J mice after intraperitoneal and oral administration of carcinogens. Toxicol Appl Pharmacol 

72:313-323. 

Styles JA, Cross MF. 1983. Activity of 2,4,6-trinitrotoluene in an in vitro mammalian gene mutation 

assay. Cancer Lett 20:103-108. 

Suzuki H, Imamura T, Koeda A, et al. 2011. Genotoxicity studies of 2,6-dinitrotoluene (2,6-DNT). J 

Toxicol Sci 36(4):499-505. 

Suzuki H, Takasawa H, Kobayashi K, et al. 2009. Evaluation of a liver micronucleus assay with 12 

chemicals using young rats (II): A study by the Collaborative Study Group for the Micronucleus 

Test/Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. Mutagenesis 

24(1):9-16. 

Sylvia JM, Janni JA, Klein JD, et al. 2000. Surface-enhanced Raman detection of 2,4-dinitrotoluene 

impurity vapor as a marker to locate landmines. Anal Chem 72(23):5834-5840. 

Takasawa H, Suzuki H, Ogawa I, et al. 2010. Evaluation of a liver micronucleus assay in young rats 

(IV): A study using a double-dosing/single-sampling method by the Collaborative Study Group for the 

Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian 

Mutagenicity Study Group (MMS). Mutat Res 698(1-2):24-29. 

Tas S, Lauwerys R, Lison D. 1996. Occupational hazards for the male reproductive system. Crit Rev 

Toxicol 26:261-307. 

Thomas K, Colborn T. 1992. Organochlorine endocrine disruptors in human tissue. In: Colborn T, 

Clement C, eds. Chemically induced alterations in sexual and functional development: The 

wildlife/human connection. Princeton, NJ: Princeton Scientific Publishing, 365-394. 

Tokiwa H, Nakagawa R, Ohnishi Y. 1981. Mutagenic assay of aromatic nitro compounds with 

Salmonella typhimurium. Mutat Res 91:321-325. 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 257 

9. REFERENCES 

TRI13. 2015. TRI explorer: Providing access to EPA’s toxics release inventory data. Washington, DC: 

U.S. Environmental Protection Agency, Office of Information Analysis and Access, Office of 

Environmental Information. Toxics Release Inventory. http://www.epa.gov/triexplorer/. April 3, 2015. 

Turner MJ. 1986. Identification and quantification of urinary metabolites of dinitrotoluenes in 

occupationally exposed humans. Chemical Industry Institute of Toxicology Activities 6:1-5. 

Turner MJ, Levine RJ, Nystrom DD, et al. 1985. Identification and quantification of urinary metabolites 

of dinitrotoluenes in occupationally exposed humans. Toxicol Appl Pharmacol 80:166-174. 

U.S. Army. 1975. Mammalian toxicity of munition compounds: Phase I. Acute oral toxicity, primary 

skin and eye irritation, dermal sensitization, and disposition and metabolism. Report No. 1. Washington, 

DC: U.S. Army, Medical Research and Development Command. ADB011150. http://www.dtic.mil/cgibin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADB011150. June 22, 2012. 

U.S. Army. 1976. Mammalian toxicity of munitions compounds. Phase II: Effects of multiple doses. 

Part III: 2,6-Dinitrotoluene. Progress report no. 4. Fort Detrick, MD: U.S. Army, Medical 

Bioengineering Research and Development Laboratory. ADA062015. http://www.dtic.mil/cgibin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADA062015. June 22, 2012. 

U.S. Army. 1978a. Mammalian toxicity of munitions compounds. Phase I. Acute oral toxicity, primary 

skin and eye irritation, dermal sensitization, disposition and metabolism and Ames tests of additional 

compounds. Progress report no. 6. Fort Detrick, MD: U.S. Army, Medical Bioengineering Research and 

Development Laboratory. ADA069333. http://www.dtic.mil/cgibin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADA069333. June 22, 2012. 

U.S. Army. 1978b. Mammalian toxicity of munitions compounds. Phase II: Effects of multiple doses. 

Part II: 2,4-Dinitrotoluene. Progress report no. 3. Fort Detrick, MD: U.S. Army, Medical 

Bioengineering Research and Development Laboratory. ADA061715. http://www.dtic.mil/cgibin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADA061715. June 22, 2012. 

U.S. Army. 1979. Mammalian toxicity of munitions compounds. Phase III: Effects of lifetime 

exposure. Part I. 2,4-Dinitrotoluene. Final report no. 7. Fort Detrick, MD: U.S. Army and Medical 

Bioengineering Research Development Laboratory. ADA077692. http://www.dtic.mil/cgibin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADA077692. June 22, 2012. 

U.S. Army. 1980. Environmental fate studies on certain munition wastewater constituents - literature 

review. U.S. Army Medical Bioengineering R&D Laboratory, Environmental Protection Research 

Division. 

U.S. Army. 1987. Water quality criteria for 2,4-dinitrotoluene and 2,6-dinitrotoluene. Fort Detrick, MD: 

U.S. Army Medical Research and Development Command. ADA187745. 

U.S. Army. 1990. An evaluation of the environmental fate and behavior munitions material (TNT, RDX) 

in soil and plant systems; environmental fate and behavior of TNT. Fort Detrick, MD: U.S. Army 

Medical Research and Development Command. ADA223546. 

U.S. Army. 1998. Analysis of explosives in plant tissues: Modifications to method 8330 for soil. 

Washington, DC: U.S. Army Corps of Engineers. IRRP-98-5. 

http://www.dtic.mil/dtic/tr/fulltext/u2/a348746.pdf. June 19, 2012. 
 

 

 

 

 

DINITROTOLUENES 258 

9. REFERENCES 

USAPHC. 2011a. Toxicology Study No. 87-XE-08G0A-08, October 2011, Toxicology Portfolio. 

Fourteen-day oral toxicity and in vivo genotoxicity of 2,3-dinitrotoluene in rats, June-July 2008. 

Aberdeen Proving Ground, Maryland: U.S. Army Public Health Command. 

USAPHC. 2011b. Toxicology Study No. 87-XE-08G0D-10, October 2011, Toxicology Portfolio. 

Fourteen-day oral toxicity and in vivo genotoxicity of 2,4-dinitrotoluene in rats, January 2009-March 

2010. Aberdeen Proving Ground, Maryland: U.S. Army Public Health Command. 

USAPHC. 2011c. Toxicology Study No. 87-XE-08G0E-10, October 2011, Toxicology Portfolio. 

Fourteen-day oral toxicity and in vivo genotoxicity of 2,5-dinitrotoluene in rats, January 2009-March 

2010. Aberdeen Proving Ground, Maryland: U.S. Army Public Health Command. 

USAPHC. 2011d. Toxicology Study No. 87-XE-08G0C-08, October 2011, Toxicology Portfolio. 

Fourteen-day oral toxicity and in vivo genotoxicity of 2,6-dinitrotoluene in rats, June-July 2008. 

Aberdeen Proving Ground, Maryland: U.S. Army Public Health Command. 

USAPHC. 2011e. Toxicology Study No. 87-XE-08G0B-08, October 2011, Toxicology Portfolio. 

Fourteen-day oral toxicity and in vivo genotoxicity of 3,4-dinitrotoluene in rats, June-July 2008. 

Aberdeen Proving Ground, Maryland: U.S. Army Public Health Command. 

USAPHC. 2011f. Toxicology Study No. 87-XE-08G0F-10, October 2011, Toxicology Portfolio. 

Fourteen-day oral toxicity and in vivo genotoxicity of 3,5-dinitrotoluene in rats, January 2009-March 

2010. Aberdeen Proving Ground, Maryland: U.S. Army Public Health Command. 

USITC. 1987. Synthetic organic chemicals: United States production and sales, 1986. Washington, DC: 

U.S. International Trade Commission, 24-42. 

U.S. Navy. 1972. Analysis of explosives in sea water and in ocean floor sediment and fauna. Naval 

Ordinance Laboratory. Ad757778. 

U.S. Navy. 1977. The effects of light on TNT and other explosives in aqueous solutions. WQEC/C 77 

32. ADA036132. 

Valli K, Brock BJ, Joshi DK et al. 1992. Degradation of 2,4-dinitrotoluene by the lignin-degrading 

fungus Phanerochaete chrysosporium. Appl Environ Microbiol 58(1):221-228. 

Vernot EH, MacEwen JD, Haun CC, et al. 1977. Acute toxicity and skin corrosion data for some organic 

and inorganic compounds and aqueous solutions. Toxicol Appl Pharmacol 42:417-423. 

Vieira I, Sonnier M, Cresteil T. 1996. Developmental expression of CYP2E1 in the human liver: 

Hypermethylation control of gene expression during the neonatal period. Eur J Biochem 238(2):476-483. 

Wallenborg SR, Marikides KE, Nyholm L. 1997. Oxidative and reductive amperometric detection of 

phenolic and nitroaromatic compounds in packed capillary column supercritical fluid chromatography. J 

Chromatogr A 785:121-128. 

West JR, Smith HW, Chasis H. 1948. Glomerular filtration rate, effective renal blood flow, and maximal 

tubular excretory capacity in infancy. J Pediatr 32:10-18. 

WHO. 2008. Guidelines for drinking-water quality. 3rd ed. Geneva, Switzerland: World Health 

Organization. http://www.who.int/entity/water_sanitation_health/dwq/fulltext.pdf. April 25, 2012. 
 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 259 

9. REFERENCES 

WHO. 2010. Guidelines for indoor air quality: Selected pollutants. Geneva, Switzerland: World Health 

Organization. http://www.euro.who.int/__data/assets/pdf_file/0009/128169/e94535.pdf. September 9, 

2014. 

WHO. 2011. Guidelines for drinking-water quality. Geneva, Switzerland: World Health Organization. 

http://whqlibdoc.who.int/publications/2011/9789241548151_eng.pdf?ua=1. September 9, 2014. 

Widdowson EM, Dickerson JWT. 1964. Chemical composition of the body. In: Comar CL, Bronner F, 

eds. Mineral metabolism: An advanced treatise. Volume II: The elements Part A. New York, NY: 

Academic Press, 1-247. 

Wilbanks MS, Gust KA, Atwa S, et al. 2014. Validation of a genomics-based hypothetical adverse 

outcome pathway: 2,4-Dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and 

exercise endurance. Toxicol Sci 141(1):44-58. 

Woodruff RC, Mason JM, Valencia R, et al. 1985. Chemical mutagenesis testing in Drosophila. V. 

Results of 53 coded compounds tested for the National Toxicology Program. Environ Mutagen 

7:677-702. 

Woollen BH, Hall MG, Craig R, et al. 1985. Dinitrotoluene: An assessment of occupational absorption 

during the manufacture of blasting explosives. Int Arch Occup Environ Health 55:319-330. 

Working PK, Butterworth BE. 1984. An assay to detect chemically induced DNA repair in rat 

spermatocytes. Environ Mutagen 6:273-286. 

Xin Y, He G, Wang Q, et al. 2011. A portable fluorescence detector for fast ultra trace detection of 

explosive vapors. Rev Sci Instrum 82(10):103102. 10.1063/1.3642661. 

Yang H, Halasz A, Zhao JS, et al. 2008. Experimental evidence for in situ natural attenuation of 2,4- and 

2,6-dinitrotoluene in marine sediment. Chemosphere 70(5):791-799. 

Yang L, Xu JB, Zhen L, et al. 2005. DNA damage of germ cell of rat induced by nitrotoluene chemicals. 

J Environ Sci (China) 17(1):84-90. 

Yinon J. 1996. Trace analysis of explosives in water by gas chromatography-mass spectrometry with a 

temperature-programmed injector. J Chromatogr A 742:205-209. 

Yook KS, Hong SM, Kim JH. 1994. Comparison of liquid-liquid extraction and solid-phase extraction 

coupled with GC/MS for determination of priority pollutants in water. Anal Sci Technol 7:441-453. 

Young WS III, Lietman PS. 1978. Chloramphenicol glucoronyl transferase: Assay, ontogeny and 

inducibility. J Pharmacol Exp Ther 204:203-211. 

Ziegler EE, Edwards BB, Jensen RL, et al. 1978. Absorption and retention of lead by infants. Pediatr 

Res 12(1):29-34. 
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10. GLOSSARY 

Absorption—The taking up of liquids by solids, or of gases by solids or liquids. 

Acute Exposure—Exposure to a chemical for a duration of 14 days or less, as specified in the 

Toxicological Profiles. 

Adsorption—The adhesion in an extremely thin layer of molecules (as of gases, solutes, or liquids) to the 

surfaces of solid bodies or liquids with which they are in contact. 

Adsorption Coefficient (Koc)—The ratio of the amount of a chemical adsorbed per unit weight of 

organic carbon in the soil or sediment to the concentration of the chemical in solution at equilibrium. 

Adsorption Ratio (Kd)—The amount of a chemical adsorbed by sediment or soil (i.e., the solid phase) 

divided by the amount of chemical in the solution phase, which is in equilibrium with the solid phase, at a 

fixed solid/solution ratio. It is generally expressed in micrograms of chemical sorbed per gram of soil or 

sediment. 

Benchmark Dose (BMD)—Usually defined as the lower confidence limit on the dose that produces a 

specified magnitude of changes in a specified adverse response. For example, a BMD10 would be the 

dose at the 95% lower confidence limit on a 10% response, and the benchmark response (BMR) would be 

10%. The BMD is determined by modeling the dose response curve in the region of the dose response 

relationship where biologically observable data are feasible. 

Benchmark Dose Model—A statistical dose-response model applied to either experimental toxicological 

or epidemiological data to calculate a BMD. 

Bioconcentration Factor (BCF)—The quotient of the concentration of a chemical in aquatic organisms 

at a specific time or during a discrete time period of exposure divided by the concentration in the 

surrounding water at the same time or during the same period. 

Biomarkers—Broadly defined as indicators signaling events in biologic systems or samples. They have 

been classified as markers of exposure, markers of effect, and markers of susceptibility. 

Cancer Effect Level (CEL)—The lowest dose of chemical in a study, or group of studies, that produces 

significant increases in the incidence of cancer (or tumors) between the exposed population and its 

appropriate control. 

Carcinogen—A chemical capable of inducing cancer. 

Case-Control Study—A type of epidemiological study that examines the relationship between a 

particular outcome (disease or condition) and a variety of potential causative agents (such as toxic 

chemicals). In a case-controlled study, a group of people with a specified and well-defined outcome is 

identified and compared to a similar group of people without outcome. 

Case Report—Describes a single individual with a particular disease or exposure. These may suggest 

some potential topics for scientific research, but are not actual research studies. 

Case Series—Describes the experience of a small number of individuals with the same disease or 

exposure. These may suggest potential topics for scientific research, but are not actual research studies. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Ceiling Value—A concentration of a substance that should not be exceeded, even instantaneously. 

Chronic Exposure—Exposure to a chemical for 365 days or more, as specified in the Toxicological 

Profiles. 

Cohort Study—A type of epidemiological study of a specific group or groups of people who have had a 

common insult (e.g., exposure to an agent suspected of causing disease or a common disease) and are 

followed forward from exposure to outcome. At least one exposed group is compared to one unexposed 

group. 

Cross-sectional Study—A type of epidemiological study of a group or groups of people that examines 

the relationship between exposure and outcome to a chemical or to chemicals at one point in time. 

Data Needs—Substance-specific informational needs that if met would reduce the uncertainties of human 

health assessment. 

Developmental Toxicity—The occurrence of adverse effects on the developing organism that may result 

from exposure to a chemical prior to conception (either parent), during prenatal development, or 

postnatally to the time of sexual maturation. Adverse developmental effects may be detected at any point 

in the life span of the organism. 

Dose-Response Relationship—The quantitative relationship between the amount of exposure to a 

toxicant and the incidence of the adverse effects. 

Embryotoxicity and Fetotoxicity—Any toxic effect on the conceptus as a result of prenatal exposure to 

a chemical; the distinguishing feature between the two terms is the stage of development during which the 

insult occurs. The terms, as used here, include malformations and variations, altered growth, and in utero 

death. 

Environmental Protection Agency (EPA) Health Advisory—An estimate of acceptable drinking water 

levels for a chemical substance based on health effects information. A health advisory is not a legally 

enforceable federal standard, but serves as technical guidance to assist federal, state, and local officials. 

Epidemiology—Refers to the investigation of factors that determine the frequency and distribution of 

disease or other health-related conditions within a defined human population during a specified period. 

Genotoxicity—A specific adverse effect on the genome of living cells that, upon the duplication of 

affected cells, can be expressed as a mutagenic, clastogenic, or carcinogenic event because of specific 

alteration of the molecular structure of the genome. 

Half-life—A measure of rate for the time required to eliminate one half of a quantity of a chemical from 

the body or environmental media. 

Immediately Dangerous to Life or Health (IDLH)—The maximum environmental concentration of a 

contaminant from which one could escape within 30 minutes without any escape-impairing symptoms or 

irreversible health effects. 

Immunologic Toxicity—The occurrence of adverse effects on the immune system that may result from 

exposure to environmental agents such as chemicals. 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 263 

10. GLOSSARY 

Immunological Effects—Functional changes in the immune response. 

Incidence—The ratio of individuals in a population who develop a specified condition to the total 

number of individuals in that population who could have developed that condition in a specified time 

period. 

Intermediate Exposure—Exposure to a chemical for a duration of 15–364 days, as specified in the 

Toxicological Profiles. 

In Vitro—Isolated from the living organism and artificially maintained, as in a test tube. 

In Vivo—Occurring within the living organism. 

Lethal Concentration(LO) (LCLO)—The lowest concentration of a chemical in air that has been reported 

to have caused death in humans or animals. 

Lethal Concentration(50) (LC50)—A calculated concentration of a chemical in air to which exposure for 

a specific length of time is expected to cause death in 50% of a defined experimental animal population. 

Lethal Dose(LO) (LDLo)—The lowest dose of a chemical introduced by a route other than inhalation that 

has been reported to have caused death in humans or animals. 

Lethal Dose(50) (LD50)—The dose of a chemical that has been calculated to cause death in 50% of a 

defined experimental animal population. 

Lethal Time(50) (LT50)—A calculated period of time within which a specific concentration of a chemical 

is expected to cause death in 50% of a defined experimental animal population. 

Lowest-Observed-Adverse-Effect Level (LOAEL)—The lowest exposure level of chemical in a study, 

or group of studies, that produces statistically or biologically significant increases in frequency or severity 

of adverse effects between the exposed population and its appropriate control. 

Lymphoreticular Effects—Represent morphological effects involving lymphatic tissues such as the 

lymph nodes, spleen, and thymus. 

Malformations—Permanent structural changes that may adversely affect survival, development, or 

function. 

Minimal Risk Level (MRL)—An estimate of daily human exposure to a hazardous substance that is 

likely to be without an appreciable risk of adverse noncancer health effects over a specified route and 

duration of exposure. 

Modifying Factor (MF)—A value (greater than zero) that is applied to the derivation of a Minimal Risk 

Level (MRL) to reflect additional concerns about the database that are not covered by the uncertainty 

factors. The default value for a MF is 1. 

Morbidity—State of being diseased; morbidity rate is the incidence or prevalence of disease in a specific 

population. 

Mortality—Death; mortality rate is a measure of the number of deaths in a population during a specified 

interval of time. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 264 

10. GLOSSARY 

Mutagen—A substance that causes mutations. A mutation is a change in the DNA sequence of a cell’s 

DNA. Mutations can lead to birth defects, miscarriages, or cancer. 

Necropsy—The gross examination of the organs and tissues of a dead body to determine the cause of 

death or pathological conditions. 

Neurotoxicity—The occurrence of adverse effects on the nervous system following exposure to a 

chemical. 

No-Observed-Adverse-Effect Level (NOAEL)—The dose of a chemical at which there were no 

statistically or biologically significant increases in frequency or severity of adverse effects seen between 

the exposed population and its appropriate control. Effects may be produced at this dose, but they are not 

considered to be adverse. 

Octanol-Water Partition Coefficient (Kow)—The equilibrium ratio of the concentrations of a chemical 

in n-octanol and water, in dilute solution. 

Odds Ratio (OR)—A means of measuring the association between an exposure (such as toxic substances 

and a disease or condition) that represents the best estimate of relative risk (risk as a ratio of the incidence 

among subjects exposed to a particular risk factor divided by the incidence among subjects who were not 

exposed to the risk factor). An OR of greater than 1 is considered to indicate greater risk of disease in the 

exposed group compared to the unexposed group. 

Organophosphate or Organophosphorus Compound—A phosphorus-containing organic compound 

and especially a pesticide that acts by inhibiting cholinesterase. 

Permissible Exposure Limit (PEL)—An Occupational Safety and Health Administration (OSHA) 

allowable exposure level in workplace air averaged over an 8-hour shift of a 40-hour workweek. 

Pesticide—General classification of chemicals specifically developed and produced for use in the control 

of agricultural and public health pests. 

Pharmacokinetics—The dynamic behavior of a material in the body, used to predict the fate 

(disposition) of an exogenous substance in an organism. Utilizing computational techniques, it provides 

the means of studying the absorption, distribution, metabolism, and excretion of chemicals by the body. 

Pharmacokinetic Model—A set of equations that can be used to describe the time course of a parent 

chemical or metabolite in an animal system. There are two types of pharmacokinetic models: data-based 

and physiologically-based. A data-based model divides the animal system into a series of compartments, 

which, in general, do not represent real, identifiable anatomic regions of the body, whereas the 

physiologically-based model compartments represent real anatomic regions of the body. 

Physiologically Based Pharmacodynamic (PBPD) Model—A type of physiologically based doseresponse model that quantitatively describes the relationship between target tissue dose and toxic end 

points. These models advance the importance of physiologically based models in that they clearly 

describe the biological effect (response) produced by the system following exposure to an exogenous 

substance. 

Physiologically Based Pharmacokinetic (PBPK) Model—Comprised of a series of compartments 

representing organs or tissue groups with realistic weights and blood flows. These models require a 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES 265 

10. GLOSSARY 

variety of physiological information: tissue volumes, blood flow rates to tissues, cardiac output, alveolar 

ventilation rates, and possibly membrane permeabilities. The models also utilize biochemical 

information, such as air/blood partition coefficients, and metabolic parameters. PBPK models are also 

called biologically based tissue dosimetry models. 

Prevalence—The number of cases of a disease or condition in a population at one point in time. 

Prospective Study—A type of cohort study in which the pertinent observations are made on events 

occurring after the start of the study. A group is followed over time. 

q1*—The upper-bound estimate of the low-dose slope of the dose-response curve as determined by the 

multistage procedure. The q1* can be used to calculate an estimate of carcinogenic potency, the 

incremental excess cancer risk per unit of exposure (usually μg/L for water, mg/kg/day for food, and 

μg/m3 for air). 

Recommended Exposure Limit (REL)—A National Institute for Occupational Safety and Health 

(NIOSH) time-weighted average (TWA) concentration for up to a 10-hour workday during a 40-hour 

workweek. 

Reference Concentration (RfC)—An estimate (with uncertainty spanning perhaps an order of 

magnitude) of a continuous inhalation exposure to the human population (including sensitive subgroups) 

that is likely to be without an appreciable risk of deleterious noncancer health effects during a lifetime. 

The inhalation reference concentration is for continuous inhalation exposures and is appropriately 

expressed in units of mg/m3 or ppm. 

Reference Dose (RfD)—An estimate (with uncertainty spanning perhaps an order of magnitude) of the 

daily exposure of the human population to a potential hazard that is likely to be without risk of deleterious 

effects during a lifetime. The RfD is operationally derived from the no-observed-adverse-effect level 

(NOAEL, from animal and human studies) by a consistent application of uncertainty factors that reflect 

various types of data used to estimate RfDs and an additional modifying factor, which is based on a 

professional judgment of the entire database on the chemical. The RfDs are not applicable to 

nonthreshold effects such as cancer. 

Reportable Quantity (RQ)—The quantity of a hazardous substance that is considered reportable under 

the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA). Reportable 

quantities are (1) 1 pound or greater or (2) for selected substances, an amount established by regulation 

either under CERCLA or under Section 311 of the Clean Water Act. Quantities are measured over a 

24-hour period. 

Reproductive Toxicity—The occurrence of adverse effects on the reproductive system that may result 

from exposure to a chemical. The toxicity may be directed to the reproductive organs and/or the related 

endocrine system. The manifestation of such toxicity may be noted as alterations in sexual behavior, 

fertility, pregnancy outcomes, or modifications in other functions that are dependent on the integrity of 

this system. 

Retrospective Study—A type of cohort study based on a group of persons known to have been exposed 

at some time in the past. Data are collected from routinely recorded events, up to the time the study is 

undertaken. Retrospective studies are limited to causal factors that can be ascertained from existing 

records and/or examining survivors of the cohort. 

Risk—The possibility or chance that some adverse effect will result from a given exposure to a chemical. 
 

 

 

 

 

 

 

DINITROTOLUENES 266 

10. GLOSSARY 

Risk Factor—An aspect of personal behavior or lifestyle, an environmental exposure, or an inborn or 

inherited characteristic that is associated with an increased occurrence of disease or other health-related 

event or condition. 

Risk Ratio—The ratio of the risk among persons with specific risk factors compared to the risk among 

persons without risk factors. A risk ratio greater than 1 indicates greater risk of disease in the exposed 

group compared to the unexposed group. 

Short-Term Exposure Limit (STEL)—The American Conference of Governmental Industrial 

Hygienists (ACGIH) maximum concentration to which workers can be exposed for up to 15 minutes 

continually. No more than four excursions are allowed per day, and there must be at least 60 minutes 

between exposure periods. The daily Threshold Limit Value-Time Weighted Average (TLV-TWA) may 

not be exceeded. 

Standardized Mortality Ratio (SMR)—A ratio of the observed number of deaths and the expected 

number of deaths in a specific standard population. 

Target Organ Toxicity—This term covers a broad range of adverse effects on target organs or 

physiological systems (e.g., renal, cardiovascular) extending from those arising through a single limited 

exposure to those assumed over a lifetime of exposure to a chemical. 

Teratogen—A chemical that causes structural defects that affect the development of an organism. 

Threshold Limit Value (TLV)—An American Conference of Governmental Industrial Hygienists 

(ACGIH) concentration of a substance to which most workers can be exposed without adverse effect. 

The TLV may be expressed as a Time Weighted Average (TWA), as a Short-Term Exposure Limit 

(STEL), or as a ceiling limit (CL). 

Time-Weighted Average (TWA)—An allowable exposure concentration averaged over a normal 8-hour 

workday or 40-hour workweek. 

Toxic Dose(50) (TD50)—A calculated dose of a chemical, introduced by a route other than inhalation, 

which is expected to cause a specific toxic effect in 50% of a defined experimental animal population. 

Toxicokinetic—The absorption, distribution, and elimination of toxic compounds in the living organism. 

Uncertainty Factor (UF)—A factor used in operationally deriving the Minimal Risk Level (MRL) or 

Reference Dose (RfD) or Reference Concentration (RfC) from experimental data. UFs are intended to 

account for (1) the variation in sensitivity among the members of the human population, (2) the 

uncertainty in extrapolating animal data to the case of human, (3) the uncertainty in extrapolating from 

data obtained in a study that is of less than lifetime exposure, and (4) the uncertainty in using lowestobserved-adverse-effect level (LOAEL) data rather than no-observed-adverse-effect level (NOAEL) data. 

A default for each individual UF is 10; if complete certainty in data exists, a value of 1 can be used; 

however, a reduced UF of 3 may be used on a case-by-case basis, 3 being the approximate logarithmic 

average of 10 and 1. 

Xenobiotic—Any chemical that is foreign to the biological system. 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-1 

APPENDIX A. ATSDR MINIMAL RISK LEVELS AND WORKSHEETS 

The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) [42 U.S.C. 

9601 et seq.], as amended by the Superfund Amendments and Reauthorization Act (SARA) [Pub. L. 99– 

499], requires that the Agency for Toxic Substances and Disease Registry (ATSDR) develop jointly with 

the U.S. Environmental Protection Agency (EPA), in order of priority, a list of hazardous substances most 

commonly found at facilities on the CERCLA National Priorities List (NPL); prepare toxicological 

profiles for each substance included on the priority list of hazardous substances; and assure the initiation 

of a research program to fill identified data needs associated with the substances. 

The toxicological profiles include an examination, summary, and interpretation of available toxicological 

information and epidemiologic evaluations of a hazardous substance. During the development of 

toxicological profiles, Minimal Risk Levels (MRLs) are derived when reliable and sufficient data exist to 

identify the target organ(s) of effect or the most sensitive health effect(s) for a specific duration for a 

given route of exposure. An MRL is an estimate of the daily human exposure to a hazardous substance 

that is likely to be without appreciable risk of adverse noncancer health effects over a specified duration 

of exposure. MRLs are based on noncancer health effects only and are not based on a consideration of 

cancer effects. These substance-specific estimates, which are intended to serve as screening levels, are 

used by ATSDR health assessors to identify contaminants and potential health effects that may be of 

concern at hazardous waste sites. It is important to note that MRLs are not intended to define clean-up or 

action levels. 

MRLs are derived for hazardous substances using the no-observed-adverse-effect level/uncertainty factor 

approach. They are below levels that might cause adverse health effects in the people most sensitive to 

such chemical-induced effects. MRLs are derived for acute (1–14 days), intermediate (15–364 days), and 

chronic (365 days and longer) durations and for the oral and inhalation routes of exposure. Currently, 

MRLs for the dermal route of exposure are not derived because ATSDR has not yet identified a method 

suitable for this route of exposure. MRLs are generally based on the most sensitive chemical-induced end 

point considered to be of relevance to humans. Serious health effects (such as irreparable damage to the 

liver or kidneys, or birth defects) are not used as a basis for establishing MRLs. Exposure to a level 

above the MRL does not mean that adverse health effects will occur. 

MRLs are intended only to serve as a screening tool to help public health professionals decide where to 

look more closely. They may also be viewed as a mechanism to identify those hazardous waste sites that 
 

 

 

 

 

 

DINITROTOLUENES A-2 

APPENDIX A 

are not expected to cause adverse health effects. Most MRLs contain a degree of uncertainty because of 

the lack of precise toxicological information on the people who might be most sensitive (e.g., infants, 

elderly, nutritionally or immunologically compromised) to the effects of hazardous substances. ATSDR 

uses a conservative (i.e., protective) approach to address this uncertainty consistent with the public health 

principle of prevention. Although human data are preferred, MRLs often must be based on animal studies 

because relevant human studies are lacking. In the absence of evidence to the contrary, ATSDR assumes 

that humans are more sensitive to the effects of hazardous substance than animals and that certain persons 

may be particularly sensitive. Thus, the resulting MRL may be as much as 100-fold below levels that 

have been shown to be nontoxic in laboratory animals. 

Proposed MRLs undergo a rigorous review process: Health Effects/MRL Workgroup reviews within the 

Division of Toxicology and Human Health Sciences, expert panel peer reviews, and agency-wide MRL 

Workgroup reviews, with participation from other federal agencies and comments from the public. They 

are subject to change as new information becomes available concomitant with updating the toxicological 

profiles. Thus, MRLs in the most recent toxicological profiles supersede previously published levels. 

For additional information regarding MRLs, please contact the Division of Toxicology and Human 

Health Sciences, Agency for Toxic Substances and Disease Registry, 1600 Clifton Road NE, Mailstop 

F-57, Atlanta, Georgia 30329-4027. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-3 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,3-DNT 

CAS Numbers: 602-01-7 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [X] Acute [ ] Intermediate [ ] Chronic 

Graph Key: 4 

Species: Rat 

Minimal Risk Level: 0.09 [X] mg/kg/day [ ] ppm 

References: Lent EM, Crouse CB, Quinn MJ, et al. 2012a. Comparison of the repeated dose toxicity of 

isomers of dinitrotoluene. Int J Toxicol 31(2):143-157. 

U.S. Army Public Health Command (USAPHC). 2011a. Toxicology Study No. 87-XE-08G0A-08, 

October 2011, Toxicology Portfolio. Fourteen-day oral toxicity and in vivo genotoxicity of 2,3­

dinitrotoluene in rats, June-July 2008. USAPHC, Aberdeen Proving Ground, Maryland. 

Experimental design: In a comparative toxicity study, groups of six male Sprague-Dawley rats were 

given 2,3-DNT via gavage doses of 0, 17, 34, 69, 138, 275, or 550 mg/kg/day for 14 days. Both untreated 

and vehicle control groups were included. Evaluations during the study included twice daily observations 

for morbidity and clinical signs and measurement of body weight and food consumption on days 0, 1, 3, 

7, and 14. Prior to sacrifice at the end of exposure, blood was collected for evaluation of hematology 

(total and differential white blood cell counts; red blood cell count; hemoglobin, hematocrit, MCV, MCH, 

mean corpuscular hemoglobin concentration [MCHC], red cell distribution width; platelet count; and 

mean platelet volume) and serum chemistry (albumin, ALP, ALT, AST, BUN, calcium, cholesterol, 

creatinine, glucose, globulin, LDH, total bilirubin, total protein, sodium, potassium, and chlorine) 

assessments. All animals received gross necropsy and organ weight determinations (brain, heart, kidney, 

epididymides, liver, spleen, and testes). Microscopic examination of organs with exposure-related 

changes in weight was performed for controls and any dose groups exhibiting changes in the 

corresponding organ weights. 

Effects noted in study and corresponding doses: All rats exposed to 550 mg/kg/day died or were 

sacrificed moribund within the first 2–3 days of dosing. Clinical signs of toxicity were observed only in 

this dose group. In the surviving dose groups, there were no statistically significant differences from 

control in body weight or food consumption, although body weight was lower (~9% less than vehicle 

controls) in the 275 mg/kg/day group. In addition, there were no significant, treatment-related changes in 

hematology or clinical chemistry parameters. The authors noted 2.3-fold increases in ALP, ALT, and 

AST in the 138 mg/kg/day group, but the difference from control was not statistically significant, and 

comparable increases were not seen at in the higher dose group (275 mg/kg/day). Significantly increased 

relative (to body weight) liver and kidney weights were observed at 275 mg/kg/day (20 and 14% higher 

than vehicle controls, respectively); lower body weights in this group likely contributed to the changes. 

Absolute organ weights and organ weights relative to brain weight were not significantly different from 

controls. There were no accompanying histopathology lesions in the liver, nor were there clinical 

chemistry changes indicative of liver or kidney injury. In animals surviving to terminal sacrifice, 

however, renal lesions were observed at 275 mg/kg/day, including trace tubular dilatation in 2/6 rats 

(compared with 1/6 controls) and trace lymphocytic infiltration in 4/6 rats (compared with 1/6 controls). 

In addition, extramedullary hematopoiesis (3/6 rats) and lymphoid hyperplasia (1/6) of the spleen, as well 

as lymphoid depletion (1/6), were observed at 275 mg/kg/day. These lesions were not seen in controls. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-4 

APPENDIX A 

Although none of these incidences was statistically significantly increased in pairwise comparisons with 

controls, the group sizes were very small, and the increases are considered to be biologically significant. 

Kidneys and spleens of animals in the lower dose groups were not examined microscopically. Results of 

this study identify an acute-duration LOAEL for kidney and splenic effects (see Table A-1) of 

275 mg/kg/day, respectively, in rats. A NOAEL for kidney and splenic effects cannot be determined in 

the absence of histopathology examinations in lower dose groups. 

Table A-1. Renal and Splenic Effects in Rats Exposed to 2,3-DNT for 14 Days 

Dose (mg/kg/day) 

End point 0 17 34 69 138 275 550 

Renal tubular dilatation 1/6a ND ND ND ND 2/6 NA 

Renal lymphocytic infiltration 1/6 ND ND ND ND 4/6 NA 

Splenic extramedullary hematopoiesis 0/6 ND ND ND ND 3/6 NA 

Splenic lymphoid hyperplasia 0/6 ND ND ND ND 1/6 NA 

Splenic lymphoid depletion 0/6 ND ND ND ND 1/6 NA 

aNumber affected/number examined. 

DNT = dinitrotoluene; NA = not applicable; all animals in this group died or were sacrificed moribund; ND = no data; 

histopathology examinations were not performed on these animals. 

Sources: Lent et al. 2012a; USAPHC 2011a 

Dose and end point used for MRL derivation: 

[] NOAEL [X] LOAEL 275 mg/kg/day was the LOAEL for renal and hematopoietic effects (renal 

tubular dilatation, renal lymphocytic infiltration, splenic extramedullary hematopoiesis and lymphoid 

hyperplasia). 

The LOAEL value of 275 mg/kg/day for renal and hematopoietic effects in rats was identified as the POD 

for derivation of the acute-duration oral MRL for 2,3-DNT (Lent et al. 2012a; USAPHC 2011a). A 

NOAEL was not determined from the study as the target organs (kidney and spleen) were not examined 

microscopically in lower dose groups. Available data were not suitable for BMD modeling, as incidence 

data were limited to the 275 mg/kg/day group and the control group. The POD of 275 mg/kg/day was 

divided by an uncertainty factor of 3,000 (10 for use of a LOAEL, 10 for extrapolation from animals to 

humans, and 10 for human variability, and 3 for database limitations), resulting in an acute-duration oral 

MRL for 2,3-DNT of 0.09 mg/kg/day. 

Uncertainty Factors used in MRL derivation: 

[X] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

[X] 3 for database limitations (the only other data available are from an acute lethality study) 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 
 

 

DINITROTOLUENES A-5 

APPENDIX A 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: The only other acuteduration oral study of 2,3-DNT was an acute lethality study, in which LD50 values of 1,120 and 

1,070 mg/kg were identified in rats and mice, respectively (Vernot et al. 1977). 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-6 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,4-DNT 

CAS Numbers: 121-14-2 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [X] Acute [ ] Intermediate [ ] Chronic 

Graph Key: 14 

Species: Dog 

Minimal Risk Level: 0.05 [X] mg/kg/day [ ] ppm 

References: Ellis HV, Hong CB, Lee CC, et al. 1985. Subchronic and chronic toxicity studies of 

2,4-dinitrotoluene. Part I. Beagle dog. J Am Coll Toxicol 4:233-242. 

U.S. Army. 1978b. Mammalian toxicity of munitions compounds. Phase II: Effects of multiple doses. 

Part II: 2,4-Dinitrotoluene. Progress report no. 3. Fort Detrick, MD: U.S. Army, Medical 

Bioengineering Research and Development Laboratory. ADA061715. 

Experimental design: In a subchronic study, beagle dogs (4/sex/group) were administered 0, 1, 5, or 

25 mg/kg/day 2,4-DNT for up to 13 weeks. 2,4-DNT was mixed with lactose and administered as 

capsules. Dogs were observed daily for behavioral changes and clinical signs of toxicity. Body weights 

were recorded weekly. Blood was taken before initiation of treatment and at 4, 8, and 13 weeks for 

evaluation of hematological parameters (erythrocyte, reticulocyte, platelet, and total and differential 

leukocyte counts; Heinz bodies; hematocrit, hemoglobin, and methemoglobin concentrations; mean cell 

volume, hemoglobin, and hemoglobin concentration) and clinical chemistry analyses (glucose, urea 

nitrogen, sodium, potassium, calcium, magnesium, and chloride; and the serum enzyme activity of 

aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase). Animals (1 sex/group) 

were sacrificed after 4 or 13 weeks of continuous treatment; an additional dog/sex/group was 

discontinued treatment after 4 or 13 weeks and sacrificed after 4 weeks of recovery. The two high-dose 

dogs removed from treatment at 4 weeks were not sacrificed until 8 months after cessation of treatment to 

test the reversibility of effects after a longer recovery period. When animals were sacrificed moribund or 

at study termination, they were examined for gross lesions. Major organs and tissues (heart, liver, spleen, 

kidneys, adrenals, and gonads) were weighed; “various” tissues (not specified) were subjected to 

histopathology. To evaluate the immunologic response to 2,4-DNT, the concentration of IgE in the serum 

was assessed after treatment for 4, 8, or 13 weeks or after treatment for 4 or 13 weeks followed by 

recovery for 4 weeks. Bone marrow and kidney cultures were also maintained and cytogenetic analyses 

(evaluation of chromosome number and morphology) were performed. 

Effects noted in study and corresponding doses: Data for acute-duration oral exposure were obtained 

from daily cageside observations for behavioral changes and clinical signs of toxicity during the first 

14 days of treatment. No mortality was observed during the first 14 days of treatment. No behavioral 

changes or clinical signs of toxicity were observed in dogs treated with 1 or 5 mg/kg/day. Neurotoxicity 

was observed at 25 mg/kg/day. Evidence of neurotoxicity, identified as loss of hind leg control, was first 

observed in a female dog on day 12 of treatment. Three additional male dogs showed similar signs on 

day 14 of treatment. All high-dose dogs showed signs of neurotoxicity after treatment for 12–22 days. 

The onset and severity of toxic signs reportedly varied among dogs within the same treatment group; 

some dogs were moribund at the same time that others began experiencing symptoms. In individual dogs, 

symptom severity varied over time, with no duration-related pattern of severity. Although incidence data 

were not reported, the study authors noted that the neurotoxic effects most often observed in the 13-week 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-7 

APPENDIX A 

study were incoordination of the hind legs and stiffness that produced an abnormal hopping gait. Some 

dogs experienced paralysis of the hind legs. In severe cases, stiffness progressed from the hind legs to the 

trunk, forelegs, neck, and head. Histopathological assessments conducted at 4 or 13 weeks showed 

lesions of the central nervous system, including generalized vacuolization, hypertrophy, mitosis of the 

endothelium and focal gliosis in the cerebellum, and/or perivascular hemorrhage in the cerebellum and 

brain stem in high-dose animals (2/2 and 3/3 animals evaluated at 4 and 13 weeks, respectively). 

However, the study authors noted that the most severe of these lesions occurred in dogs that developed 

toxic signs of neurotoxicity late in the study (time to onset of symptoms not reported). Results of this 

study identify acute-duration NOAEL and LOAEL values for neurotoxicity in dogs of 5 and 

25 mg/kg/day, respectively. 

Dose and end point used for MRL derivation: 

[X] NOAEL [ ] LOAEL [] 5 mg/kg/day was the NOAEL for 

neurological effects (loss of hind leg control). 

The NOAEL value of 5 mg/kg/day for neurotoxicity in dogs was identified as the POD for derivation of 

the acute-duration oral MRL for 2,4-DNT (Ellis et al. 1985; U.S. Army 1978b). Neurotoxicity data were 

not suitable for BMD modeling, since effects were only observed at the highest dose tested. Therefore, 

the NOAEL value for 5 mg/kg/day was used at the POD. This value was divided by an uncertainty factor 

of 100 (10 for extrapolation from animals to humans and 10 for human variability), resulting in an acuteduration oral MRL for 2,4-DNT of 0.05 mg/kg/day. 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: No other acuteduration studies were located in which neurotoxicity was reported after oral exposure to 2,4-DNT. Other 

acute-duration studies in rodents identified hepatic effects (single cell necrosis and glycogen deposition) 

and hematological effects (splenic extramedullary hematopoesis) in male Sprague-Dawley rats treated at 

36 and 71 mg/kg/day (respectively) via gavage for 14 days (Lent et al. 2012a; USAPHC 2011b), slight 

cyanosis in male Sprague-Dawley rats treated at 60 mg/kg (the lowest tested dose) for 5 days (Lane et al. 

1985), and decreased fertility in CD-1 female mice dosed with 250 mg/kg 2,4-DNT for 2 days (Soares 

and Lock 1980). Neurotoxicity was observed in beagle dogs after subchronic or chronic treatment with 

2,4-DNT. Clinical signs of neurotoxicity (including incoordination and paralysis), sometimes 

accompanied by central nervous system lesions (generalized vacuolization, hypertrophy, mitosis of the 

endothelium and focal gliosis in the cerebellum, and perivascular hemorrhages of the cerebellum and 

brain stem) were reported in dogs (4/sex/group) dosed with 25 mg/kg 2,4-DNT for up to 13 weeks (Ellis 

et al. 1985; U.S. Army 1978b) and in dogs (6 sex/group) treated at 1.5 (one dog) or 10 mg/kg/day (all 

dogs) for up to 24 months (Ellis et al. 1985; U.S. Army 1979). Dogs appear to be the most sensitive 

species for 2,4-DNT-induced neurotoxicity; in CD rats and CD-1 mice treated with 2,4-DNT for up to 

24 months, neurotoxic effects were absent or occurred at much higher doses in similarly designed studies 
 

DINITROTOLUENES A-8 

APPENDIX A 

(U.S. Army 1978b, 1979). Neuromuscular effects similar to those observed in dogs occurred in rats 

administered 2,4-DNT at 266 or 145 mg/kg/day (for males and females, respectively) for up to 13 weeks, 

but not in rats treated with 2,4-DNT at up to 34 or 45 mg/kg/day (for males and females, respectively) for 

24 months. Mice treated with 2,4-DNT at 413 or 468 mg/kg/day (for males and females, respectively) for 

up to 13 weeks or 898 mg/kg/day for 24 months did not show clinical signs of neurotoxicity (U.S. Army 

1978b, 1979). 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-9 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,4-DNT 

CAS Numbers: 121-14-2 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [ ] Acute [X] Intermediate [ ] Chronic 

Graph Key: 32 

Species: Dog 

Minimal Risk Level: 0.007 [X] mg/kg/day [ ] ppm 

References: U.S. Army. 1979. Mammalian toxicity of munitions compounds. Phase III: Effects of 

lifetime exposure. Part I. 2,4-Dinitrotoluene. Final report no. 7. Fort Detrick, MD: U.S. Army and 

Medical Bioengineering Research Development Laboratory. ADA077692. 

Ellis HV, Hong CB, Lee CC, et al. 1985. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. 

Part I. Beagle dog. J Am Coll Toxicol 4:233-242. 

Experimental design: Young beagle dogs (6 dogs/sex/group; age not specified) were administered 0, 0.2, 

1.5, or 10 mg/kg 2,4-DNT in capsules for 24 months. Dogs were observed daily for behavioral changes 

and clinical signs of toxicity. Body weights were recorded weekly. Feed consumption was measured 

during 1 week each month starting in month 6. Blood was taken before initiation of treatment and after 3, 

6, 9, 12, 18, and 24 months of exposure for assessment of hematological parameters (erythrocyte, 

reticulocyte, platelet, and total and differential leukocyte counts; Heinz bodies, clotting time, hematocrit, 

hemoglobin, and methemoglobin concentrations; and mean cell volume, hemoglobin, and hemoglobin 

concentration) and clinical chemistry (fasting glucose, urea nitrogen, levels of sodium, potassium, 

calcium, magnesium, chloride, and bilirubin [high-dose dogs with toxic signs], and the serum enzyme 

activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) analyses. 

Animals (one male and one female/group) were sacrificed after 12 or 24 months of continuous treatment; 

an additional dog/sex/group was discontinued from treatment at these time points and were sacrificed 

after a 4-week recovery period to evaluate the reversibility of effects (including clinical signs, hematology 

and clinical chemistry, organ weights, and histopathological effects). Animals that were moribund during 

the study and those that survived to study termination were sacrificed and examined for gross lesions; 

major organs and tissues (including the brain, heart, liver, spleen, kidneys, adrenals, thyroids, pituitary, 

and gonads) were weighed, and comprehensive histopathological analyses (35 tissues) were performed. 

Effects noted in study and corresponding doses: Intermediate-duration oral exposure of dogs to 2,4-DNT 

produced methemoglobinemia, anemia, and compensatory hematopoiesis (Ellis et al. 1985; U.S. Army 

1979). Hematological effects of 2,4-DNT are initiated by methemoglobin production, which occurs when 

the ferrous iron in complex with the heme groups of hemoglobin is oxidized to ferric iron. Ferric iron 

does not bind oxygen, resulting in anemia. Ferric iron also contributes to the denaturation of hemoglobin 

and subsequent removal of erythrocytes from the blood. Heinz bodies are also detected as granules in 

erythrocytes resulting from denatured hemoglobin. Increased hematopoiesis is typically observed as a 

compensatory response to decreased erythrocyte count. Hematological effects consistent with 

development of methemoglobinemia were observed in dogs administered oral 2,4-DNT at all intermediate 

duration time points (3, 6, and 9 months). Although effects at all time points were qualitatively similar, 

hematological changes observed after 9 months of exposure were more consistent and pronounced than 

those observed at the 3- and 6-month time periods. Therefore, only data from the 9-month evaluation 

were considered for derivation of the intermediate-duration oral MRL. The only significant effects 
 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-10 

APPENDIX A 

observed at the intermediate-duration time points were changes to hematological parameters. At the midand low dose, no clinical signs of toxicity, behavioral changes, or effects on clinical chemistry parameters 

were observed at the intermediate-duration time points at any of the doses tested. At the high dose, 

clinical signs of neurotoxicity (decreased muscle control and incoordination), sometimes accompanied by 

decreased body weight, were observed; these effects contributed to the death of four of six dogs within 

the first 20 weeks of the study. 

Effects on hematological parameters in male and female dogs administered oral 2,4-DNT for 9 months 

are summarized in Table A-2. Male and female dogs exposed to 2,4-DNT for 9 months at doses of 

1.5 and 10 mg/kg/day showed detectable amounts of methemoglobin in the serum (the initiating 

hematological effect), with changes reaching statistical significance in males and females in the 

10 mg/kg/day group. In female dogs administered 10 mg/kg/day, statistically significant decreases in 

erythrocyte count, hematocrit, and hemoglobin, a statistically significant increase in reticulocyte count, 

and the presence of Heinz bodies in serum were observed. Similar hematological effects were observed 

in female dogs administered 0.2 and 1.5 mg/kg/day, although effects did not reach statistical significance, 

most likely because the power of the study to detect statistically significant changes was compromised by 

the small number of dogs per treatment group. However, based on a clinically significant increase in 

methemoglobin levels of 225% in female dogs administered 1.5 mg/kg/day, the NOAEL and LOAEL 

values for hematological effects in this study are 0.2 and 1.5 mg/kg/day, respectively. Effects on 

hematological parameters in male dogs were similar to those in female dogs, although changes did not 

reach statistical significance in the 10 mg/kg/day group, possibly due to low numbers of male dogs 

evaluated (hematological data available for only two males in the 10 mg/kg/day group). After treatment 

for 18 or 24 months, slight or no anemia, near-normal reticulocyte levels, no Heinz bodies, and minimal 

amounts of methemoglobin were detected, likely reflective of an adaptive response. Recovery from 

hematological effects also occurred in dogs allowed to recover for 4 weeks after dosing for 12 or 

24 months. 
 

 

DINITROTOLUENES A-11 

APPENDIX A 

Table A-2. Hematological Effects in Beagle Dogs Exposed to 2,4-DNT for 

9 Months 

Dose (mg/kg/day) 

End point 0 0.2 1.5 10 

Males 

Erythrocyte count 

(x10/mm) 

6.51±0.12 (6)a 6.23±0.18 (6) 

[↓4] 

6.36±0.13 (6) 

[↓2] 

6.36±0.13 (2) 

[↓2] 

Heinz bodies (%) 0.0±0.0(6) 0.0±0.0(6) 

[NA] 

0.0±0.0(6) 

[NA] 

1.8±0.6(2)b 

[NA] 

Reticulocytes (%) 0.69±0.15 (6) 0.55±0.09 (6) 

[↓20] 

0.57±0.11 (6) 

[↓17] 

1.39±0.47 (2) 

[↑101] 

Hematocrit (%) 46.7±1.0 (6) 45.3±1.1 (6) 

[↓3] 

45.0±0.9 (6) 

[↓4] 

42.0±3.0 (2) 

[↓10] 

Hemoglobin (%) 16.6±0.3 (6) 15.4±0.4 (6) 15.7±0.3 (6) 15.7±0.3 (6) 

Methemoglobin (%) 0.0±0.0 (6) 0.0±0.0 (6) 

[NA] 

0.9±0.6 (6) 

[NA] 

2.8±0.3 (2)b 

[NA] 

Females 

Erythrocyte count 

(x10/mm) 

6.49±0.24 (6)a 5.90±0.17 (6) 

[↓9] 

5.78±0.21 (6) 

[↓11] 

5.05±0.17 (6)b 

[↓22] 

Heinz bodies (%) 0.0±0.0(6) 0.0±0.0(6) 

[NA] 

0.0±0.0(6) 

[NA] 

0.84±0.21 (6)b 

[NA] 

Reticulocytes (%) 0.60±0.08 (6) 0.70±0.17 (6) 

[↑17] 

0.45±0.10 (6) 

[↓25] 

1.33±0.19 (6)b 

[↑122] 

Hematocrit (%) 46.0±1.3 (6) 42.8±1.7 (6) 

[↓7] 

42.5±1.2 (6) 

[↓8] 

37.2±1.6 (6)b 

[↓19] 

Hemoglobin (%) 16.1±0.5 (6) 14.7±0.6 (6) 

[↓9] 

14.8±0.5 (6) 

[↓8] 

13.3±0.5 (6)b 

[↓17] 

Methemoglobin (%) 0.4±0.4 (6) 0.0±0.0 (6) 

[↓100] 

1.3±0.6 (6) 

[↑225] 

2.8±0.7 (6)b 

[↑600] 

aValues are means±standard error (number of animals) [percent change from controls]. bStatistically significant based on analyses performed by the study authors (Dunnett’s multiple comparison
 

procedure).
 

DNT = dinitrotoluene; NA = not applicable 

Sources: Ellis et al. 1985; U.S. Army 1979 

Dose and end point used for MRL derivation: 

[ ] NOAEL [ ] LOAEL [X] BMDL 0.67 mg/kg/day as a BMDL1SD for hematological effects (decreased 

hematocrit) 

Results of hematology assessments show that intermediate-duration, oral exposure of dogs to 2,4-DNT 

induced methemoglobinemia, anemia, and compensatory hematopoiesis (Ellis et al. 1985; U.S. Army 

1979). Changes in several hematological parameters, including decreased erythrocyte count, hematocrit, 

and hemoglobin and increased reticulocytes, methemoglobin, and Heinz bodies were observed after 

treatment with 2,4-DNT for 3, 6, and 9 months. However, hematological effects at 9 months were more 

pronounced and consistent than those observed at 3 and 6 months; therefore, hematological effects 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-12 

APPENDIX A 

observed at 9 months were identified as the critical effect for derivation of the intermediate-duration oral 

MRL. To determine the POD, hematological data from female dogs treated with 2,4-DNT for 9 months 

were further evaluated by BMD analysis. The following data sets in female dogs were selected for BMD 

modeling: erythrocyte count, reticulocytes, hematocrit, hemoglobin, and methemoglobin. Data on Heinz 

bodies in serum were not selected for BMD modeling, since these granules were detected in high-dose 

animals only (i.e., all-or-nothing response); the absence of changes at lower dose levels suggests that 

these data would not be suitable for modeling. Hematological data from male dogs were not considered 

for additional BMD analyses due to the low number of dogs evaluated in the 10 mg/kg/day group (for 

most hematological parameters, data were available for only two dogs). 

To determine the POD for derivation of the intermediate-duration oral MRL, all available continuousvariable models in the EPA BMDS (version 2.1) were fit to the data for increased methemoglobin, 

increased reticulocytes, decreased hemoglobin, decreased erythrocyte count, and decreased hematocrit 

(Ellis et al. 1985; U.S. Army 1979). The BMD and the 95% lower confidence limit (BMDL) were 

estimated for doses associated with a change of 1 standard deviation from the controls, and are in units of 

mg/kg/day. For continuous data, in the absence of a clear criteria as to what level of change should be 

considered adverse, the BMR is defined as a change equal to 1 standard deviation from the control mean 

(EPA 2000a). Adequate model fit is judged by three criteria: goodness-of-fit (p>0.1), visual inspection 

of the dose-response curve, and scaled residual at the data point (except the control) closest to the 

predefined BMR. Among all of the models providing adequate fit to the data, the lowest benchmark dose 

(BMDL, the lower limit of a one-sided 95% CI on the BMD) is selected as the POD when differences 

between the BMDLs estimated from these models are >3-fold; otherwise, the BMDL from the model with 

the lowest AIC is chosen. 

Neither the constant nor the non-constant variance model provided an adequate fit to the data for 

decreased erythrocyte count or increased methemoglobin; therefore, these data were not considered 

suitable for BMD modeling. BMD model prediction for increased reticulocytes, decreased hemoglobin, 

and decreased hematocrit are shown in Tables A-3, A-4, and A-5, respectively. Of models meeting 

adequate fit criteria for each hematological parameter, the lowest BMDL1SD values were 5.64 mg/kg/day 

for increased reticulocytes (polynomial 3-degree; Figure A-1), 3.66 mg/kg/day for decreased hemoglobin 

(exponential model 2; Figure A-2), and 0.67 mg/kg/day for decreased hematocrit (exponential model 4; 

Figure A-3). Of these, the lowest BMDL1SD of 0.67 mg 2,4-DNT/kg/day for decreased hematocrit was 

selected as the POD for derivation of the intermediate-duration oral MRL for 2,4-DNT. This value was 

divided by an uncertainty factor of 100 (10 for animals to human extrapolation and 10 for human 

variability), resulting in an intermediate-duration oral MRL of 0.007 mg/kg/day. 
 

 

 

DINITROTOLUENES A-13 

APPENDIX A 

Table A-3. Model Predictions for 2,4-DNT for Increased Reticulocytes (%) in Female 

Dogs Following 9 Months of Exposure (U.S. Army 1979)
 

Test for Scaled residualsc 

significant Dose Dose BMD1SD BMDL1SD 

Model 

difference 

p-valuea 

Variance 

p-valueb 

Means 

p-valueb 

below 

BMD 

above 

BMD AIC 

Overall 

largest 

(mg/kg/ 

day) 

(mg/kg/ 

day) 

Constant variance 

Exponential (model 2)d 0.0007 0.12 0.23 -1.32 0.09 -1.32 -21.60 5.55 4.35 

Exponential (model 3)d 0.0007 0.12 0.18 -0.98 5.59x10-8 -0.98 -20.75 9.40 4.58 

Exponential (model 4)d 0.0007 0.12 0.06 -1.51 0.21 -1.50 -18.93 4.61 3.19 

Exponential (model 5)d 0.0007 0.12 NA -0.98 8.61x10-8 -0.98 -18.75 9.17 1.59 

Hilld 0.0007 0.12 NA -0.98 -9.04x10-7 -0.98 -18.75 9.10 1.60 

Lineare 0.0007 0.12 0.17 -1.51 0.21 -1.51 -20.93 4.61 3.19 

Polynomial (2-degree)e 0.0007 0.12 0.36 -1.06 0.02 -1.06 -22.50 6.69 5.60 

Polynomial 

(3-degree)e,f 

0.0007 0.12 0.40 -0.99 0.003 -0.99 -22.72 7.64 5.64 

Powerd 0.0007 0.12 0.18 -0.98 3.56x10-10 -0.98 -20.75 9.31 3.68 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the BMD; also the largest residual at any dose.

dPower restricted to ≥1. 

eCoefficients restricted to be positive. f

Selected model. Constant variance model provided adequate fit to variance data. With constant variance model 

applied, all models, except for the Exponential 4 and 5 and Hill models, provided adequate fit to means. BMDLs for 

models providing adequate fit were considered to be sufficiently close (differed by <2–3-fold), so the model with the 

lowest AIC was selected. 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated with 

the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark 

response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom; 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed); SD = standard deviation 
 

DINITROTOLUENES A-14 

APPENDIX A 

Figure A-1. Fit of Polynomial 3-Degree Model to Data on 2,4-DNT for
 

Increased Reticulocytes (%) in Female Dogs Following 

9 Months of Exposure (U.S. Army 1979)

Polynomial Model with 0.95 Confidence Level 

0.2

 0.4

 0.6

 0.8

 1

 1.2

 1.4

 1.6

 1.8

Mean Response 

BMDL BMD

Polynomial 

0 2 4 6 8 10
 

dose
 

16:04 04/02 2012
 
 

 

 

 

DINITROTOLUENES A-15 

APPENDIX A 

Table A-4. Model Predictions for 2,4-DNT for Decreased Hemoglobin (%) in 

Female Dogs Following 9 Months of Exposure (U.S. Army 1979)
 

Test for Scaled residualsc 

significant Dose Dose BMD1SD BMDL1SD 

Model 

difference 

p-valuea 

Variance 

p-valueb 

Means 

p-valueb AIC 

below 

BMD 

above 

BMD 

Overall 

largest 

(mg/kg/ 

day) 

(mg/kg/ 

day) 

Constant variance 

Exponential 

(model 2)d,e 

0.04 0.92 0.14 -0.40 0.10 1.46 41.47 5.90 3.66 

Exponential (model 3)d 0.04 0.92 0.14 -0.40 0.10 1.46 41.47 5.90 3.66 

Exponential (model 4)d 0.04 0.92 0.07 0.29 -0.03 -1.36 42.88 2.43 0.01 

Exponential (model 5)d 0.04 0.92 0.07 0.29 -0.03 -1.36 42.88 2.43 0.01 

Hilld NA NA NA NA NA NA NA NA NA 

Linearf 0.04 0.92 0.14 -0.43 0.09 1.48 41.52 6.12 3.94 

Polynomial (2-degree)f 0.04 0.92 0.14 -0.43 0.09 1.48 41.52 6.12 3.94 

Polynomial (3-degree)f 0.04 0.92 0.14 -0.43 0.09 1.48 41.52 6.12 3.94 

Powerd 0.04 0.92 0.14 -0.43 0.09 1.48 41.52 6.12 3.94 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the BMD; also the largest residual at any dose.

dPower restricted to ≥1. 

eSelected model. Constant variance model provided adequate fit to variance data. With constant variance model 

applied, all models, except for the Exponential 4 and 5 and Hill model (computation failed), provided adequate fit to 

means. BMDLs for models providing adequate fit were considered to be sufficiently close (differed by <2–3-fold), so 

the model with the lowest AIC was selected (the Exponential 3 model converged on to the Exponential 2). 

f

Coefficients restricted to be negative. 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated 

with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote 

benchmark response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom; 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed); SD = standard deviation 
 

 

DINITROTOLUENES A-16 

APPENDIX A 

Figure A-2. Fit of the Exponential 2 Model to Data on 2,4-DNT for 

Decreased Hemoglobin (%) in Female Dogs Following 9 Months 

of Exposure (U.S. Army 1979)

Exponential Model 2 with 0.95 Confidence Level 

12

 13

 14

 15

 16

 17

Mean Response 

BMDL BMD

Exponential 

0 2 4 6 8 10
 

dose
 

08:20 04/12 2012
 
 

 

 

 

DINITROTOLUENES A-17 

APPENDIX A 

Table A-5. Model Predictions for 2,4-DNT for Decreased Hematocrit (%) in Female 

Dogs Following 9 Months of Exposure (U.S. Army 1979)
 

Test for Scaled residualsc 

significant Dose Dose BMD1SD BMDL1SD 

Model 

difference 

p-valuea 

Variance 

p-valueb 

Means 

p-valueb AIC 

below 

BMD 

above 

BMD 

Overall 

largest 

(mg/kg/ 

day) 

(mg/kg/ 

day) 

Constant variance 

Exponential (model 2)d 0.01 0.79 0.26 -0.42 0.09 1.24 89.65 4.60 3.04 

Exponential (model 3)d 0.01 0.79 0.26 -0.42 0.09 1.24 89.65 4.60 3.04 

Exponential 

(model 4)d,e 0.01 0.79 0.14 0.21 -0.01 -1.10 91.12 2.19 0.67 

Exponential (model 5)d 0.01 0.79 0.14 0.21 -0.01 -1.10 91.12 2.19 0.67 

Hilld NA NA NA NA NA NA NA NA NA 

Linearf 0.01 0.79 0.25 -0.45 0.09 1.26 89.71 4.86 3.32 

Polynomial (2-degree)f 0.01 0.79 0.25 -0.45 0.09 1.26 89.71 4.86 3.32 

Polynomial (3-degree)f 0.01 0.79 0.25 -0.45 0.09 1.26 89.71 4.86 3.32 

Powerd 0.01 0.79 0.25 -0.45 0.09 1.26 89.71 4.86 3.32 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the BMD; also the largest residual at any dose.

dPower restricted to ≥1. 

eSelected model. Constant variance model provided adequate fit to variance data. With constant variance model 

applied, all models, except for the Hill model (computation failed), provided adequate fit to means. BMDLs for 

models providing adequate fit were not considered to be sufficiently close (differed by >2–3-fold), so the model with 

the lowest BMDL was selected (the Exponential 5 model converged on to the Exponential 4). 

f

Coefficients restricted to be negative. 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated 

with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote 

benchmark response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom; 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed); SD = standard deviation 
 

 

 

 

 

DINITROTOLUENES A-18 

APPENDIX A 

Figure A-3. Fit of the Exponential 4 Model to Data on 2,4-DNT for
 

Decreased Hematocrit (%) in Female Dogs Following 9 Months
 

of Exposure (U.S. Army 1979)

Exponential Model 4 with 0.95 Confidence Level 

50

 48

 46

 44

 42

 40

 38

 36

 34

 32

Mean Response 

BMDL BMD

Exponential 

0 2 4 6 8 10 

dose 

08:07 04/12 2012 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: The hematological 

effects observed in this study are consistent with well-characterized effects observed after exposure to 

aromatic amines and with effects observed at higher doses in other studies of intermediate duration (Hong 

et al. 1985; Lee et al. 1985; Kozuka et al. 1979; U.S. Army 1978b). Similar hematological effects 

(anemia, accompanied by the presence of Heinz bodies) were observed in beagle dogs treated at 

25 mg/kg/day (but not 5 mg/kg/day) for up to 13 weeks (Ellis et al. 1985; U.S. Army 1978b). Dogs 
 

 

 

 

 

 

DINITROTOLUENES A-19 

APPENDIX A 

appear to be the most sensitive species. In Wistar rats, methemoglobin was increased substantially after 

treatment with 2,4-DNT at 347 mg/kg/day for 6 months (Kozuka et al. 1979). Milder hematological 

effects (mild reticulocytosis and hemosiderosis of the spleen) were also observed in CD rats treated at 

93 or 108 mg/kg/day (for males or females, respectively) for up to 13 weeks (Lee et al. 1985; U.S. Army 

1978b). Mice treated with 2,4-DNT for up to 13 weeks showed evidence of hematological effects (mild 

anemia, characterized by increased reticulocytes and decreased hematocrit and hemoglobin) only at the 

highest tested dose (413 mg/kg/day for males or 468 mg/kg/day for females) (Ellis et al. 1985; U.S. Army 

1978b). Hematological effects were also observed in 2-year studies in beagle dogs, CD rats, and CD-1 

mice, with dogs being the most sensitive species. Female dogs treated with 2,4-DNT at 1.5 mg/kg/day 

showed decreased erythrocyte count, hematocrit, and hemoglobin after 12 months; similar effects were 

observed in dogs of both sexes at 10 mg/kg/day (U.S. Army 1979). Anemia occurred at higher doses in 

2-year studies in rats (≥3.9 mg/kg/day) and mice (898 mg/kg/day). 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-20 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,4-DNT 

CAS Numbers: 121-14-2 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [ ] Acute [ ] Intermediate [X] Chronic 

Graph Key: 57 

Species: Dog 

Minimal Risk Level: 0.001 [X] mg/kg/day [ ] ppm 

References: U.S. Army. 1979. Mammalian toxicity of munitions compounds. Phase III: Effects of 

lifetime exposure. Part I. 2,4-Dinitrotoluene. Final report no. 7. Fort Detrick, MD: U.S. Army and 

Medical Bioengineering Research Development Laboratory. ADA077692. 

Ellis HV, Hong CB, Lee CC, et al. 1985. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. 

Part I. Beagle dog. J Am Coll Toxicol 4:233-242. 

Experimental design: Young beagle dogs (6 dogs/sex/group; age not specified) were administered 0, 0.2, 

1.5, or 10 mg/kg/day 2,4-DNT in capsules for 24 months. Dogs were observed daily for behavioral 

changes and clinical signs of toxicity. Body weights were recorded weekly. Feed consumption was 

measured during 1 week each month starting in month 6. Blood was taken before initiation of treatment 

and after 3, 6, 9, 12, 18, and 24 months of exposure for assessment of hematological parameters 

(erythrocyte, reticulocyte, platelet, and total and differential leukocyte counts; Heinz bodies; clotting time, 

hematocrit, hemoglobin, and methemoglobin concentrations; and mean cell volume, hemoglobin, and 

hemoglobin concentration) and clinical chemistry (fasting glucose, urea nitrogen, sodium, potassium, 

calcium, magnesium, chloride, and bilirubin [high-dose dogs with toxic signs], and the serum enzyme 

activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) analyses. 

Animals (one male and one female/group) were sacrificed after 12 or 24 months of continuous treatment; 

an additional dog/sex/group was discontinued from treatment at these time points and were sacrificed 

after a 4-week recovery period to evaluate the reversibility of effects (including clinical signs, hematology 

and clinical chemistry, organ weights, and histopathological analyses). Animals that were moribund 

during the study and those that survived to study termination were sacrificed and examined for gross 

lesions; major organs and tissues (including the brain, heart, liver, spleen, kidneys, adrenals, thyroids, 

pituitary, and gonads) were weighed, and comprehensive histopathological analyses (35 tissues) were 

performed. 

Effects noted in study and corresponding doses: Chronic-duration oral exposure of dogs to 2,4-DNT at 

≥1.5 mg/kg/day produced anemia and compensatory hematopoiesis (Ellis et al. 1985; U.S. Army 1979). 

Hematological effects of 2,4-DNT are initiated by methemoglobin production, which occurs when the 

ferrous iron in complex with the heme groups of hemoglobin is oxidized to ferric iron. Ferric iron does 

not bind oxygen, resulting in anemia. Ferric iron also contributes to the denaturation of hemoglobin and 

subsequent removal of erythrocytes from the blood. Heinz bodies (granules of denatured hemoglobin) are 

also detected within erythrocytes. Increased hematopoiesis is typically observed as a compensatory 

response to decreased erythrocyte count. 

Hematological effects consistent with the development of methemoglobin-induced anemia and 

compensatory hematopoiesis were observed after dosing for 12 months in dogs administered 1.5 and 

10 mg/kg/day (Table A-6). Female dogs administered 2,4-DNT at 1.5 mg/kg/day for 12 months showed 
 

 

 

 

 

 

 

DINITROTOLUENES A-21 

APPENDIX A 

statistically significant reductions in erythrocyte count, hematocrit, and hemoglobin concentration after 

treatment. At 10 mg/kg/day, more pronounced changes in these hematological parameters were observed, 

with statistically significant reductions in erythrocyte count, hematocrit, and hemoglobin and a 

statistically significant increase in reticulocyte count. In the low-dose group, similar hematological 

effects (decreased erythrocyte count and decreased hematocrit) were observed in female dogs, but these 

changes were not statistically significant. Effects on hematological parameters in male dogs were similar 

to those seen in female dogs, although many changes (with the exception of reticulocytes) did not reach 

statistical significance in the 10 mg/kg/day group, possibly due to low numbers of male dogs evaluated 

(hematological data available for only two males in the 10 mg/kg/day group). After treatment for 18 or 

24 months in both males and females, only slight or no anemia, near normal reticulocyte levels, no Heinz 

bodies, and minimal amounts of methemoglobin were detected, likely reflective of an adaptive response. 

Therefore, only data from the 12-month evaluation were considered for derivation of the chronic-duration 

oral MRL. 

No clinical signs of toxicity, behavioral changes, or effects on hematological or clinical chemistry 

parameters were observed in dogs administered 2,4-DNT at 0.2 mg/kg/day. Four high-dose dogs (three 

males and one female) exhibited severe signs of neurotoxicity (characterized by decreased muscle control 

and incoordination), sometimes accompanied by a reduction in body weight. These effects contributed to 

the death of three of six high-dose dogs (all males) prior to study termination (study weeks 8–20). 

Clinical signs of neurotoxicity were also noted intermittently in one male dog administered 2,4-DNT at 

1.5 mg/kg/day. Although biliary hyperplasia was noted at necropsy in male and female dogs 

administered 2,4-DNT, the frequency of the response did not exhibit dose-dependence. 
 

 

 

 

 

 

DINITROTOLUENES A-22 

APPENDIX A 

Table A-6. Hematological Effects in Beagle Dogs Exposed to 2,4-DNT for 

12 Months 

Dose (mg/kg/day) 

End point 0 0.2 1.5 10 

Males 

Erythrocyte count 

(x10/mm) 

5.96±0.22 (6)a 5.33±0.16 (6) 

[↓11] 

5.69±0.19 (6) 

[↓5] 

5.22±0.19 (2) 

[↓12] 

Heinz bodies (%) 0.0±0.0(6) 0.0±0.0(6) 

[NA] 

0.0±0.0(6) 

[NA] 

0.52±0.37 (2)b 

[NA] 

Reticulocytes (%) 0.40±0.09 (6) 0.78±0.13 (6) 

[↑95] 

0.66±0.11 (6) 

[↑65] 

1.23±0.23 (2)b 

[↑208] 

Hematocrit (%) 45.2±0.9 (6) 41.8±1.2 (6) 

[↓8] 

44.7±0.8 (6) 

[↓1] 

44.0±4.0 (2) 

[↓3] 

Hemoglobin (%) 15.1±0.4 (6) 14.1±0.3 (6) 

[↓7] 

14.8±0.4 (6) 

[↓2] 

14.2±1.2 (2) 

[↓6] 

Methemoglobin (%) 0.0±0.0 (6) 0.0±0.0 (6) 

[NA] 

0.0±0.0 (6) 

[NA] 

0.0±0.0 (2) 

[NA] 

Females 

Erythrocyte count 

(x10/mm) 

5.87±0.20 (6)a 5.54±0.14 (6) 

[↓6] 

4.69±0.25 (6)b 

[↓20] 

4.45±0.26 (6)b 

[↓24] 

Heinz bodies (%) 0.0±0.0(6) 0.0±0.0(6) 

[NA] 

0.0±0.0(6) 

[NA] 

0.5±0.2 (6)b 

[NA] 

Reticulocytes (%) 0.39±0.04 (6) 0.84±0.07 (6)b 

[↑115] 

0.45±0.06 (6) 

[↑15] 

1.59±0.18 (6)b 

[↑308] 

Hematocrit (%) 45.8±1.1 (6) 44.7±1.3 (6) 

[↓2] 

41.8±1.2 (6)b 

[↓9] 

40.8±0.6 (6)b 

[↓11] 

Hemoglobin (%) 15.1±0.4 (6) 15.1±0.4 (6) 

[0] 

13.7±0.8 (6)b 

[↓9] 

12.9±0.2 (6)b 

[↓15] 

Methemoglobin (%) 0.0±0.0 (6) 0.6±0.6 (6) 

[NA] 

0.0±0.0 (6) 

[NA] 

0.0±0.0 (6) 

[NA] 

aValues are means±standard error (number of animals) [percent change from controls].
 bStatistically significant based on analyses performed by the study authors (Dunnett’s multiple comparison
 

procedure).
 

DNT = dinitrotoluene; NA = not applicable 

Sources: Ellis et al. 1985; U.S. Army 1979 

Dose and end point used for MRL derivation: 

[ ] NOAEL [ ] LOAEL [X] BMDL 0.12 mg/kg was the BMDL1SD for hematological effects (decreased 

erythrocyte count). 

Results of hematology assessments show that chronic-duration, oral exposure of dogs to 2,4-DNT 

induced anemia and compensatory hematopoiesis (Ellis et al. 1985; U.S. Army 1979). Changes in 

several hematological parameters, including decreased erythrocyte count, hematocrit, and hemoglobin 

were observed after treatment with 2,4-DNT at 1.5 mg/kg/day for 12 months (Table A-6). Hematological 

effects were selected as the critical effect rather than neurotoxicity, which was observed only 

intermittently in one of six dogs exposed to 1.5 mg/kg/day. Hematological data are expressed as group 

means; therefore, these data are considered more robust than observations of intermittent neurotoxicity in 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-23 

APPENDIX A 

a single animal. To determine the POD for derivation of the chronic-duration oral MRL for 2,4-DNT, 

hematological data from female dogs treated with 2,4-DNT for 12 months were further evaluated by 

BMD analysis. The following data sets in female dogs were selected for BMD modeling: erythrocyte 

count, hematocrit, and hemoglobin. Hematological data from male dogs were not considered for 

additional BMD analyses due to the low number of dogs evaluated in the 10 mg/kg/day group (data were 

available for only two dogs). All available continuous-variable models in the EPA BMDS (version 2.1) 

were fit to the data. The BMD and the 95% lower confidence limit (BMDL) were estimated for doses 

associated with a change of 1 standard deviation from the controls, and are in units of mg/kg/day. For 

continuous data, in the absence of a clear criteria as to what level of change should be considered adverse, 

the BMR is defined as a change equal to 1 standard deviation from the control mean (EPA 2000a). 

Adequate model fit is judged by three criteria: goodness-of-fit (p>0.1), visual inspection of the doseresponse curve, and scaled residual at the data point (except the control) closest to the predefined BMR. 

Among all of the models providing adequate fit to the data, the lowest benchmark dose (BMDL, the lower 

limit of a one-sided 95% CI on the BMD) is selected as the POD when differences between the BMDLs 

estimated from these models are >3-fold; otherwise, the BMDL from the model with the lowest AIC is 

chosen. 

Neither the constant nor the non-constant variance model provided an adequate fit to the data for 

decreased hemoglobin; therefore, these data were not considered suitable for BMD modeling. BMD 

model prediction for hematocrit and erythrocyte count are shown in Tables A-7 and A-8, respectively. Of 

models meeting adequate fit criteria, the lowest BMDL1SD values for each hematological end point were 

0.13 mg/kg/day for decreased hematocrit (exponential 4 model; Figure A-4) and 0.12 mg/kg/day for 

decreased erythrocyte count (exponential 4 model; Figure A-5). Of these, the lowest BMDL1SD of 0.12 

mg/kg/day for decreased erythrocyte count was selected as the POD for derivation of the intermediateduration oral MRL for 2,4-DNT. This value was divided by an uncertainty factor of 100 (10 for animals 

to human extrapolation and 10 for human variability), resulting in a chronic-duration oral MRL of 0.001 

mg/kg/day. 
 

 

DINITROTOLUENES A-24 

APPENDIX A 

Table A-7. Model Predictions for 2,4-DNT for Decreased Hematocrit (%) in Female 

Dogs Following 12 Months of Exposure (U.S. Army 1979)
 

Test for Scaled residualsc 

significant Dose Dose BMD1SD BMDL1SD 

Model 

difference 

p-valuea 

Variance 

p-valueb 

Means 

p-valueb 

below 

BMD 

above 

BMD 

Overall 

largest AIC 

(mg/kg/ 

day) 

(mg/kg/ 

day) 

Constant variance 

Exponential (model 2)d 0.01 0.34 0.06 -1.82 0.29 -1.82 78.08 6.77 4.09 

Exponential (model 3)d 0.01 0.34 0.06 -1.82 0.29 -1.82 78.08 6.77 4.09 

Exponential 

(model 4)d,e 

0.01 0.34 0.92 -0.07 0.04 -0.07 74.50 0.61 0.13 

Exponential (model 5)d 0.01 0.34 0.92 -0.07 0.04 -0.07 74.50 0.61 0.13 

Hilld NA NA NA NA NA NA NA NA NA 

Linearf 0.01 0.34 0.06 -1.83 0.27 -1.83 78.15 6.96 4.31 

Polynomial (2-degree)f 0.01 0.34 0.06 -1.83 0.27 -1.83 78.15 6.96 4.31 

Polynomial (3-degree)f 0.01 0.34 0.06 -1.83 0.27 -1.83 78.15 6.96 4.31 

Powerd 0.01 0.34 0.06 -1.83 0.27 -1.83 78.15 6.96 4.31 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the BMD; also the largest residual at any dose.
 dPower restricted to ≥1. 

eSelected model. Constant variance model provided adequate fit to variance data. With constant variance model
 

applied, the only models that provided adequate fit to the means were the Exponential 4 and 5 models (the Exponential
 

5 converged on to the Exponential 4).
 f

Coefficients restricted to be negative.
 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated with 

the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark
 

response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom;
 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed); SD = standard deviation
 
 

 

DINITROTOLUENES A-25 

APPENDIX A 

Figure A-4. Fit of Exponential 4 Model to Data on 2,4-DNT for Decreased
 

Hematocrit (%) in Female Dogs Following 12 Months of Exposure
 

(U.S. Army 1979)

Exponential Model 4 with 0.95 Confidence Level 

38

 40

 42

 44

 46

 48

Mean Response 

BMDL BMD

Exponential 

0 2 4 6 8 10
 

dose
 

15:12 04/12 2012
 
 

 

DINITROTOLUENES A-26 

APPENDIX A 

Table A-8. Model Predictions for Decreased Erythrocyte Count in Dogs Treated with 

2,4-DNT for 12 Months 

Test for Scaled residualsc 

significant Dose Dose BMD1SD BMDL1SD 

difference Variance Means below above Overall (mg/kg/ (mg/kg/ 

Model p-valuea p-valueb p-valueb BMD BMD largest AIC day) day) 

Constant variance 

Exponential (model 2)d 0.0005 0.46 0.005 -2.40 0.43 -2.40 6.16 5.11 3.21 

Exponential (model 3)d 0.0005 0.46 0.005 -2.40 0.43 -2.40 6.16 5.11 3.21 

Exponential 0.0005 0.46 0.91 -0.08 0.05 -0.08 -2.55 0.35 0.12 

(model 4)d,e 

Exponential (model 5)d 0.0005 0.46 0.91 -0.08 0.05 -0.08 -2.55 0.35 0.12 

Hilld NA NA NA NA NA NA NA NA NA 

Linearf 0.0005 0.46 0.004 -2.41 0.35 -2.41 6.47 5.58 3.69 

Polynomial (2-degree)f 0.0005 0.46 0.004 -2.41 0.35 -2.41 6.47 5.58 3.69 

Polynomial (3-degree)f 0.0005 0.46 0.004 -2.41 0.35 -2.41 6.47 5.58 3.69 

Powerd 0.0005 0.46 0.004 -2.41 0.35 -2.41 6.47 5.58 3.69 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the BMD; also the largest residual at any dose.
 dPower restricted to ≥1. 

eSelected model. Constant variance model provided adequate fit to variance data. With constant variance model
 

applied, the only models that provided adequate fit to the means were the Exponential 4 and 5 models (the Exponential
 

5 converged on to the Exponential 4).
 f

Coefficients restricted to be negative.
 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated with 

the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark
 

response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom;
 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed); SD = standard deviation
 
 

 

 

DINITROTOLUENES A-27 

APPENDIX A 

Figure A-5. Fit of Exponential Model 4 to Data on Decreased Erythrocyte Count in 

Female Dogs Treated with 2,4-DNT for 12 Months

Exponential Model 4 with 0.95 Confidence Level 

6.5

 6

 5.5

 5

 4.5

 4

Mean Response 

BMDL BMD

Exponential 

0 2 4 6 8 10 

dose 

12:56 04/11 2012 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: The hematological 

effects observed in this study are consistent with effects observed at higher doses in studies of 

intermediate duration (Hong et al. 1985; Lee et al. 1985; Kozuka et al. 1979; U.S. Army 1978b). Dogs 

were the most sensitive species in studies of chronic duration. In a 2-year study, male CD rats 

administered 2,4-DNT at 3.9 mg/kg/day showed decreased erythrocyte count after treatment for 

12 months. Additional evidence for anemia, including further reductions in red blood cell count, 
DINITROTOLUENES A-28 

APPENDIX A 

decreased hematocrit, decreased hemoglobin, and a compensatory increase in reticulocytes, was observed 

in male and female rats administered high-dose 2,4-DNT (34 and 45 mg/kg/day for males and females, 

respectively) for 12 or 18 months (Lee et al. 1985; U.S. Army 1979). CD-1 mice administered 2,4-DNT 

for 2 years showed no evidence of methemoglobin-induced anemia or compensatory reticulocytosis, 

except for decreased erythrocyte count and hemoglobin, and increased numbers of reticulocytes at the 

highest tested dose (898 mg/kg/day) (Lee et al. 1985; U.S. Army 1979). 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-29 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,5-DNT 

CAS Numbers: 619-15-8 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [X] Acute [ ] Intermediate [ ] Chronic 

Graph Key: 4 

Species: Rat 

Minimal Risk Level: 0.007 [X] mg/kg/day [ ] ppm 

References: Lent EM, Crouse CB, Quinn MJ, et al. 2012a. Comparison of the repeated dose toxicity of 

isomers of dinitrotoluene. Int J Toxicol 31(2):143-157. 

U.S. Army Public Health Command (USAPHC). 2011c. Toxicology Study No. 87-XE-08G0E-10, 

October 2011, Toxicology Portfolio. Fourteen-day oral toxicity and in vivo genotoxicity of 2,5­

dinitrotoluene in rats, January 2009-March 2010. USAPHC, Aberdeen Proving Ground, Maryland. 

Experimental design: In a comparative toxicity study, groups of six male Sprague-Dawley rats were 

given 2,5-DNT via gavage doses of 0, 10, 19, 39, 77, 154, or 308 mg/kg/day for 14 days. Both untreated 

and vehicle control groups were included. Evaluations during the study included twice daily observations 

for morbidity and clinical signs and measurement of body weight and food consumption on days 0, 1, 3, 

7, and 14. Prior to sacrifice at the end of exposure, blood was collected for evaluation of hematology 

(total and differential white blood cell counts; red blood cell count; hemoglobin, hematocrit, MCV, MCH, 

MCHC, red cell distribution width; platelet count; and mean platelet volume) and serum chemistry 

(albumin, ALP, ALT, AST, BUN, calcium, cholesterol, creatinine, glucose, globulin, LDH, total 

bilirubin, total protein, sodium, potassium, and chlorine) assessments. All animals received gross 

necropsy and organ weight determinations (brain, heart, kidney, epididymides, liver, spleen, and testes). 

Microscopic examination of organs with exposure-related changes in weight was performed for controls 

and any dose groups exhibiting changes in the corresponding organ weights. 

Effects noted in study and corresponding doses: One rat exposed to 308 mg/kg/day died prematurely; the 

timing of death was not reported. Clinical signs of toxicity consisting of cyanosis, squinting, prostrate 

posture, rapid or labored breathing, hunched posture, dark urine, red discharge around the nose, and 

discolored or soft feces were observed at ≥154 mg/kg/day. There were no differences from control in 

body weight or food consumption of exposed groups. Dose-related hematology changes were noted, 

including decreased red blood cells at ≥77 mg/kg/day; increased MCV and MCH at ≥154 mg/kg/day; and 

increased red blood cell distribution width (at 77 and 154 mg/kg/day, but not at 308 mg/kg/day). Total 

white blood cell count was increased at 308 mg/kg/day, resulting from a nonsignificant increase in 

lymphocytes; in addition, the percent of eosinophils was decreased at this dose. There were no clinically 

significant, dose-related changes in serum chemistry parameters. At gross necropsy, dark and enlarged 

spleens were observed in 6/6 rats in the 154 mg/kg/day group and in 4/5 surviving rats exposed to 

308 mg/kg/day. In addition, dark spleens were noted in 3/6 and 1/6 rats of the 77 and 39 mg/kg/day dose 

groups. Absolute and relative organ weight increases occurred in the heart at the highest dose (~70% 

higher than controls). Marked, statistically significant increases (≥90%) in absolute and relative spleen 

weight were observed at doses ≥77 mg/kg/day. In addition, nonsignificant, but dose-related, increases of 

20% or more were seen at ≥19 mg/kg/day. No other organ weight changes were observed. Histopathology findings in the spleen consisted of mild-to-moderate extramedullary hematopoiesis in 3/6 rats 

exposed to 39 mg/kg/day and all rats exposed to higher doses; one vehicle control exhibited trace 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-30 

APPENDIX A 

extramedullary hematopoiesis. Table A-9 shows the splenic effects. The authors also reported mild-tomoderate fibrosis (4/5 rats given 308 mg/kg/day) and trace-to-moderate inflammation (2/5 rats given 308 

mg/kg/day) in the heart. Results of this study identify an acute-duration LOAEL of 39 mg/kg/day for 

hematopoietic system effects in rats. A NOAEL cannot be defined in the absence of spleen 

histopathology at lower doses, particularly in light of the spleen weight increases at lower doses. 

Table A-9. Splenic Effects in Rats Exposed to 2,5-DNT for 14 Days 

Dose (mg/kg/day) 

End point 0 10 19 39 77 154 308 

Mild-to-moderate 

extramedullary 

hematopoiesis 

(number exposed/ 

number examined) 

Absolute spleen 

weight (g) 

Spleen weight relative 

to body weight 

Spleen weight relative 

to brain weight 

0/6 

0.6533± 

0.07016 (6)a 

0.1851± 

0.01497 (6) 

39.4777± 

13.39556 

(6) 

ND 

0.7185± 

0.0.1107 (6) 

[↑10] 

0.2159± 

0.03228 (6) 

[↑17] 

45.1837± 

14.36118 

(6) [↑14] 

ND 

0.7733± 

0.010433 

(6) [↑18] 

0.2223± 

0.03484 (6) 

[↑20] 

39.8441± 

5.59536 (6) 

[↑1] 

3/6 

0.9203± 

0.12979 (6) 

[↑41] 

0.259± 

0.04213 (6) 

[↑40] 

46.0439± 

6.72756 (6) 

[↑17] 

5/5 

1.2428± 

016262b (6) 

[↑90] 

0.3591± 

0.03192b (6) 

[↑94] 

62.8775± 

8.01491b (6) 

[↑59] 

6/6 

0.1.8722± 

0.22994b (6) 

[↑187] 

0.546± 

0.0586b (6) 

[↑195] 

95.9326± 

14.5203b (6) 

[↑143] 

5/5 

2.2824± 

0.46711b (5) 

[↑249] 

0.6721± 

0.10433b (5) 

[↑263] 

122.6007± 

27.29763b 

(5) [↑211] 

aValues are means±standard deviation (number of animals) [percent change from controls].

bStatistically significant based on analyses performed by the study authors (Tukey’s or Dunnett’s multiple comparison test).
 

DNT = dinitrotoluene; ND = no data; histopathology examination was not performed for these animals 

Sources: Lent et al. 2012a; USAPHC 2011c 

Dose and end point used for MRL derivation: 

[] NOAEL [ ] LOAEL [X] BMDL 2.05 mg/kg/day was the BMDL10 for hematopoietic effects (mild-tomoderate extramedullary hematopoiesis). 

Hematopoietic system toxicity was identified as the most sensitive effect of acute-duration oral exposure 

to 2,5-DNT, based on the LOAEL value of 39 mg/kg/day in rats administered 2,5-DNT via gavage for 

14 days (Lent et al. 2012a; USAPHC 2011c). To determine the POD, the incidence of extramedullary 

hematopoiesis and the absolute and relative spleen weights (relative to body weight and relative to brain 

weight) were subjected to BMD modeling. For quantal data (incidence of extramedullary hematopoiesis), 

the BMD and the 95% lower confidence limit (BMDL) were estimated for doses associated with a 10% 

increase in incidence over the control incidence, and are in units of mg/kg/day. For continuous data 

(spleen weights), the BMD and the 95% lower confidence limit (BMDL) were estimated for doses 

associated with a 1 standard deviation increase over the control value, and are also in units of mg/kg/day. 

Adequate model fit is judged by three criteria: goodness-of-fit (p>0.1), visual inspection of the doseresponse curve, and scaled residual at the data point (except the control) closest to the predefined BMR. 

Among all of the models providing adequate fit to the data, the model with the lowest benchmark dose 

(BMDL, the lower limit of a one-sided 95% CI on the BMD) is selected when differences between the 

BMDLs estimated from these models are >3-fold; otherwise, the model with the lowest AIC is chosen. 
 

 

 

 

 

 

 

DINITROTOLUENES A-31 

APPENDIX A 

No model fit was achieved with the data on spleen weight relative to brain weight, so this end point was 

not considered further. BMD model predictions for incidence of extramedullary hematopoiesis and 

increases in absolute and relative spleen weights are shown in Tables A-10, A-11 and A-12, respectively. 

The BMDLs from the best-fitting models for each parameter were 2.05 mg/kg/day (BMDL10) for 

increased incidence of extramedullary hematopoiesis (multistage one-degree; Figure A-6); 7.71 

mg/kg/day (BMDL1SD) for increased absolute spleen weight (Exponential Model 5; Figure A-7) and 8.26 

mg/kg/day (BMDL1SD) for increased relative spleen weight (Exponential model 5; Figure A-8). The 

lowest BMDL of 2.05 mg/kg/day was selected as the POD. The POD of 2.05 mg/kg/day was divided by 

an uncertainty factor of 300 (10 for extrapolation from animals to humans, 10 for human variability, and 3 

for database limitations), resulting in an acute-duration oral MRL for 2,5-DNT of 0.007 mg/kg/day. 

Table A-10. Incidence of Mild-to-Moderate Extramedullary Hematopoiesis
 

in the Spleen of Male Sprague-Dawley Rats Administered 2,5-DNT via
 

Gavage for 14 Days
 

χ2 Scaled residualsb 

Goodness- Dose Dose 

of-fit below above Overall BMD10 BMDL10 

Model DF χ2 p-valuea BMD BMD largest AIC (mg/kg) (mg/kg) 

Gammac 4 0.00 1.00 0.00 -0.01 0.06 10.32 28.28 2.77 

Logistic 3 0.00 1.00 0.00 0.00 0.00 12.32 34.60 9.23 

LogLogisticd 4 0.00 1.00 0.00 0.00 0.00 10.32 34.52 7.43 

LogProbitd 3 0.00 1.00 0.00 0.00 0.00 12.32 33.45 6.14 

Multistage (1-degree)e,f 4 1.50 0.83 0.00 -0.90 -0.90 12.40 3.69 2.05 

Multistage (2-degree)e 4 0.32 0.99 0.00 -0.35 0.44 10.82 13.76 2.52 

Multistage (3-degree)e 4 0.02 1.00 0.00 -0.06 0.14 10.36 20.56 2.74 

Multistage (4-degree)e 3 0.01 1.00 0.00 -0.02 0.08 12.33 21.54 2.71 

Probit 3 0.00 1.00 0.00 0.00 0.00 12.32 30.40 8.31 

Weibullc 3 0.00 1.00 0.00 0.00 0.00 12.32 25.68 2.77 

aValues <0.1 fail to meet conventional goodness-of-fit criteria.
 bScaled residuals at doses immediately below and above the benchmark dose; also the largest residual at any dose.
 cPower restricted to ≥1. 

dSlope restricted to ≥1. 

eBetas restricted to ≥0. 

f

Selected model. All models provided adequate fit to the data. BMDLs for models providing adequate fit were not 

sufficiently close (differed by >3-fold), so the model with the lowest BMDL was selected (Multistage 1-degree). 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected 

benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 

10 = dose associated with 10% extra risk); DF= degrees of freedom; DNT = dinitrotoluene 
 

DINITROTOLUENES A-32 

APPENDIX A 

Figure A-6. Fit of Multistage (1-Degree) Model to Data on 2,5-DNT, Incidence of
 

Mild-to-Moderate Extramedullary Hematopoiesis in the Spleen of Rats 

Exposed for 14 Days (Lent et al. 2012a; USAPHC 2011c)

Multistage Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BM 

Fraction Affected 

1

 0.8

 0.6

 0.4

 0.2

 0

BMDL BMD

Multistage 

0 50 100 150 200 250 300 

dose 

13:07 12/23 2014 
 

 

 

 

 

 

DINITROTOLUENES A-33 

APPENDIX A 

Table A-11. Model Predictions for Increased Absolute Spleen Weight in Male
 

Sprague-Dawley Rats Administered 2,5-DNT via Gavage for 14 Days
 

Test for Scaled residualsc 

Model 

significant 

difference 

p-valuea 

Variance 

p-valueb 

Means 

p-valueb 

Dose 

below 

BMD AIC 

Dose 

above 

BMD 

Overall 

largest 

BMD1SD 

(mg/kg) 

BMDL1SD 

(mg/kg) 

All doses 

Constant variance 

Lineard <0.0001 <0.0001 0.005 -0.24 0.88 2.92 -72.15 41.35 34.02 

Non-constant variance 

Exponential 

(model 2)e 

<0.0001 0.87 <0.0001 -0.53 -0.14 -2.47 -87.77 18.74 13.56 

Exponential 

(model 3)e 

<0.0001 0.87 <0.0001 -0.53 -0.14 -2.47 -87.77 18.74 13.56 

Exponential 

(model 4)e 

<0.0001 0.87 0.51 0.62 -0.07 1.39 -108.51 8.50 6.15 

Exponential 

(model 5)e,f 

<0.0001 0.87 0.88 0.32 0.13 0.52 -109.15 15.01 7.78 

Hille <0.0001 0.87 0.78 0.36 0.16 0.72 -108.73 15.46 NA 

Lineard <0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 NA 

Polynomial 

(2-degree)d 

<0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 7.81 

Polynomial 

(3-degree)d 

<0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 7.81 

Polynomial 

(4-degree)d 

<0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 7.81 

Polynomial 

(5-degree)d 

<0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 7.81 

Polynomial 

(6-degree)d 

<0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 7.81 

Powere <0.0001 0.87 0.21 -0.15 -0.32 -1.90 -106.70 10.58 7.81 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the benchmark dose; also the largest residual at any dose. dCoefficients restricted to be positive.
 ePower restricted to ≥1
	 f

Selected model. Constant variance model did not fit the variance data, but non-constant variance model did. With
 

non-constant variance model applied, all models, except for the Exponential 2 and 3, the Hill and the Linear models,
 

provided adequate to the data (BMDL computation failed for the Hill and the Linear). BMDLs for models providing 

adequate fit were sufficiently close (differed by <2–3-fold), so the model with the lowest AIC was selected
 

(exponential model 5).
 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated 

with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote 

benchmark response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom;
 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose 

tested); SD = standard deviation
 
 

DINITROTOLUENES A-34 

APPENDIX A 

Figure A-7. Fit of Exponential Model 5 to Data on 2,5-DNT for Increased Absolute
 

Spleen Weight in Rats Exposed for 14 Days (Lent et al. 2012a; USAPHC 2011c)

Exponential Model 5, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Level for BMD 

0.5

 1

 1.5

 2

 2.5

 3

Mean Response 

BMDL BMD

Exponential 

0 50 100 150 200 250 300 

dose 

14:21 12/23 2014 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-35 

APPENDIX A 

Table A-12. Model Predictions for Model Predictions for Increased Relative 

Spleen Weight in Male Sprague-Dawley Rats Administered 2,5-DNT via
 

Gavage for 14 Days
 

Test for Scaled residualsc 

significant Dose Dose 

difference Variance Means below above Overall BMD1SD BMDL1SD 

Model p-valuea p-valueb p-valueb BMD BMD largest AIC (mg/kg) (mg/kg) 

All doses 

Constant variance 

Lineard <0.0001 0.0007 0.0005 -0.68 -0.55 3.28 -183.44 35.89 29.69 

Non-constant variance 

Exponential (model <0.0001 0.37 <0.0001 -0.25 0.38 -2.52 -190.27 19.37 14.06 

2)e 

Exponential (model <0.0001 0.37 <0.0001 -0.25 0.38 -2.52 -190.27 19.37 14.06 

3)e 

Exponential (model <0.0001 0.37 0.22 0.34 0.78 1.60 -213.48 8.74 6.32 

4)e 

Exponential (model 

5)e,f 

<0.0001 0.37 0.39 1.07 0.14 1.07 -214.23 17.64 8.26 

Hille <0.0001 0.37 0.29 1.11 0.18 -0.83 -213.46 18.31 NA 

Lineard <0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 NA 

Polynomial 

(2-degree)d 

<0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 8.13 

Polynomial 

(3-degree)d 

<0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 8.13 

Polynomial 

(4-degree)d 

<0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 8.13 

Polynomial 

(5-degree)d 

<0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 8.13 

Polynomial 

(6-degree)d 

<0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 8.13 

Powere <0.0001 0.37 0.06 0.66 -0.42 -1.98 -210.46 11.04 8.13 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the benchmark dose; also the largest residual at any dose.

dCoefficients restricted to be positive.
 ePower restricted to ≥1
	 f

Selected model. Constant variance model did not fit the variance data, but non-constant variance model did. With
 

non-constant variance model applied, the Exponential 4 and 5 models provided adequate to the data (BMDL 

computation failed for the Hill and the Linear models). BMDLs for models providing adequate fit were sufficiently
 

close (differed by <3-fold), so the model with the lowest AIC was selected (Exponential model 5).
 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated 

with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote 

benchmark response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom;
 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose 

tested); SD = standard deviation
 
 

 

 

 

 

 

 

 

DINITROTOLUENES A-36 

APPENDIX A 

Figure A-8. Fit of Exponential Model 5 to Data on 2,5-DNT for Increased Relative 

Spleen Weight in Rats Exposed for 14 Days (Lent et al. 2012a; USAPHC 2011c)

Exponential Model 5, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Level for BMD 

Mean Response 

0.8

 0.7

 0.6

 0.5

 0.4

 0.3

 0.2

BMDL BMD

Exponential 

0 50 100 150 200 250 300 

dose 

14:40 12/23 2014 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

[X] 3 for database limitations (the only other data available are from an acute lethality study) 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: LD50 values of 

710 and 1,230 mg/kg/day, respectively, were reported in male Sprague-Dawley rats and male CF-1 mice 

exposed to 2,5-DNT (Vernot et al. 1977); no additional information was provided. No other acuteduration oral studies of 2,5-DNT were located, nor were there any studies of longer durations. 
DINITROTOLUENES A-37 

APPENDIX A 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-38 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,6-DNT 

CAS Numbers: 606-20-2 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [X] Acute [ ] Intermediate [ ] Chronic 

Graph Key: 8 

Species: Dog 

Minimal Risk Level: 0.09 [X] mg/kg/day [ ] ppm 

References: U.S. Army. 1976. Mammalian toxicity of munitions compounds. Phase II: Effects of 

multiple doses. Part III: 2,6-Dinitrotoluene. Progress report no. 4. Fort Detrick, MD: U.S. Army, 

Medical Bioengineering Research and Development Laboratory. ADA062015. 

Experimental design: Young beagle dogs (4 dogs/sex/group; age not specified) were administered 0, 4, 

20, or 100 mg/kg 2,6-DNT in capsules for 13 weeks (U.S. Army 1976). Dogs were observed daily for 

behavioral changes and clinical signs of toxicity. Body weights were recorded weekly. Feed 

consumption was measured daily. Blood was taken before initiation of treatment and at 2, 4, 8, and 

13 and/or 17 weeks (4-week post-treatment recovery period) for hematological (erythrocyte, reticulocyte, 

platelet, and total and differential leukocyte counts; Heinz bodies; hematocrit, hemoglobin, and 

methemoglobin concentrations; and mean cell volume, hemoglobin, and hemoglobin concentration) and 

clinical chemistry (glucose, urea nitrogen, levels of sodium, potassium, calcium, magnesium, and 

chloride; and serum enzyme activities of aspartate aminotransferase, alanine aminotransferase, and 

alkaline phosphatase) analyses. No additional assessments were conducted for acute-duration exposure. 

Assessments conducted for intermediate-duration exposure (4–13 weeks and 4-week post-treatment 

recovery period) included hematology and clinical chemistry; gross pathological examination, organ 

weights (heart, liver, spleen, kidneys, adrenals, and gonads), and microscopic examination of tissues 

(“various” tissues, not specified) were assessed in animals dying before the end of treatment, and at the 

end of the 13-week treatment period and the 4-week post-treatment recovery period. Bone marrow and 

kidney cultures were also maintained and cytogenetic analyses (of chromosome number and morphology) 

were performed. 

Effects noted in study and corresponding doses: Acute-duration oral exposure of dogs to 2,6-DNT at 

≥20 mg/kg/day show the development of anemia and compensatory hematopoiesis (U.S. Army 1976) 

(data summarized in Table A-13). Hematological effects of 2,6-DNT are initiated by methemoglobin 

production, which occurs when the ferrous iron in complex with the heme groups of hemoglobin is 

oxidized to ferric iron. Ferric iron does not bind oxygen, resulting in anemia. Ferric iron also contributes 

to the denaturation of hemoglobin and subsequent removal of erythrocytes from the blood. Heinz bodies 

(granules of denatured hemoglobin) are also detected within erythrocytes. Increased hematopoiesis is 

typically observed as a compensatory response to decreased erythrocyte count. Since immature 

erythrocytes are typically larger, mean cell volume and mean cell hemoglobin tend to be increased. Dogs 

treated at 20 mg/kg/day showed a statistically significant decrease in erythrocyte count and a significant 

increase in mean cell hemoglobin. At 100 mg/kg/day, more pronounced changes in these hematological 

parameters were observed; dogs showed statistically significant reductions in erythrocyte count, 

hematocrit, and hemoglobin and a statistically significant increase in reticulocyte count. In the low-dose 

group, similar hematological effects (decreased erythrocyte count, hemoglobin, and hematocrit, and 

increased reticulocytes) were observed, but these changes did not achieve statistical significance. Midand high-dose dogs (but not low-dose dogs) continued to show signs of anemia and compensatory 
 

 

 

 

 

 

 

DINITROTOLUENES A-39 

APPENDIX A 

hematopoiesis (decreased hematocrit and hemoglobin, and increased numbers of reticulocytes) for the 

duration of the 13-week study. Dogs treated at 20 mg/kg/day for 4 or 13 weeks and then removed from 

treatment showed recovery from hematological effects after 4 weeks; dogs treated at 100 mg/kg/day for 

4 weeks did not show complete recovery until 19 weeks after cessation of treatment. 

Table A-13. Hematological effects in Beagle Dogs Exposed to 2,6-DNT for 

2 Weeks 

Dose (mg/kg/day) 

End point 0 4 20 100 

Erythrocyte count (x10/mm) 5.62±0.16 (8)a 5.06±0.10 (8) 

[↓10] 

4.73±0.20 (8)b 

[↓16] 

1.85±0.28 (7)b 

[↓67] 

Heinz bodies (%) 0.00±0.00 (8) 0.00±0.00 (8) 

[NA] 

0.00±0.00 (8) 

[NA] 

0.00±0.00 (7) [NA] 

Reticulocytes (%) 0.76±0.07 (8) 1.10±0.13 (8) 

[↑45] 

1.43±0.32 (8) 

[↑88] 

16.99±3.33 (7)b 

[↑2,136] 

Hematocrit (%) 42.1±1.7 (8) 38.9±0.6 (8) 

[↓8] 

39.3±1.2 (8) 

[↓7] 

22.9±2.8 (7)b 

[↓46] 

Hemoglobin (%) 14.8±0.5 (8) 13.3±0.2 (8) 

[↓10] 

13.0±0.4 (8) 

[↓12] 

6.3±0.9 (7)b 

[↓57] 

Methemoglobin (%) 0.0±0.0 (8) 0.0±0.0 (8) 

[NA] 

0.0±0.0 (8) 

[NA] 

0.0±0.0 (7) 

[NA] 

Mean cell hemoglobin (micro μg) 26.3±0.2 (8) 26.3±0.3 (8) 

[0] 

27.7±0.3 (8)b 

[↑5] 

34.4±1.2 (7)b 

[↑31] 

aValues are means±standard error (number of animals) [percent change from controls].

bStatistically significant based on analyses performed by the study authors (Dunnett’s multiple comparison
 

procedure).
 

DNT = dinitrotoluene; NA = not applicable 

Source: U.S. Army 1976 

Although incidence data were not reported, the study authors noted that at least three dogs (sex not 

specified) administered 2,6-DNT at 100 mg/kg/day showed clinical signs of toxicity (listlessness, 

incoordination, lack of balance, pale gums, dark urine, and weakness, particularly of the hind limbs) 

within the first 2 weeks of the study,. One dogs (a male) died during week 2. Similar (but milder) 

symptoms were reported in mid-dose dogs starting in week 4. No clinical signs of toxicity were observed 

in dogs administered the low dose of 2,6-DNT. 

Histopathological assessments of tissues were not conduced in animals exposed for only 2 weeks. 

However, after 13-week of treatment, mild splenic hematopoiesis was noted in low-dose animals. 

Numerous lesions were detected in mid- and high-dose dogs; the number and severity of these lesions was 

increased at the high-dose. Affected organs included the liver (bile duct hyperplasia, degeneration, 

inflammation, and/or extramedullary hematopoiesis), kidney (degeneration and inflammation, dilated 

tubules), spleen (extramedullary hematopoiesis and lymphoid depletion), and testes (degeneration and 

atrophy of spermatogenic cells. 

Dose and end point used for MRL derivation: 

[ ] NOAEL [ ] LOAEL [X] BMDL 9.31 mg/kg was the BMDL1SD for hematological effects (decreased 

erythrocyte count). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-40 

APPENDIX A 

Results of hematology assessments show that acute-duration, oral exposure of dogs to 2,6-DNT induced 

anemia and compensatory hematopoiesis (U.S. Army 1976). Statistically significant changes in 

hematological parameters, including decreased erythrocyte count and increased mean cell hemoglobin, 

were observed after treatment with 2,6-DNT at 20 mg/kg/day for 2 weeks (Table A-13). Changes to other 

hematological parameters only reached statistical significance at 100 mg/kg/day. Therefore, the most 

sensitive hematological parameters were erythrocyte count and mean cell hemoglobin. To determine the 

POD for derivation of the acute-duration oral MRL for 2,6-DNT, erythrocyte count and mean cell 

hemoglobin further evaluated by BMD analysis. All available continuous-variable models in the EPA 

BMDS (version 2.1) were fit to the data. The BMD and the 95% lower confidence limit (BMDL) were 

estimated for doses associated with a change of 1 standard deviation from the controls, and are in units of 

mg/kg/day. For continuous data, in the absence of a clear criteria as to what level of change should be 

considered adverse, the BMR is defined as a change equal to 1 standard deviation from the control mean 

(EPA 2000a). Adequate model fit is judged by three criteria: goodness-of-fit (p>0.1), visual inspection 

of the dose-response curve, and scaled residual at the data point (except the control) closest to the 

predefined BMR. Among all of the models providing adequate fit to the data, the lowest benchmark dose 

(BMDL, the lower limit of a one-sided 95% CI on the BMD) is selected as the POD when differences 

between the BMDLs estimated from these models are >3-fold; otherwise, the BMDL from the model with 

the lowest AIC is chosen. 

Neither the constant nor the non-constant variance model provided an adequate fit to the data for mean 

cell hemoglobin; therefore, these data were not considered suitable for BMD modeling. With nonconstant variance model applied, the linear, polynomial, and power models provided an adequate fit to the 

data for erythrocyte count (Table A-14). The polynomial and power models converged to the linear 

model. The figure shown from the linear model (Figure A-9) is representative of figures from the 

polynomial and power models (not shown). The BMDL1SD value of 9.31 mg/kg/day derived from this 

model was selected as the POD. This value was divided by an uncertainty factor of 100 (10 for animals 

to human extrapolation and 10 for human variability), resulting in an acute-duration oral MRL of 0.09 

mg/kg/day. 
 

 

DINITROTOLUENES A-41 

APPENDIX A 

Table A-14. Model Predictions for Decreased Erythrocyte Count in Dogs Treated 

with 2,6-DNT for 2 Weeks 

Scaled residualsc Test for 

significant Dose Dose BMD1SD BMDL1SD 

difference Variance Means below above Overall (mg/kg/ (mg/kg/ 

Model p-valuea p-valueb p-valueb BMD BMD largest AIC day) day) 

Constant variance 

Lineard <0.0001 0.08 0.25 -1.22 0.12 -1.22 -4.12 14.39 11.60 

Non Constant variance 

Exponential (model 2)e <0.0001 0.18 0.09 -1.55 1.26 -1.55 -3.38 8.15 6.00 

Exponential (model 3)e <0.0001 0.18 0.04 -1.60 0.64 -1.60 -1.90 11.15 6.17 

Exponential (model 4)e <0.0001 0.18 0.09 -1.55 1.26 -1.55 -3.38 8.15 5.84 

Exponential (model 5)e <0.0001 0.18 0.04 -1.60 0.64 -1.60 -1.90 11.15 6.07 

Hille NA NA NA NA NA NA NA NA NA 

Lineard,f <0.0001 0.18 0.16 -1.42 0.13 -1.42 -4.47 12.21 9.31 

Polynomial (2-degree)d <0.0001 0.18 0.16 -1.42 0.13 -1.42 -4.47 12.21 9.31 

Polynomial (3-degree)d <0.0001 0.18 0.16 -1.42 0.13 -1.42 -4.47 12.21 9.31 

Powere <0.0001 0.18 0.16 -1.42 0.13 -1.42 -4.47 12.21 9.31 

aValues >0.05 fail to meet conventional goodness-of-fit criteria. bValues <0.10 fail to meet conventional goodness-of-fit criteria.
 cScaled residuals at doses immediately below and above the BMD; also the largest residual at any dose.
 dCoefficients restricted to be negative.
 ePower restricted to ≥1.
	 f

Selected model. Constant variance model did not provide adequate fit to variance data, but non-homogenous variance 

model did. With non-constant variance model applied, all models, except for the Exponential (means <0.1) and Hill
 

models (computation failed), provided adequate fit to the means. The polynomial and power models all converged to 

the linear model.
 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the exposure concentration associated with 

the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark
 

response: i.e., 10 = exposure concentration associated with 10% extra risk); DF = degrees of freedom;
 

DNT = dinitrotoluene; NA = not applicable (BMDL computation failed); SD = standard deviation
 
 

 

DINITROTOLUENES A-42 

APPENDIX A 

Figure A-9. Fit of Linear Model to Data on Decreased Erythrocyte Count in Dogs
 

Treated with 2,6-DNT for 2 Weeks

Linear Model with 0.95 Confidence Level 

Mean Response 

6

 5

 4

 3

 2

 1

BMDL BMD

Linear 

0 20 40 60 80 100 

dose 

08:35 04/06 2012 

BMDs and BMDLs indicated are associated with a change of 1 standard deviation from the control, and are in units 

of mg/kg/day. 

Source: U.S. Army 1976 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable.
 

Was a conversion used from intermittent to continuous exposure? No.
 
 

 

 

 

 

 

 

 

DINITROTOLUENES A-43 

APPENDIX A 

Other additional studies or pertinent information which lend support to this MRL: Hematological effects 

consistent with methemoglobinemia-induced anemia and compensatory hematopoiesis have been 

observed in laboratory animals orally exposed to 2,6-DNT for acute and intermediate durations. No 

chronic-duration studies were identified that evaluated hematological effects after exposure to 2,6-DNT. 

Hematological effects (increased hemoglobin, hematocrit, and increased erythrocyte count, granulocyte, 

and reticulocyte counts) were observed in female Sprague-Dawley rats administered 2,6-DNT at 

199 mg/kg/day via gavage for 48 hours (Deng et al. 2011) and in male Sprague-Dawley rats exposed to 

134 mg/kg/day via gavage for 14 days (Lent et al. 2012a; USAPHC 2011d). In intermediate-duration 

studies, dogs appear to more sensitive than rats or mice. In dogs orally exposed to 2,6-DNT at 

4 mg/kg/day for 4 or 13 weeks, extramedullary erythropoiesis in the spleen secondary to methemoglobinemia and anemia was observed (U.S. Army 1976). Changes in hematological parameters associated with 

anemia and compensatory hematopoiesis (including decreased hematocrit and hemoglobin, and increased 

numbers of reticulocytes) occurred at 20 and 100 mg/kg/day. The incidence and severity of these effects 

were more pronounced at 100 mg/kg/day. Similar effects were observed in rats (U.S. Army 1976). In 

CD rats administered 2,6-DNT at ≥7 mg/kg/day and sacrificed after treatment for 4 or 13 weeks, 

increased incidences of extramedullary hematopoiesis and/or splenic hemosiderosis (increased iron 

accumulation) were observed. However, changes in hematological parameters (measured at 4, 8, and 

13 weeks) indicative of anemia and compensatory hematopoiesis (including significant decreases in 

erythrocyte count, hematocrit, hemoglobin, and increased reticulocytes) were observed only at the highest 

tested dose (145 and 155 mg/kg/day for male and female rats, respectively); these effects were most 

pronounced after treatment for 4 weeks. Although histopathological effects (extramedullary 

hematopoiesis) were observed in CD-1 mice administered 2,6-DNT at ≥51 mg/kg/day (but not 

11 mg/kg/day) for 4 or 13 weeks, no statistically significant changes in hematological parameters were 

seen. The study authors indicated that some blood samples clotted, making hematological analyses 

impossible to perform. The small number of animals evaluated likely contributed to the identification of 

histopathological findings of the spleen in the apparent absence of 2,6-DNT-induced hematological 

effects. The results of intermediate-duration studies indicate that hematological effects (or 

histopathological effects secondary to methemoglobinemia and anemia) are the most sensitive effects 

after exposure to 2,6-DNT; additional effects observed in intermediate-duration studies occurred at higher 

doses than hematological effects. 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-44 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 2,6-DNT 

CAS Numbers: 606-20-2 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [ ] Acute [X] Intermediate [ ] Chronic 

Graph Key: 18 

Species: Dog 

Minimal Risk Level: 0.004 [X] mg/kg/day [ ] ppm 

Reference: U.S. Army. 1976. Mammalian toxicity of munitions compounds. Phase II: Effects of 

multiple doses. Part III: 2,6-Dinitrotoluene. Progress report no. 4. Fort Detrick, MD: U.S. Army, 

Medical Bioengineering Research and Development Laboratory. ADA062015 

Experimental design: Young beagle dogs (4 dogs/sex/group; age not specified) were administered 0, 4, 

20, or 100 mg/kg 2,6-DNT in capsules for 13 weeks (U.S. Army 1976). Dogs were observed daily for 

behavioral changes and clinical signs of toxicity. Body weights were recorded weekly. Feed 

consumption was measured daily. Blood was taken before initiation of treatment and at 2, 4, 8, and 

13 and/or 17 weeks (4-week post-treatment recovery period) for hematological (erythrocyte, reticulocyte, 

platelet, and total and differential leukocyte counts; Heinz bodies; hematocrit, hemoglobin, and 

methemoglobin concentrations; and mean cell volume, hemoglobin, and hemoglobin concentration) and 

clinical chemistry (glucose, urea nitrogen, levels of sodium, potassium, calcium, magnesium, and 

chloride; and serum enzyme activities of aspartate aminotransferase, alanine aminotransferase, and 

alkaline phosphatase) analyses. Animals (1 sex/group) were sacrificed after 4 or 13 weeks of continuous 

treatment; an additional dog/sex/group was discontinued treatment after 4 or 13 weeks and sacrificed after 

4 weeks of recovery (week 8 or 17) to evaluate the reversibility of effects. The two high-dose dogs 

removed from treatment at 4 weeks were not sacrificed until 19 weeks after cessation of treatment to test 

the reversibility of effects after a longer recovery period. When animals were moribund or at study 

termination, they were examined for gross lesions; major organs and tissues were weighed (heart, liver, 

spleen, kidneys, adrenals, and gonads); and “various” tissues (number not specified) were subjected to 

histopathological examinations. Bone marrow and kidney cultures were also maintained and cytogenetic 

analyses (of chromosome number and morphology) were performed. 

Effects noted in study and corresponding doses: Intermediate-duration oral exposure of dogs to 2,6-DNT 

at 4 mg/kg/day produced extramedullary erythropoiesis (formation of erythrocytes outside of the bone 

marrow) in the spleen secondary to methemoglobinemia and anemia (U.S. Army 1976). Hematological 

effects and compensatory erythropoiesis induced by 2,6-DNT are initiated by methemoglobin production, 

which occurs when the ferrous iron in complex with the heme groups of hemoglobin is oxidized to ferric 

iron. Ferric iron does not bind oxygen, resulting in anemia. Ferric iron also contributes to the 

denaturation of hemoglobin and subsequent removal of erythrocytes from the blood. Increased 

erythropoiesis is typically observed as a compensatory response to decreased erythrocyte count. 

Mortality occurred in dogs administered 20 and 100 mg/kg/day. Two mid-dose female dogs died in 

week 9; all high-dogs died by week 8. Effects observed in dogs treated at 20 and 100 mg/kg/day were 

clinical signs of neurotoxicity (listlessness, incoordination, and lack of balance), decreased feed 

consumption and subsequent reductions in body weight, and changes in hematological parameters 

associated with anemia and compensatory hematopoiesis (including decreased hematocrit and 

hemoglobin, and increased numbers of reticulocytes). The incidence and severity of these effects were 
 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-45 

APPENDIX A 

more pronounced at 100 mg/kg/day relative to 20 mg/kg/day. Dogs administered 2,6-DNT at 

4 mg/kg/day showed no clinical signs of toxicity, and although similar hematological effects occurred, 

these changes were not statistically significant. No significant effects on clinical chemistry end points 

were observed in any 2,6-DNT treatment group. In general, dogs treated at 20 mg/kg/day for 4 or 

13 weeks and then removed from treatment showed recovery from neurotoxicity and hematological 

effects after 4 weeks; dogs treated at 100 mg/kg/day for 4 weeks did not show complete recovery until 

19 weeks after cessation of treatment. 

Histopathological evaluation of the spleen showed an increased incidence of extramedullary 

erythropoiesis, an adaptive response to 2,6-DNT-induced methemoglobinemia and anemia, in dogs 

administered ≥4 mg/kg/day for 4 or 13 weeks (Table A-15). The incidence and severity of this lesion was 

dose-related. Additional histopathological changes observed dogs administered 2,6-DNT at 20 or 

100 mg/kg/day for 4 or 13 weeks included effects on the thymus (involution), liver (extramedullary 

hematopoiesis, bile duct hyperplasia, degeneration, and inflammation), kidneys (degeneration, 

inflammation, and diluted tubules), and testes (degeneration and/or decreased spermatogenesis). Highdose dogs also showed evidence of lymphoid depletion in the spleen and lymph nodes. No other 

treatment-related histopathological changes were observed in dogs dosed with 2,6-DNT at 4 mg/kg/day 

for 13 weeks. 

Table A-15. Extramedullary Erythropoiesis of the Spleen in Beagle Dogs Exposed 

to 2,6-DNT for 4 or 13 Weeks 

Timepoint Dose (mg/kg/day) 

(weeks) 0 4 20 100 

4 0/2a 1/2 2/2 2/2 

(mild) (moderate) (1 mild; 1 markedly severe) 

13 0/2 2/2 3/3 4/4 

(1 minimal; 1 mild) (1 minimal, 1 mild, 1 moderate) (2 marked, 2 markedly severe) 

aNumber examined/number affected (severity of lesion). 

DNT = dinitrotoluene 

Source: U.S. Army 1976 

Dose and end point used for MRL derivation: 

[ ] NOAEL [X] LOAEL 4 mg/kg/day for mild extramedullary erythropoiesis in the spleen. 

The LOAEL value of 4 mg/kg/day for an increased incidence of extramedullary erythropoiesis in the 

spleens of dogs was identified as the POD for derivation of the intermediate-duration oral MRL for 

2,6-DNT (U.S. Army 1976). Histopathology data were not suitable for BMD modeling, since the number 

of animals evaluated at each dose and time was small (n=2 animals). Therefore, the LOAEL value for 

4 mg/kg/day was used at the POD. This value was divided by an uncertainty factor of 1,000 (10 for use 

of a LOAEL, 10 for extrapolation from animals to humans, and 10 for human variability), resulting in an 

acute-duration oral MRL for 2,6-DNT of 0.004 mg/kg/day. 

Uncertainty Factors used in MRL derivation: 

[X] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 
 

 

 

 

 

 

DINITROTOLUENES A-46 

APPENDIX A 

[X] 10 for human variability 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: Similar 

histopathological effects (extramedullary hematopoiesis and/or splenic hemosiderosis), indicative of an 

adaptive response to anemia and compensatory erythropoiesis, were observed in CD rats administered 

2,6-DNT at ≥7 mg/kg/day and in CD-1 mice administered 2,6-DNT at ≥51 mg/kg/day (but not 

11 mg/kg/day) for 4 or 13 weeks (U.S. Army 1976). Hematological effects were also identified in 

intermediate-duration studies. In these studies, dogs appear to be the most sensitive species. Dogs treated 

at 20 mg/kg/day showed a statistically significant decrease in erythrocyte count and a significant increase 

in mean cell hemoglobin at 2 weeks. At 100 mg/kg/day, more pronounced changes in these 

hematological parameters were observed; statistically significant reductions in erythrocyte count, 

hematocrit, and hemoglobin and a statistically significant increase in reticulocyte count were observed. In 

CD rats, changes in hematological parameters indicative of anemia (significant decreases in erythrocyte 

count, hematocrit, and hemoglobin) and compensatory hematopoiesis (increased reticulocytes) were 

observed only at the highest tested dose (145 and 155 mg/kg/day for male and female rats, respectively). 

Although histopathological effects (extramedullary hematopoiesis) was observed in CD-1 mice 

administered 2,6-DNT at 51 mg/kg/day (but not 11 mg/kg/day), no statistically significant changes in 

hematological parameters were seen after treatment for 4 or 13 weeks. The study authors indicated that 

some blood samples clotted, making hematological analyses impossible to perform. The small number of 

animals evaluated likely contributed to the identification of histopathological findings of the spleen in the 

apparent absence of 2,6-DNT-induced hematological effects. 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-47 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 3,4-DNT 

CAS Numbers: 610-39-9 

Date: February 2016 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [X] Acute [ ] Intermediate [ ] Chronic 

Graph Key: 1 

Species: Rat 

Minimal Risk Level: 0.03 [X] mg/kg/day [ ] ppm 

References: Lent EM, Crouse CB, Quinn MJ, et al. 2012a. Comparison of the repeated dose toxicity of 

isomers of dinitrotoluene. Int J Toxicol 31(2):143-157. 

U.S. Army Public Health Command (USAPHC). 2011e. Toxicology Study No. 87-XE-08G0B-08, October 2011, 

Toxicology Portfolio. Fourteen-day oral toxicity and in vivo genotoxicity of 3,4-dinitrotoluene in rats, June-July 

2008. USAPHC, Aberdeen Proving Ground, Maryland. 

Experimental design: In a comparative toxicity study, groups of six male Sprague-Dawley rats were 

given 3,4-DNT via gavage doses of 0, 7, 14, 28, 57, 113, or 227 mg/kg/day for 14 days. Both untreated 

and vehicle control groups were included. Evaluations during the study included twice daily observations 

for morbidity and clinical signs and measurement of body weight and food consumption on days 0, 1, 3, 

7, and 14. Prior to sacrifice at the end of exposure, blood was collected for evaluation of hematology 

(total and differential white blood cell counts; red blood cell count; hemoglobin, hematocrit, MCV, MCH, 

MCHC, red cell distribution width; platelet count; and mean platelet volume) and serum chemistry 

(albumin, ALP, ALT, AST, BUN, calcium, cholesterol, creatinine, glucose, globulin, LDH, total 

bilirubin, total protein, sodium, potassium, and chlorine) assessments. All animals received gross 

necropsy and organ weight determinations (brain, heart, kidney, epididymides, liver, spleen, and testes). 

Microscopic examination of organs with exposure-related changes in weight was performed for controls 

and any dose groups exhibiting changes in the corresponding organ weights. 

Effects noted in study and corresponding doses: All rats in the study of 3,4-DNT survived until 

scheduled sacrifice. Rats in the highest dose group (227 mg/kg/day) exhibited clinical signs of toxicity 

including cyanosis, lethargy, dark urine, facial twitching, and hypoactivity or staring behavior; one rat 

exposed to 113 mg/kg/day exhibited dark urine. High-dose rats had lower body weights than controls 

throughout the study, and a biologically significant (but not statistically significant) 10% reduction in 

terminal body weight was observed at this dose. Food intake was also decreased at this dose, albeit not at 

a level that reached statistical significance. Both hemoglobin and hematocrit were lower than controls at 

all doses of 3,4-DNT, with statistically significant reductions at doses of 14, 28, and 113 mg/kg/day. The 

authors indicated that both parameters remained within reference ranges at all doses. White blood cell 

counts were not affected by treatment, but the percent of neutrophils was increased and percent of 

lymphocytes was decreased at 14, 28, and 227 mg/kg/day; a clear dose-response relationship was not 

evident. Other hematology parameters were not affected by treatment, and none of the serum chemistry 

parameters exhibited a dose-related change. There were no gross necropsy findings of note. The only 

treatment-related organ weight change was an increase (≥13% compared with vehicle controls) in relative 

liver-to-body weight at the two highest doses (≥113 mg/kg/day); absolute liver weight was not affected, 

and there were no treatment-related histopathology findings in the liver. Relative liver weights may have 

been altered as a consequence of the lower body weights seen at the highest doses. Microscopic lesions 

were noted in the heart, spleen, and kidney. One rat in the highest dose group exhibited mild myocardial 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-48 

APPENDIX A 

fibrosis, inflammation, and necrosis. Splenic lesions, of trace-to mild severity, were observed at doses 

≥57 mg/kg/day, including extramedullary hematopoiesis (2/6, 2/5, and 3/6 rats at the three highest doses) 

and lymphoid hyperplasia (3/6, 2/5, 2/6 at 57, 113, and 227 mg/kg/day respectively) (see Table A-16). 

Spleens were not examined in lower dose animals. Microscopic lesions of the kidney occurred in the high 

dose group, and included trace-to-moderate proximal tubule degeneration (1/6 rats), renal tubular 

basophilia (4/6), and lymphocytic infiltration (5/6); kidneys from animals in lower dose groups were not 

examined. Results of this study identify an acute-duration LOAEL value for hematopoietic system 

effects of 57 mg/kg/day in rats. A NOAEL for hematopoietic system effects cannot be determined in the 

absence of histopathology examinations in lower dose groups. 

Table A-16. Splenic Lesions in Rats Exposed to 3,4-DNT for 14 Days 

Dose (mg/kg/day) 

End point 0 7 14 28 57 113 227 

Extramedullary hematopoiesis 0/6a ND ND ND 2/6 2/5 3/6 

Lymphoid hyperplasia 0/6 ND ND ND 3/6 2/5 2/6 

aNumber affected/number examined 

DNT = dinitrotoluene; ND = no data; histopathology examination was not performed for these animals. 

Sources: Lent et al. 2012a; USAPHC 2011e 

Dose and end point used for MRL derivation: 

[] NOAEL [ ] LOAEL [X] BMDL 8.05 mg/kg/day was the BMDL10 for hematopoietic effects (traceto-mild extramedullary hematopoiesis and lymphoid hyperplasia). 

Hematopoietic system toxicity was identified as the most sensitive effect of acute-duration oral exposure 

to 3,4-DNT, based on the LOAEL value of 57 mg/kg/day in rats administered 3,4-DNT via gavage for 

14 days (Lent et al. 2012a; USAPHC 2011e). To select a POD, the following data sets were subjected to 

BMD modeling: incidences of extramedullary hematopoiesis and lymphoid hyperplasia of the spleen. The 

BMD and the 95% lower confidence limit (BMDL) were estimated for doses associated with a 10% 

increase in incidence over the control incidence, and are in units of mg/kg/day. Adequate model fit is 

judged by three criteria: goodness-of-fit (p>0.1), visual inspection of the dose-response curve, and scaled 

residual at the data point (except the control) closest to the predefined BMR. Among all of the models 

providing adequate fit to the data, the model with the lowest benchmark dose (BMDL, the lower limit of a 

one-sided 95% CI on the BMD) is selected when differences between the BMDLs estimated from these 

models are >3-fold; otherwise, the model with the lowest AIC is chosen. 

BMD model predictions for incidences of extramedullary hematopoiesis and lymphoid hyperplasia of the 

spleen are shown in Tables A-17 and A-18, respectively. The BMDL10 values from the best-fitting 

models for were the same (8.05 mg/kg/day) for both increased incidence of extramedullary hematopoiesis 

(log-logistic; Figure A-10) and increased incidence of lymphoid hyperplasia of the spleen (log-logistic; 

Figure A-11). Thus, the BMDL10 of 8.05 mg/kg/day was selected as the POD. The POD of 

8.05 mg/kg/day was divided by an uncertainty factor of 300 (10 for extrapolation from animals to 

humans, 10 for human variability, and 3 for database limitations), resulting in an acute-duration oral MRL 

for 3,4-DNT of 0.03 mg/kg/day. 
 

 

DINITROTOLUENES A-49 

APPENDIX A 

Table A-17. Model Predictions for Incidence of Extramedullary
 

Hematopoiesis in the Spleen of Male Sprague-Dawley Rats
 

Administered 3,4-DNT via Gavage for 14 Days
 

χ2 Scaled residualsb 

Goodness- Dose Dose 

of-fit below above Overall BMD10 BMDL10 

Model DF χ2 p-valuea BMD BMD largest AIC (mg/kg) (mg/kg) 

Gammac 3 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Logistic 2 1.80 0.41 0.76 0.44 -0.96 29.20 64.08 37.29 

LogLogisticd,e 3 0.34 0.95 0.00 0.44 0.44 25.01 18.56 8.05 

LogProbitd 3 1.86 0.60 0.00 1.02 1.02 26.32 40.34 24.07 

Multistage (1-degree)f 3 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Multistage (2-degree)f 3 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Multistage (3-degree)f 3 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Probit 2 1.77 0.41 0.79 0.43 -0.91 29.10 60.21 36.19 

Weibullc 3 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

aValues <0.1 fail to meet conventional goodness-of-fit criteria. bScaled residuals at doses immediately below and above the benchmark dose; also the largest residual at any dose.
 cPower restricted to ≥1. 

dSlope restricted to ≥1. 

eSelected model. All models provided adequate fit to the data. BMDLs for models providing adequate fit were not 

sufficiently close (differed by >3-fold), so the model with the lowest BMDL was selected (LogLogistic; also has the 

lowest AIC). 

f

Betas restricted to ≥0. 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected 

benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: 

i.e., 10 = dose associated with 10% extra risk); DF = degrees of freedom; DNT = dinitrotoluene 
 

 

DINITROTOLUENES A-50 

APPENDIX A 

Figure A-10. Fit of Log-logistic Model to Data on Incidence of Extramedullary
 

Hematopoiesis in Rats Treated with 3,4-DNT for 14 Days

Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B 

0

 0.2

 0.4

 0.6

 0.8

Fraction Affected 

BMDL BMD 

Log-Logistic 

0 50 100 150 200 

dose 

13:22 12/23 2014 
 

DINITROTOLUENES A-51 

APPENDIX A 

Table A-18. Model Predictions for Incidence of Lymphoid Hyperplasia in 

the Spleen of Male Sprague-Dawley Rats Administered 3,4-DNT via 

Gavage for 14 Days 

χ2 Scaled residualsb 

Goodness- Dose Dose 

of-fit below above Overall BMD10 BMDL10 

Model DF χ2 p-valuea BMD BMD largest AIC (mg/kg) (mg/kg) 

Gammac 2 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Logistic 2 1.80 0.41 0.76 0.44 -0.96 29.20 64.08 37.29 

LogLogisticd,e 3 0.34 0.95 0.00 0.44 0.44 25.01 18.56 8.05 

LogProbitd 2 1.86 0.60 0.00 1.02 1.02 26.32 40.34 24.07 

Multistage (1-degree)f 2 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Multistage (2-degree)f 2 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Multistage (3-degree)f 2 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

Probit 2 1.77 0.41 0.79 0.43 -0.91 29.10 60.21 36.19 

Weibullc 2 0.86 0.83 0.00 0.74 0.74 25.48 25.51 14.32 

aValues <0.1 fail to meet conventional goodness-of-fit criteria. bScaled residuals at doses immediately below and above the benchmark dose; also the largest residual at any dose.
 cPower restricted to ≥1. 

dSlope restricted to ≥1. 

eSelected model. All models provided adequate fit to the data. BMDLs for models providing adequate fit were not 

sufficiently close (differed by >3-fold), so the model with the lowest BMDL was selected (LogLogistic). f

Betas restricted to ≥0. 

AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected 

benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: 

i.e., 10 = dose associated with 10% extra risk); DF = degrees of freedom; DNT = dinitrotoluene 
 

 

 

 

DINITROTOLUENES A-52 

APPENDIX A 

Figure A-11. Fit of Log-logistic Model to Data on Incidence of Lymphoid 

Hyperplasia in Rats Treated with 3,4-DNT for 14 Days

Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B 

Fraction Affected 

0.8

 0.6

 0.4

 0.2

 0

BMDL BMD 

Log-Logistic 

0 50 100 150 200 

dose 

13:35 12/23 2014 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

[X] 3 for database limitations (no other toxicological studies of this isomer are available) 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable.
 

Was a conversion used from intermittent to continuous exposure? No.
 

Other additional studies or pertinent information which lend support to this MRL: No other acuteduration oral studies of 3,4-DNT were located, nor were there any studies of longer durations. 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-53 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

Chemical Name: 3,5-DNT 

CAS Numbers: 618-85-9 

Date: February 2015 

Profile Status: Final 

Route: [ ] Inhalation [X] Oral 

Duration: [X] Acute [ ] Intermediate [ ] Chronic 

Graph Key: 4 

Species: Rat 

Minimal Risk Level: 0.03 [X] mg/kg/day [ ] ppm 

References: Lent EM, Crouse CB, Quinn MJ, et al. 2012a. Comparison of the repeated dose toxicity of 

isomers of dinitrotoluene Int J Toxicol 31(2):143-157. 

U.S. Army Public Health Command (USAPHC). 2011f. Toxicology Study No. 87-XE-08G0F-10, 

October 2011, Toxicology Portfolio. Fourteen-day oral toxicity and in vivo genotoxicity of 

3,5-dinitrotoluene in rats, January 2009-March 2010. USAPHC, Aberdeen Proving Ground, Maryland. 

Experimental design: In a comparative toxicity study, groups of six male Sprague-Dawley rats were 

given 3,5-DNT via gavage doses of 0, 5, 10, 19, 39, 77, or 155 mg/kg/day for 14 days. Both untreated 

and vehicle control groups were included. Evaluations during the study included twice daily observations 

for morbidity and clinical signs and measurement of body weight and food consumption on days 0, 1, 3, 

7, and 14. Prior to sacrifice at the end of exposure, blood was collected for evaluation of hematology 

(total and differential red blood cell counts; red blood cell count; hemoglobin, hematocrit, MCV, MCH, 

MCHC, red cell distribution width; platelet count; and mean platelet volume) and serum chemistry 

(albumin, ALP, ALT, AST, BUN, calcium, cholesterol, creatinine, glucose, globulin, LDH, total 

bilirubin, total protein, sodium, potassium, and chlorine) assessments. All animals received gross 

necropsy and organ weight determinations (brain, heart, kidney, epididymides, liver, spleen, and testes). 

Microscopic examination of organs with exposure-related changes in weight was performed for controls 

and any dose groups exhibiting changes in the corresponding organ weights. 

Effects noted in study and corresponding doses: All rats exposed to 77 and 155 mg/kg/day 3,5-DNT died 

or were sacrificed moribund within the first 8 days on study, as did one rat in the 39 mg/kg/day group. 

Clinical signs of toxicity were observed at the lethal doses; in addition, animals exposed to 

≥39 mg/kg/day were reported to exhibit neurological signs progressing from facial twitching to paralysis 

of the forelimbs. It is not clear whether these signs were seen only in the animals that were moribund or 

if some neurological signs were seen in animals that survived treatment. Mean terminal body weight was 

decreased by 13% at 39 mg/kg/day; treatment did not affect food intake. The only hematology or clinical 

chemistry changes possibly attributable to treatment were increased albumin, total protein, and total 

bilirubin at 39 mg/kg/day. Gross necropsy findings among animals surviving to termination included 

small testis size in three rats given 19 mg/kg/day and in five rats given 39 mg/kg/day. These findings 

were confirmed by organ weight measurements indicating significant reductions (≥40%) in absolute and 

relative testes weights at these doses. Brain weight relative to body weight was increased by 30% 

(relative to vehicle controls) in the 39 mg/kg/day group; no other organ weight changes were reported. 

Testicular histopathology changes were observed in all rats exposed to doses ≥19 mg/kg/day (see Table 

A-19); these included mild-to-severe tubular degeneration and trace-to-severe multinucleated giant cell 

formation. Trace lymphoid depletion was observed in the spleens of 1/6 rats in each of the groups 

exposed to 10 and 19 mg/kg/day; no lesions were seen at 39 mg/kg/day. Finally, mild or moderate 
 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES A-54 

APPENDIX A 

inflammatory infiltrates were observed in the brains of 3/6 rats exposed to 39 mg/kg/day. Results of this 

study identify acute-duration NOAEL and LOAEL values for testicular lesions of 10 and 19 mg/kg/day, 

respectively, in rats. 

Table A-19. Testes Effects in Rats Exposed to 3,5-DNT for 14 Days 

Dose (mg/kg/day) 

End point 0 5 10 19 39a 77 155 

Tubular degeneration 0/6b 0/6 0/6 6/6c 6/6c NA NA 

Multinucleated giant cell formation 0/6 0/6 0/6 6/6c 5/6c NA NA 

aIncludes one animal that died or was sacrificed moribund before the end of the study. bNumber affected/number examined.
 cStatistically significant at p<0.05 based on Fisher’s exact test performed for this review.
 

DNT = dinitrotoluene; NA = not applicable; all rats died or were sacrificed moribund at these doses 

Sources: Lent et al. 2012a; USAPHC 2011f 

Dose and end point used for MRL derivation: 

[X] NOAEL [ ] LOAEL [ ] BMDL 10 mg/kg/day was the NOAEL for testicular effects (mild-to-severe 

tubular degeneration and trace-to-severe multinucleated giant cell formation). 

Testicular toxicity was identified as the most sensitive effect of acute-duration oral exposure to 3,5-DNT, 

based on the LOAEL value of 19 mg/kg/day in rats administered 3,5-DNT via gavage for 14 days (Lent et 

al. 2012a; USAPHC 2011f). The NOAEL value of 10 mg/kg/day was used as the POD. Available data 

were not suitable for BMD modeling, as the incidences of testicular lesions increased from 0/6 in the 

control group to 6/6 at the LOAEL. The POD of 10 mg/kg/day was divided by an uncertainty factor of 

300 (10 for extrapolation from animals to humans, 10 for human variability, and 3 for database 

limitations), resulting in an acute-duration oral MRL for 3,5-DNT of 0.03 mg/kg/day. 

Uncertainty Factors used in MRL derivation: 

[ ] 10 for use of a LOAEL 

[X] 10 for extrapolation from animals to humans 

[X] 10 for human variability 

[X] 3 for database limitations (no other toxicological studies of this isomer are available) 

Was a conversion factor used from ppm in food or water to a mg/body weight dose? No. 

If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Not 

applicable. 

Was a conversion used from intermittent to continuous exposure? No. 

Other additional studies or pertinent information which lend support to this MRL: No other acuteduration oral studies of 3,5-DNT were located, nor were there any studies of longer durations. 

Agency Contact (Chemical Manager): Carolyn Harper 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES B-1 

APPENDIX B. USER'S GUIDE 

Chapter 1 

Public Health Statement 

This chapter of the profile is a health effects summary written in non-technical language. Its intended 

audience is the general public, especially people living in the vicinity of a hazardous waste site or 

chemical release. If the Public Health Statement were removed from the rest of the document, it would 

still communicate to the lay public essential information about the chemical. 

The major headings in the Public Health Statement are useful to find specific topics of concern. The 

topics are written in a question and answer format. The answer to each question includes a sentence that 

will direct the reader to chapters in the profile that will provide more information on the given topic. 

Chapter 2 

Relevance to Public Health 

This chapter provides a health effects summary based on evaluations of existing toxicologic, 

epidemiologic, and toxicokinetic information. This summary is designed to present interpretive, weightof-evidence discussions for human health end points by addressing the following questions: 

1.	 What effects are known to occur in humans? 

2.	 What effects observed in animals are likely to be of concern to humans? 

3.	 What exposure conditions are likely to be of concern to humans, especially around hazardous 

waste sites? 

The chapter covers end points in the same order that they appear within the Discussion of Health Effects 

by Route of Exposure section, by route (inhalation, oral, and dermal) and within route by effect. Human 

data are presented first, then animal data. Both are organized by duration (acute, intermediate, chronic). 

In vitro data and data from parenteral routes (intramuscular, intravenous, subcutaneous, etc.) are also 

considered in this chapter. 

The carcinogenic potential of the profiled substance is qualitatively evaluated, when appropriate, using 

existing toxicokinetic, genotoxic, and carcinogenic data. ATSDR does not currently assess cancer 

potency or perform cancer risk assessments. Minimal Risk Levels (MRLs) for noncancer end points (if 

derived) and the end points from which they were derived are indicated and discussed. 

Limitations to existing scientific literature that prevent a satisfactory evaluation of the relevance to public 

health are identified in the Chapter 3 Data Needs section. 

Interpretation of Minimal Risk Levels 

Where sufficient toxicologic information is available, ATSDR has derived MRLs for inhalation and oral 

routes of entry at each duration of exposure (acute, intermediate, and chronic). These MRLs are not 

meant to support regulatory action, but to acquaint health professionals with exposure levels at which 

adverse health effects are not expected to occur in humans. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES B-2 

APPENDIX B 

MRLs should help physicians and public health officials determine the safety of a community living near 

a chemical emission, given the concentration of a contaminant in air or the estimated daily dose in water. 

MRLs are based largely on toxicological studies in animals and on reports of human occupational 

exposure. 

MRL users should be familiar with the toxicologic information on which the number is based. Chapter 2, 

"Relevance to Public Health," contains basic information known about the substance. Other sections such 

as Chapter 3 Section 3.9, "Interactions with Other Substances,” and Section 3.10, "Populations that are 

Unusually Susceptible" provide important supplemental information. 

MRL users should also understand the MRL derivation methodology. MRLs are derived using a 

modified version of the risk assessment methodology that the Environmental Protection Agency (EPA) 

provides (Barnes and Dourson 1988) to determine reference doses (RfDs) for lifetime exposure. 

To derive an MRL, ATSDR generally selects the most sensitive end point which, in its best judgement, 

represents the most sensitive human health effect for a given exposure route and duration. ATSDR 

cannot make this judgement or derive an MRL unless information (quantitative or qualitative) is available 

for all potential systemic, neurological, and developmental effects. If this information and reliable 

quantitative data on the chosen end point are available, ATSDR derives an MRL using the most sensitive 

species (when information from multiple species is available) with the highest no-observed-adverse-effect 

level (NOAEL) that does not exceed any adverse effect levels. When a NOAEL is not available, a 

lowest-observed-adverse-effect level (LOAEL) can be used to derive an MRL, and an uncertainty factor 

(UF) of 10 must be employed. Additional uncertainty factors of 10 must be used both for human 

variability to protect sensitive subpopulations (people who are most susceptible to the health effects 

caused by the substance) and for interspecies variability (extrapolation from animals to humans). In 

deriving an MRL, these individual uncertainty factors are multiplied together. The product is then 

divided into the inhalation concentration or oral dosage selected from the study. Uncertainty factors used 

in developing a substance-specific MRL are provided in the footnotes of the levels of significant exposure 

(LSE) tables. 

Chapter 3 

Health Effects 

Tables and Figures for Levels of Significant Exposure (LSE) 

Tables and figures are used to summarize health effects and illustrate graphically levels of exposure 

associated with those effects. These levels cover health effects observed at increasing dose 

concentrations and durations, differences in response by species, MRLs to humans for noncancer end 

points, and EPA's estimated range associated with an upper- bound individual lifetime cancer risk of 1 in 

10,000 to 1 in 10,000,000. Use the LSE tables and figures for a quick review of the health effects and to 

locate data for a specific exposure scenario. The LSE tables and figures should always be used in 

conjunction with the text. All entries in these tables and figures represent studies that provide reliable, 

quantitative estimates of NOAELs, LOAELs, or Cancer Effect Levels (CELs). 

The legends presented below demonstrate the application of these tables and figures. Representative 

examples of LSE Table 3-1 and Figure 3-1 are shown. The numbers in the left column of the legends 

correspond to the numbers in the example table and figure. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES B-3 

APPENDIX B 

LEGEND 

See Sample LSE Table 3-1 (page B-6) 

(1)	 Route of Exposure. One of the first considerations when reviewing the toxicity of a substance 

using these tables and figures should be the relevant and appropriate route of exposure. Typically 

when sufficient data exist, three LSE tables and two LSE figures are presented in the document. 

The three LSE tables present data on the three principal routes of exposure, i.e., inhalation, oral, 

and dermal (LSE Tables 3-1, 3-2, and 3-3, respectively). LSE figures are limited to the inhalation 

(LSE Figure 3-1) and oral (LSE Figure 3-2) routes. Not all substances will have data on each 

route of exposure and will not, therefore, have all five of the tables and figures. 

(2)	 Exposure Period. Three exposure periods—acute (less than 15 days), intermediate (15– 

364 days), and chronic (365 days or more)—are presented within each relevant route of exposure. 

In this example, an inhalation study of intermediate exposure duration is reported. For quick 

reference to health effects occurring from a known length of exposure, locate the applicable 

exposure period within the LSE table and figure. 

(3)	 Health Effect. The major categories of health effects included in LSE tables and figures are 

death, systemic, immunological, neurological, developmental, reproductive, and cancer. 

NOAELs and LOAELs can be reported in the tables and figures for all effects but cancer. 

Systemic effects are further defined in the "System" column of the LSE table (see key number 

18). 

(4)	 Key to Figure. Each key number in the LSE table links study information to one or more data 

points using the same key number in the corresponding LSE figure. In this example, the study 

represented by key number 18 has been used to derive a NOAEL and a Less Serious LOAEL 

(also see the two "18r" data points in sample Figure 3-1). 

(5)	 Species. The test species, whether animal or human, are identified in this column. Chapter 2, 

"Relevance to Public Health," covers the relevance of animal data to human toxicity and 

Section 3.4, "Toxicokinetics," contains any available information on comparative toxicokinetics. 

Although NOAELs and LOAELs are species specific, the levels are extrapolated to equivalent 

human doses to derive an MRL. 

(6)	 Exposure Frequency/Duration. The duration of the study and the weekly and daily exposure 

regimens are provided in this column. This permits comparison of NOAELs and LOAELs from 

different studies. In this case (key number 18), rats were exposed to “Chemical x” via inhalation 

for 6 hours/day, 5 days/week, for 13 weeks. For a more complete review of the dosing regimen, 

refer to the appropriate sections of the text or the original reference paper (i.e., Nitschke et al. 

1981). 

(7)	 System. This column further defines the systemic effects. These systems include respiratory, 

cardiovascular, gastrointestinal, hematological, musculoskeletal, hepatic, renal, and 

dermal/ocular. "Other" refers to any systemic effect (e.g., a decrease in body weight) not covered 

in these systems. In the example of key number 18, one systemic effect (respiratory) was 

investigated. 

(8)	 NOAEL. A NOAEL is the highest exposure level at which no harmful effects were seen in the 

organ system studied. Key number 18 reports a NOAEL of 3 ppm for the respiratory system, 

which was used to derive an intermediate exposure, inhalation MRL of 0.005 ppm (see 

footnote "b"). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DINITROTOLUENES B-4 

APPENDIX B 

(9)	 LOAEL. A LOAEL is the lowest dose used in the study that caused a harmful health effect. 

LOAELs have been classified into "Less Serious" and "Serious" effects. These distinctions help 

readers identify the levels of exposure at which adverse health effects first appear and the 

gradation of effects with increasing dose. A brief description of the specific end point used to 

quantify the adverse effect accompanies the LOAEL. The respiratory effect reported in key 

number 18 (hyperplasia) is a Less Serious LOAEL of 10 ppm. MRLs are not derived from 

Serious LOAELs. 

(10)	 Reference. The complete reference citation is given in Chapter 9 of the profile. 

(11)	 CEL. A CEL is the lowest exposure level associated with the onset of carcinogenesis in 

experimental or epidemiologic studies. CELs are always considered serious effects. The LSE 

tables and figures do not contain NOAELs for cancer, but the text may report doses not causing 

measurable cancer increases. 

(12)	 Footnotes. Explanations of abbreviations or reference notes for data in the LSE tables are found 

in the footnotes. Footnote "b" indicates that the NOAEL of 3 ppm in key number 18 was used to 

derive an MRL of 0.005 ppm. 

LEGEND 

See Sample Figure 3-1 (page B-7) 

LSE figures graphically illustrate the data presented in the corresponding LSE tables. Figures help the 

reader quickly compare health effects according to exposure concentrations for particular exposure 

periods. 

(13)	 Exposure Period. The same exposure periods appear as in the LSE table. In this example, health 

effects observed within the acute and intermediate exposure periods are illustrated. 

(14)	 Health Effect. These are the categories of health effects for which reliable quantitative data 

exists. The same health effects appear in the LSE table. 

(15)	 Levels of Exposure. Concentrations or doses for each health effect in the LSE tables are 

graphically displayed in the LSE figures. Exposure concentration or dose is measured on the log 

scale "y" axis. Inhalation exposure is reported in mg/m3 or ppm and oral exposure is reported in 

mg/kg/day. 

(16)	 NOAEL. In this example, the open circle designated 18r identifies a NOAEL critical end point in 

the rat upon which an intermediate inhalation exposure MRL is based. The key number 18 

corresponds to the entry in the LSE table. The dashed descending arrow indicates the 

extrapolation from the exposure level of 3 ppm (see entry 18 in the table) to the MRL of 

0.005 ppm (see footnote "b" in the LSE table). 

(17)	 CEL. Key number 38m is one of three studies for which CELs were derived. The diamond 

symbol refers to a CEL for the test species-mouse. The number 38 corresponds to the entry in the 

LSE table. 
 

 

 

DINITROTOLUENES B-5 

APPENDIX B 

(18)	 Estimated Upper-Bound Human Cancer Risk Levels. This is the range associated with the upperbound for lifetime cancer risk of 1 in 10,000 to 1 in 10,000,000. These risk levels are derived 

from the EPA's Human Health Assessment Group's upper-bound estimates of the slope of the 

cancer dose response curve at low dose levels (q1*). 

(19)	 Key to LSE Figure. The Key explains the abbreviations and symbols used in the figure. 
SAMPLE

1 → Table 3-1. Levels of Significant Exposure to [Chemical x] – Inhalation 

LOAEL (effect) Exposure 

Key to Less serious Serious (ppm) frequency/ NOAEL (ppm) figurea Species duration System (ppm) Re

2 3 4 

→ INTERMEDIATE EXPOSURE 

5 

→ Systemic ↓

18 Rat 

→

CHRONIC EXPOSURE Cancer 38 Rat 39 Rat 40 Mouse 

6 ↓13 wk 5 d/wk 6 hr/d 18 mo 5 d/wk 7 hr/d 89–104 wk 5 d/wk 6 hr/d 79–103 wk 5 d/wk 6 hr/d 

7 8 9 ↓ ↓ ↓Resp 3b 10 (hyperplasia) 

11 ↓20 (CEL, multiple 

organs) 

10 (CEL, lung tumors, 

nasal tumors) 

10 (CEL, lung tumors, 

hemangiosarcomas) 

10 

↓

NitWoNTNT

12 → a The number corresponds to entries in Figure 3-1. 

b Used to derive an intermediate inhalation Minimal Risk Level (MRL) of 5x10-3 ppm; dose adjusted for intermittent exby an uncertainty factor of 100 (10 for extrapolation from animal to humans, 10 for human variability). 
Intermediate (15-364 days) Chronic (≥ 365 days) 
DINITROTOLUENES B-8 

APPENDIX B 

This page is intentionally blank. 
 

DINITROTOLUENES C-1 

APPENDIX C. ACRONYMS, ABBREVIATIONS, AND SYMBOLS 

ACGIH American Conference of Governmental Industrial Hygienists 

ACOEM American College of Occupational and Environmental Medicine 

ADI acceptable daily intake 

ADME absorption, distribution, metabolism, and excretion 

AED atomic emission detection 

AFID alkali flame ionization detector 

AFOSH Air Force Office of Safety and Health 

ALT alanine aminotransferase 

AML acute myeloid leukemia 

AOAC Association of Official Analytical Chemists 

AOEC Association of Occupational and Environmental Clinics 

AP alkaline phosphatase 

APHA American Public Health Association 

AST aspartate aminotransferase 

atm atmosphere 

ATSDR Agency for Toxic Substances and Disease Registry 

AWQC Ambient Water Quality Criteria 

BAT best available technology 

BCF bioconcentration factor 

BEI Biological Exposure Index 

BMD/C benchmark dose or benchmark concentration 

BMDX dose that produces a X% change in response rate of an adverse effect 

BMDLX 95% lower confidence limit on the BMDX 

BMDS Benchmark Dose Software 

BMR benchmark response 

BSC Board of Scientific Counselors 

C centigrade 

CAA Clean Air Act 

CAG Cancer Assessment Group of the U.S. Environmental Protection Agency 

CAS Chemical Abstract Services 

CDC Centers for Disease Control and Prevention 

CEL cancer effect level 

CELDS Computer-Environmental Legislative Data System 

CERCLA Comprehensive Environmental Response, Compensation, and Liability Act 

CFR Code of Federal Regulations 

Ci curie 

CI confidence interval 

CL ceiling limit value 

CLP Contract Laboratory Program 

cm centimeter 

CML chronic myeloid leukemia 

CPSC Consumer Products Safety Commission 

CWA Clean Water Act 

DHEW Department of Health, Education, and Welfare 

DHHS Department of Health and Human Services 

DNA deoxyribonucleic acid 

DOD Department of Defense 

DOE Department of Energy 

DOL Department of Labor 
 

DINITROTOLUENES C-2 

APPENDIX C 

DOT Department of Transportation 

DOT/UN/ Department of Transportation/United Nations/ 

NA/IMDG North America/Intergovernmental Maritime Dangerous Goods Code 

DWEL drinking water exposure level 

ECD electron capture detection 

ECG/EKG electrocardiogram 

EEG electroencephalogram 

EEGL Emergency Exposure Guidance Level 

EPA Environmental Protection Agency 

F Fahrenheit 

F1 first-filial generation 

FAO Food and Agricultural Organization of the United Nations 

FDA Food and Drug Administration 

FEMA Federal Emergency Management Agency 

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act 

FPD flame photometric detection 

fpm feet per minute 

FR Federal Register 

FSH follicle stimulating hormone 

g gram 

GC gas chromatography 

gd gestational day 

GLC gas liquid chromatography 

GPC gel permeation chromatography 

HPLC high-performance liquid chromatography 

HRGC high resolution gas chromatography 

HSDB Hazardous Substance Data Bank 

IARC International Agency for Research on Cancer 

IDLH immediately dangerous to life and health 

ILO International Labor Organization 

IRIS Integrated Risk Information System 

Kd adsorption ratio 

kg kilogram 

kkg metric ton 

Koc organic carbon partition coefficient 

Kow octanol-water partition coefficient 

L liter 

LC liquid chromatography 

LC50 lethal concentration, 50% kill 

LCLo lethal concentration, low 

LD50 lethal dose, 50% kill 

LDLo lethal dose, low 

LDH lactic dehydrogenase 

LH luteinizing hormone 

LOAEL lowest-observed-adverse-effect level 

LSE Levels of Significant Exposure 

LT50 lethal time, 50% kill 

m meter 

MA trans,trans-muconic acid 

MAL maximum allowable level 

mCi millicurie 
 

 

 

DINITROTOLUENES C-3 

APPENDIX C 

MCL maximum contaminant level 

MCLG maximum contaminant level goal 

MF modifying factor 

MFO mixed function oxidase 

mg milligram 

mL milliliter 

mm millimeter 

mmHg millimeters of mercury 

mmol millimole 

mppcf millions of particles per cubic foot 

MRL Minimal Risk Level 

MS mass spectrometry 

NAAQS National Ambient Air Quality Standard 

NAS National Academy of Science 

NATICH National Air Toxics Information Clearinghouse 

NATO North Atlantic Treaty Organization 

NCE normochromatic erythrocytes 

NCEH National Center for Environmental Health 

NCI National Cancer Institute 

ND not detected 

NFPA National Fire Protection Association 

ng nanogram 

NHANES National Health and Nutrition Examination Survey 

NIEHS National Institute of Environmental Health Sciences 

NIOSH National Institute for Occupational Safety and Health 

NIOSHTIC NIOSH's Computerized Information Retrieval System 

NLM National Library of Medicine 

nm nanometer 

nmol nanomole 

NOAEL no-observed-adverse-effect level 

NOES National Occupational Exposure Survey 

NOHS National Occupational Hazard Survey 

NPD nitrogen phosphorus detection 

NPDES National Pollutant Discharge Elimination System 

NPL National Priorities List 

NR not reported 

NRC National Research Council 

NS not specified 

NSPS New Source Performance Standards 

NTIS National Technical Information Service 

NTP National Toxicology Program 

ODW Office of Drinking Water, EPA 

OERR Office of Emergency and Remedial Response, EPA 

OHM/TADS Oil and Hazardous Materials/Technical Assistance Data System 

OPP Office of Pesticide Programs, EPA 

OPPT Office of Pollution Prevention and Toxics, EPA 

OPPTS Office of Prevention, Pesticides and Toxic Substances, EPA 

OR odds ratio 

OSHA Occupational Safety and Health Administration 

OSW Office of Solid Waste, EPA 

OTS Office of Toxic Substances 
 

 

 

DINITROTOLUENES C-4 

APPENDIX C 

OW Office of Water 

OWRS Office of Water Regulations and Standards, EPA 

PAH polycyclic aromatic hydrocarbon 

PBPD physiologically based pharmacodynamic 

PBPK physiologically based pharmacokinetic 

PCE polychromatic erythrocytes 

PEL permissible exposure limit 

pg picogram 

PHS Public Health Service 

PID photo ionization detector 

pmol picomole 

PMR proportionate mortality ratio 

ppb parts per billion 

ppm parts per million 

ppt parts per trillion 

PSNS pretreatment standards for new sources 

RBC red blood cell 

REL recommended exposure level/limit 

RfC reference concentration 

RfD reference dose 

RNA ribonucleic acid 

RQ reportable quantity 

RTECS Registry of Toxic Effects of Chemical Substances 

SARA Superfund Amendments and Reauthorization Act 

SCE sister chromatid exchange 

SGOT serum glutamic oxaloacetic transaminase 

SGPT serum glutamic pyruvic transaminase 

SIC standard industrial classification 

SIM selected ion monitoring 

SMCL secondary maximum contaminant level 

SMR standardized mortality ratio 

SNARL suggested no adverse response level 

SPEGL Short-Term Public Emergency Guidance Level 

STEL short term exposure limit 

STORET Storage and Retrieval 

TD50 toxic dose, 50% specific toxic effect 

TLV threshold limit value 

TOC total organic carbon 

TPQ threshold planning quantity 

TRI Toxics Release Inventory 

TSCA Toxic Substances Control Act 

TWA time-weighted average 

UF uncertainty factor 

U.S. United States 

USDA United States Department of Agriculture 

USGS United States Geological Survey 

VOC volatile organic compound 

WBC white blood cell 

WHO World Health Organization 
 

DINITROTOLUENES C-5 

APPENDIX C 

> greater than 

≥ greater than or equal to 

= equal to 

< less than 

≤ less than or equal to 

% percent 

α alpha 

β beta 

γ gamma 

δ delta 

μm micrometer 

μg microgram q1

* cancer slope factor 

– negative 

+ positive 

(+) weakly positive result 

(–) weakly negative result