TOXICOLOGICAL PROFILE FOR

2,4- and 2,6-DINITROTOLUENE

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Agency for Toxic Substances and Disease Registry

December 1998
2,4- and 2,6-DINITROTOLUENE ii

DISCLAIMER

The use of company or product name(s) is for identification only and does not imply endorsement by the

Agency for Toxic Substances and Disease Registry.
2,4- and 2,6-DINITROTOLUENE iii

UPDATE STATEMENT

A Toxicological Profile for 2,4- and 2,6-dinitrotoluene was released in September 1997. This edition

supersedes any previously released draft or final profile.

Toxicological profiles are revised and republished as necessary, but no less than once every three years. For

information regarding the update status of previously released profiles, contact ATSDR at:

Agency for Toxic Substances and Disease Registry

Division of Toxicology/Toxicology Information Branch

1600 Clifton Road NE, E-29

Atlanta, Georgia 30333


vi

*Legislative Background

The toxicological profiles are developed in response to the Super-fund Amendments and

Reauthorization Act (SARA) of 1986 (Public Law 99-499) which amended the Comprehensive

Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or Superfund). This public

law directed ATSDR to prepare 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. The availability of the revised priority list of 275

hazardous substances was announced in the Federal Register on November 17, 1997 (62 FR 61332). For

prior versions of the list of substances, see Federal Register notices dated April 29, 1996 (61 FR 18744);

April 17, 1987 (52 FR 12866); October 20, 1988 (53 FR 41280); October 26, 1989 (54 FR 43619);

October 17,199O (55 FR 42067); October 17, 1991 (56 IX 52166); October 28, 1992 (57 FR 48801); and

February 28, 1994 (59 FR 9486). Section 104(i)(3) of CERCLA, as amended, directs the Administrator

of ATSDR to prepare a toxicological profile for each substance on the list.
2,4- and 2,6-DINITROTOLUENE 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: Health Effects: Specific health effects of a given hazardous compound are reported by route

of exposure, by type of health effect (death, systemic, immunologic, reproductive), 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:

Section 1.6 How Can (Chemical X) Affect Children?

Section 1.7 How Can Families Reduce the Risk of Exposure to (Chemical X)?

Section 2.6 Children’s Susceptibility

Section 5.6 Exposures of Children

Other Sections of Interest:

Section 2.7 Biomarkers of Exposure and Effect

Section 2.10 Methods for Reducing Toxic Effects

 _________________________________________________________________________________

ATSDR Information Center

Phone: l-800-447-1544 (to be replaced by l-888-42-ATSDR in 1999)

or 404-639-6357 Fax: 404-639-6359

E-mail: atsdric@cdc.gov Internet: http://atsdrl.atsdr.cdc.gov:8080

The following additional material can be ordered through the ATSDR Information Center:

Case Studies in Environmental Medicine: Taking an Exposure History-The importance of taking an

exposure history and how to conduct one are described, and an example of a thorough exposure

history is provided. Other case studies of interest include Reproductive and Developmental

Hazards; Skin Lesions and Environmental Exposures; Cholinesterase-Inhibiting Pesticide

Toxicity; and numerous chemical-specific case studies.
2,4- and 2,6-DINITROTOLUENE 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 (ToxFAQs) 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 l Phone:

770-488-7000 l 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, 200 Independence Avenue, SW,

Washington, DC 20201 l Phone: 800-356-4674 or NIOSH Technical Information Branch, Robert

A. Taft Laboratory, Mailstop C-19,4676 Columbia Parkway, Cincinnati, OH 45226-1998 l Phone:

800-35-NIOSH.

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 l Phone: 919-541-3212.

 _____________________________________________________________________________________

Referrals

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 l Phone: 202-347-4976 l FAX: 202-

347-4950 l e-mail: aoec@dgs.dgsys.com l AOEC Clinic Director: http://occ-envmed.

mc.duke.edu/oem/aoec.htm.

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, 55 West Seegers Road, Arlington Heights, IL 60005 l Phone: 847-

228-6850 l FAX: 847-228-1856.
2,4- and 2,6-DINITROTOLUENE ix

CONTRIBUTORS

CHEMICAL MANAGER/AUTHORS:

Zemoria A. Rosemond, B.A.

ATSDR, Division of Toxicology, Atlanta, GA

Diana Wong, Ph.D., DABT

Sciences International, Inc., Alexandria, VA

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 Research Implementation Branch reviews data needs sections to assure

consistency across profiles and adherence to instructions in the Guidance.

4- and 2,6-DINITROTOLUENE xi

PEER REVIEW

A peer review panel was assembled for 2,4- and 2,6-dinitrotoluene. The panel consisted of the following

members:

1 . G.A. Shakeel Ansari, Ph.D., Professor, Department of Human Biological Chemistry and Genetics, and

Department of Pathology, University of Texas Medical Branch, Galveston, TX.

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

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

3 . Gary Stoner, Ph.D., Professor and Chair, Division of Environmental Health Sciences, School of Public

Health, Ohio State University, Columbus, OH.

4 . William J. George, Ph.D., Professor and Director of Toxicology, Tulane University School of

Medicine, New Orleans, LA.

5 . Gerald Kennedy, DuPont/Haskell Lab., Newark, DE.

These experts collectively have knowledge of 2,4- and 2,6-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. A list of databases reviewed and a

list of unpublished documents cited are also included in the administrative record.

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.









2,4- and 2,6-DINITROTOLUENE 1

1. PUBLIC HEALTH STATEMENT

This public health statement tells you about 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene

(2,6-DNT) and the effects of exposure.

The 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 the sites targeted for

long-term federal cleanup activities. 2,4-DNT and 2,6-DNT have been found in at least 122 of

the 1,467 current or former NPL sites. However, the total number of NPL sites evaluated for this

substance is not known. As more sites are evaluated, the sites at which 2,4-DNT and 2,6-DNT is

found may increase. This information is important because exposure to this substance may harm

you and because these sites may be sources of exposure.

When a substance is released from a large area, such as an industrial plant, or from a container,

such as a drum or bottle, it enters the environment. This release does not always lead to

exposure. You are exposed to a substance only when you come in contact with it. You may be

exposed by breathing, eating, or drinking the substance or by skin contact.

If you are exposed to 2,4-DNT or 2,6-DNT, many factors determine whether you’ll be harmed.

These factors include the dose (how much), the duration (how long), and how you come in

contact with it. You must also consider the other chemicals you’re exposed to and your age, sex,

diet, family traits, lifestyle, and state of health.

1.1 WHAT ARE 2,4- AND 2,6-DINITROTOLUENE?

2,4-DNT and 2,6-DNT are pale yellow solids with a slight odor and are two of the six forms of

the chemical called dinitrotoluene (DNT). The other four forms (2,3-DNT, 2,5-DNT, 3,4-DNT,

and 3,5-DNT) only make up about 5% of the technical grade DNT. DNT is not a natural

substance but rather is usually made by reacting toluene (a solvent) with mixed nitric and sulfuric
2,4- and 2,6-DINITROTOLUENE 2

1. PUBLIC HEALTH STATEMENT

acids, which are strong acids. DNT is used to produce flexible polyurethane foams used in the

bedding and furniture industry. DNT is also used to produce ammunition and explosives and to

make dyes. It is also used in the air bags of automobiles. It has been found in the soil, surface

water, and groundwater of at least 122 hazardous waste sites that contain buried ammunition

wastes and wastes from manufacturing facilities that release DNT. DNT does not usually

evaporate and is found in the air only in manufacturing plants. DNT also does not usually remain

in the environment for a long time because it is broken down by sunlight and bacteria into

substances such as carbon dioxide, water, and nitric acid. More detailed information on the

chemical and physical properties of DNT and its uses is provided in Chapters 3 and 4.

1.2 WHAT HAPPENS TO 2,4- AND 2,6-DINITROTOLUENE WHEN THEY ENTER

 THE ENVIRONMENT?

DNT can be found in air, surface water, groundwater, and soil. Releases to the air are usually in

the form of dusts or aerosols from manufacturing plants. Evaporation from water containing

DNT is not a likely means of release to the air. DNT is thought to break down in air by a variety

of chemical reactions that take place upon exposure to sunlight.

In water, DNT can be broken down by sunlight. Under conditions without oxygen or without

light, DNT may be broken down by biological degradation, whereby microbes utilize the chemical

as a source of energy and convert it into chemicals such as carbon dioxide and water. DNT in

surface water from rivers and streams and groundwater from wells can result from releases of

waste water from trinitrotoluene (TNT) manufacturing facilities and from buried munition wastes.

No information was located regarding the changing of DNT to other chemical substances in soil.

DNT is unlikely to build up in animal tissues after animals are exposed by eating impacted soil,

water, or vegetation, or by inhaling contaminated air. However, since DNT is quite soluble in

water, it can be transferred to plants via root uptake from soil or irrigation with contaminated

water, although no direct measurements have been found. It is, however, expected to accumulate

readily in plant materials, although no direct measurements have been found.
2,4- and 2,6-DINITROTOLUENE 3

1. PUBLIC HEALTH STATEMENT

For more information on what happens to DNT in the environment, see Chapters 4 and 5.

1.3 HOW MIGHT I BE EXPOSED TO 2,4- AND 2,6-DINITROTOLUENE?

Members of the general population are likely to be exposed only if they are near a DNT

contaminated waste site or manufacturing facilities that release DNT.

2,4- or 2,6-DNT may enter the environment from waste waters that industries discharge into

rivers and streams or from the improper disposal of wastes. However, regular testing of water in

the United States shows that 2,4- and 2,6-DNT were found in less than 2% of the water samples.

Testing of hazardous waste sites shows that 2,4- and 2,6-DNT are present at less than 8.5% of

these sites. Available information indicates that DNT does not appear to be widespread in the

environment. More information on how people might be exposed to 2,4- or 2,6-DNT is given in

Chapter 5.

1.4 HOW CAN 2,4- AND 2,6-DINITROTOLUENE ENTER AND LEAVE MY BODY?

When industrial workers are exposed to 2,4- or 2,6-dinitrotoluene, the major ways that these

chemicals enter their bodies are by breathing or absorbing small amounts of the chemical through

the skin. Some ingestion may also occur as the result of eating or smoking without prior

handwashing.

After individuals breathe air, drink water, or eat food contaminated with 2,4- or 2,6-DNT, these

chemicals are changed into different substances by the liver and in the intestines. After this, most

of these chemicals leave the body within 24 hours in the urine, with a small amount in the feces.

This information comes from animal experiments done in laboratories and from studies of

industrial workers. More detailed information is given in Chapter 2.
2,4- and 2,6-DINITROTOLUENE 4

1. PUBLIC HEALTH STATEMENT

1.5 HOW CAN 2,4- AND 2,6-DINITROTOLUENE AFFECT MY HEALTH?

To protect the public from the harmful effects of toxic chemicals and to find ways to treat people

who have been harmed, scientists use many tests.

One way to see if a chemical will hurt people is to learn how the chemical is absorbed, used, and

released by the body; for some chemicals, animal testing may be necessary. Animal testing may

also be used to identify health effects such as cancer or birth defects. Without laboratory animals,

scientists would lose a basic method to get information needed to make wise decisions to protect

public health. Scientists have the responsibility to treat research animals with care and

compassion. Laws today protect the welfare of research animals, and scientists must comply with

strict animal care guidelines.

Increases in death rate due to heart disease have been seen in workers exposed to 2,4-DNT or

technical grade DNT (Tg-DNT), but these workers may also have been exposed to other

chemicals. 2,4- and 2,6-DNT may also affect the nervous system and the blood of exposed

workers. One study showed that male workers exposed to 2,4- and 2,6-DNT had reduced levels

of sperm, but later studies did not confirm the finding.

Exposure to high levels of these compounds in animals regularly causes lowered numbers of

sperm and reduced fertility. Studies of animals have also shown that nervous system disorders,

liver damage, and kidney damage can occur, as well as a reduction in the numbers of red blood

cells. Both 2,4- and 2,6-DNT can cause liver cancer in laboratory rats and may produce the same

effect in humans. More information on the health effects of DNT is given in Chapter 2. The

International Agency for Research on Cancer (IARC) has determined that 2,4- and 2,6-DNT are

possibly carcinogenic to humans.
2,4- and 2,DINITROTOLUENE 5

1. PUBLIC HEALTH STATEMENT

1.6 HOW CAN 2,4- AND 2,6-DINITROTOLUENE AFFECT CHILDREN?

This section discusses potential health effects from exposures during the period from conception

to maturity at 18 years of age in humans. Potential effects on children resulting from exposures of

the parents are also considered.

2,4- and 2,6-DNT are not widespread throughout the environment. If you do not live near a plant

or a waste site that contains DNT, it is unlikely that your children would be exposed to DNT.

DNT is normally associated with industrial or military production plants or munitions storage

sites. DNT is water soluble, so if contamination has occurred it will usually be carried in water.

Children are at risk for exposure if DNT has leached into a community’s drinking water supply

from a nearby hazardous site since they drink more fluid in proportion to their body weight than

adults. Children playing in DNT-contaminated surface water might be more exposed than adults,

both because of this behavior and because of their larger skin area in proportion to their body

weight.

Since DNT exposure is usually related to adult workers, health effects on chiklren have not been

studied. It is not known if DNT affects children differently than adults, or what long term effects

might appear in adults exposed as children.

No studies have investigated effects of DNT on the developmental process in humans, and few

studies have focused on animals. No studies have been done to see if DNT or its toxic breakdown

products cross the placenta, or get into breast milk.

1.7 HOW CAN FAMILIES REDUCE THE RISK OF EXPOSURE TO 2,4- AND

2,6-DINITROTOLUENE?

If your doctor finds that you have been exposed to significant amounts of 2,4-DNT or 2,6-DNT,

ask if children may also be exposed. When necessary your doctor may need to ask your State

Department of Public Health to investigate.
2,4- and 2,6-DINITROTOLUENE 6

1. PUBLIC HEALTH STATEMENT

If you live near a site that could be contaminated with DNT you should discourage your children

from putting foreign objects, groundwater, and dirt in their mouths since DNT is quite water

soluble. Make sure they wash their hands frequently and before eating. Discourage your children

both from putting their hands in their mouths and horn other hand-to-mouth activities.

1.8 IS THERE A MEDICAL TEST TO DETERMINE WHETHER I HAVE BEEN

EXPOSED TO 2,4- AND 2,6-DINITROTOLUENE?

Both 2,4- and 2,6-DNT and the chemicals they are changed into by the body can be measured in

the blood and urine of exposed individuals (if urine is collected within 24 hours). The tests cannot

show how much 2,4- or 2,6-DNT an individual has been exposed to. These tests are not usually

available in doctors’ offices but can be performed by special laboratories. More detailed

information on medical tests is presented in Chapter 6.

1.9 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 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 in not-to-exceed levels in air, water, soil, or

food that are usually based on levels that affect animals; then they are adjusted to help protect

people. Sometimes these not-to-exceed levels differ among federal organizations because of

different exposure times (an 8-hour workday or a 24-hour day), the use of different animal

studies, or other factors.
2,4- and 2,6-DINITROTOLUENE 7

1. PUBLIC HEALTH STATEMENT

Recommendations and regulations are also periodically updated as more information becomes

available. For the most current information, check with the federal agency or organization that

provides it. Some regulations and recommendations for 2,4- and 2,6-DNT include the following:

EPA recommendations define the safe lifetime daily maximum oral dose for 2,4-DNT as

0.002 milligram per kilogram per day for 2,4-DNT, and 0.001 milligram per kilogram per day

for 2,6-DNT. In addition to EPA recommendations, OSHA regulations state that an average

8-hour exposure to total DNT in workplace air should not be more than 1.5 milligrams per cubic

meter of air (mg/m3

). NIOSH has published a Recommended Exposure Limit (REL) guideline

of 1.5 mg/m3

. An REL is an average concentration for a 10-hour workday over a 40-hour

workweek. Spills or releases to the environment of more than 1,000 pounds of DNT must be

reported immediately to the federal government. More information on regulations and advisories

for DNT is given in Chapter 7.

1.10 WHERE CAN I GET MORE INFORMATION?

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

environmental quality department or

Agency for Toxic Substances and Disease Registry

Division of Toxicology

1600 Clifton Road NE, Mailstop E-29

Atlanta, GA 30333

* Information line and technical assistance

Phone: l-800-447-1544

Fax: (404) 639-6359

ATSDR can also tell you the location of occupational and environmental health clinics. These

clinics specialize in recognizing, evaluating, and treating illnesses resulting from exposure to

hazardous substances.
2,4- and 2,6-DINITROTOLUENE 8

1. PUBLIC HEALTH STATEMENT

* To order toxicological profiles, contact

 National Technical Information Service

 5285 Port Royal Road

 Springfield, VA 22161

 Phone: (800) 553-6847 or (703) 487-4650
2,4- and 2,6-DINITROTOLUENE 9

2. HEALTH EFFECTS

2.1 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 of the toxicology of 2,4- and 2,6-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.

A glossary and list of acronyms, abbreviations, and symbols can be found at the end of this profile.

2.2 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).

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 lowest-observed-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
2,4- and 2,6-DINITROTOLUENE 10

2. HEALTH EFFECTS

determining whether or not 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 (LOAEL) 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.

Levels of exposure associated with carcinogenic effects (Cancer Effect Levels, CELs) of 2,4-DNT are

indicated in Table 2-l. Because cancer effects could occur at lower exposure levels, Figure 2-l also shows a

range for the upper bound of estimated excess risks, ranging from a risk of 1 in 10,000 to 1 in 10,000,000

( 10-4 to 10-7), as developed by EPA.

Estimates of exposure levels posing minimal risk to humans (Minimal Risk Levels or MRLs) have been made

for 2,4- and 2,6-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.

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.
2,4- and 2,6-DINITROTOLUENE 11

2. HEALTH EFFECTS

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.

Although the primary foci of this document are 2,4-DNT and 2,6-DNT, some information on technical grade

DNT (Tg-DNT) is also provided. Tg-DNT contains approximately 76% 2,4-DNT, 19% 2,6-DNT, and other

isomers. Unless specified otherwise, 2,4-DNT and 2,6-DNT are abbreviated generically as DNT in this

document.

2.2.1 Inhalation Exposure

Most of the data on human health effects associated with exposure to 2,4-DNT or 2,6-DNT are derived from

studies of workers in occupational settings. Exposure monitoring of workers in the past has generally been

inadequate. Consequently, few dose-response data based on human exposure to 2,4- or 2,6-DNT are

available.

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 DNT makes inhalation of

vapors unlikely, it can occur when contaminated particulate material is in the air. In addition, some dermal

exposure is probable, and some ingestion may also occur as the result of eating or smoking without prior

handwashing.

2.2.1.l Death

In a retrospective cohort mortality study of 457 munitions workers who were exposed to either 2,4-DNT or

Tg-DNT at 2 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 (Standard Mortality Rates

[SMR] of 126 and 143; 95% confidence intervals [CI] 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 more than 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 more than 15 years for a significant increase in mortality due to ischemic
2,4- and 2,6-DINITROTOLUENE 12

2. HEALTH EFFECTS

heart disease. There also appeared to be a relationship between heart disease and the intensity of exposure to

dinitrotoluenes. 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

DNT 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 located regarding death in animals after inhalation exposure to 2,4- or 2,6-DNT.

2.2.1.2 Systemic Effects

No studies were located regarding respiratory, dermal, or ocular effects in humans or animals after inhalation

exposure to 2,4- or 2,6-DNT.

Cardiovascular Effects. As described in Section 2.2.1.1, 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 inhalation exposure to 2,4- or

2,6-DNT.

Gastrointestinal Effects. Vomiting and nausea were mentioned as health complaints in a survey of

154 male workers involved in the production of smokeless gunpowders during World War II (McGee et al.

1942). The exposure concentrations of 2,4-DNT were not specified but may have been relatively high

because of the lack of modem industrial hygiene practices. These symptoms were still mentioned in a followup

survey of workers that was undertaken at the same facility after the installation of safeguards to reduce
2,4- and 2,6-DINITROTOLUENE 13

2. HEALTH EFFECTS

DNT exposure (McGee et al. 1947). Since exposure to other compounds cannot be ruled out, attribution of

these symptoms to DNT cannot be verified.

No studies were located regarding gastrointestinal effects in animals after inhalation exposure to 2,4- or

2,6-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. 1942, 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, the 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.

No studies were located regarding hematological effects in animals after inhalation exposure to 2,4- or

2,6-DNT.

Musculoskeletal Effects. Men who worked in a munitions plant during World War II mentioned

muscular weakness as a complaint in a survey of these workers (McGee et al. 1942). However, the lack of

2,4-DNT exposure data and lack of an adequate control population prevent these data from being useful for

anything other than qualitative description. Joint pain, especially in the knees, and other incapacitating

symptoms were found in unspecified numbers of French workers in a plant that produced DNT 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.
2,4- and 2,6-DINITROTOLUENE 14

2. HEALTH EFFECTS

No studies were located regarding musculoskeletal effects in animals after inhalation exposure to 2,4- or

2.6-DNT.

Hepatic Effects. Two of 154 workers at a munitions plant suffered from acute hepatitis with jaundice but

recovered soon after they were no longer exposed to 2,4-DNT (McGee et al. 1942). Although the exposure

concentrations were not known, they were assumed to be high based upon the industrial hygiene practices

prevalent at that time. Furthermore, since an adequate control population was not used, this study provides

only a qualitative description of symptoms, at best. A later study of 714 workers at this same plant found

that 29 experienced liver tenderness (McGee et al. 1947). This incidence would indicate an increased effect

on the liver from the 1942 study, despite improvements in industrial hygiene and engineering practices

designed to decrease worker exposure in the interval between the two studies. 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 (Ahrenholz and Meyer 1982). Air

samples contained concentrations ranging from 0.026 to 0.890 mg/m3 Tg-DNT (mean 0.207 mg/m3

Tg-DNT).

No studies were located regarding hepatic effects in animals after inhalation exposure to 2,4- or 2,6-DNT.

Renal Effects. No effects were observed on either of the renal parameters (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 (Ahrenholz and Meyer 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 Tg-DNT). The study was limited by a

small exposure population and lack of historical individual exposure monitoring.

No studies were located regarding renal effects in animals after inhalation exposure to 2,4- or 2,6-DNT.

2.2.1.3 Immunological and Lymphoreticular Effects

No studies were located regarding immunological or lymphoreticular effects in humans or animals after

inhalation exposure to 2,4- or 2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 15

2. HEALTH EFFECTS

2.2.1.4 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. (1942,1947). Neurological signs reported by these workers included headache, dizziness,

insomnia, unpleasant taste in the mouth, 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 animals after inhalation exposure to 2,4- or

2,6-DNT.

2.2.1.5 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 in the incidence of

miscarriage in their wives compared to controls (Ahrenholz and Meyer 1982; Hamill et al. 1982). In the

Ahrenholz and Meyer (1982) study, DNT concentrations ranged from 0.026 to 0.890 mg/m3

 (mean

0.207 mg/m3

 Tg-DNT). Interpretation of these studies is somewhat confounded by the lack of distinction

between DNT and TDA exposure and by 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 (Ahrenholz and Meyer 1982; Hamill et al. 1982; Levine et al. 1985a). 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.
2,4-and 2,6-DINITROTOLUENE 16

2. HEALTH EFFECTS

One study by the CDC (1981) noted that sperm counts were decreased by more than 50% in workers in a

Kentucky chemical plant exposed to DNT and TDA compared to workers unexposed 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 DNT/TDA.

No studies were located regarding reproductive effects in animals after inhalation exposure to 2,4-DNT or

2,6-DNT.

2.2.1.6 Developmental Effects

No studies were located regarding developmental effects in humans or animals after inhalation exposure to

2,4- or 2,6-DNT.

2.2.1.7 Genotoxic Effects

No studies were located regarding genotoxic effects in humans or animals after inhalation exposure to 2,4- or

2,6-DNT.

Genotoxicity studies are discussed in Section 2.5.

2.2.1.8 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

unexposed 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 exposure to DNT, and possible exposure of

the workers to other chemicals. However, no significant increases in mortality from malignant neoplasms as
2,4- and 2,6-DINITROTOLUENE 17

2. HEALTH EFFECTS

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 again 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.

No studies were located regarding cancer in animals following inhalation exposure to 2,4- or 2,6-DNT.

2.2.2 Oral Exposure

No studies were located regarding health effects in humans following oral exposure to 2,4- or 2,6-DNT.

However, it is assumed that oral ingestion could be a secondary route for occupationally exposed humans.

2.2.2.1 Death

No studies were located regarding death in humans after oral exposure to 2,4- or 2,6-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

LD,,s that have been determined for rats after gavage dosing with 2,4-DNT range from 270 to 650 mgkg

(Ellis et al. 1978; Lee et al. 1975; Vernot et al. 1977); in mice, LD50s were reported to be between 1,340 and

1,954 mg/kg after 2,4-DNT administration (Ellis et al. 1978; Lee et al. 1975; Vernot et al. 1977). In a

dominant lethal study by Lane et al. (1985), 8 of 15 male Sprague-Dawley rats died after receiving 5 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).

Death has been reported after intermediate- and chronic-duration exposure to 2,4-DNT in numerous studies.

One of 8 male and 8 of 8 female CD rats died after 3-13 weeks of ingesting 2,4-DNT in the diet (Lee et al.

1978,1985). 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 347-413 mg/kg 2,4-DNT in the diet for up to 6 months (Hong et al.

1985; Kozuka et al. 1979; Lee et al. 1978). 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
2,4- and 2,6-DINITROTOLUENE 18

2. HEALTH EFFECTS

22 mg/kg/day 2,4-DNT or mice fed up to 76 mg/kg/day 2,4-DNT for 78 weeks (NCI 1978). In a

3-generation reproductive study, there appeared to be an increased incidence of death among F,, dams during

parturition after receiving 45.3 mg/kg/day 2,4-DNT in the diet for 6 months (Ellis et al. 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 (Ellis et al. 1979).

In a 13-week study, some dogs fed 25 mg/kg/day became moribund after 22 or more days and had to be

terminated, whereas no treatment-related deaths were reported in dogs fed 5 mg/kg/day (Ellis et al. 1985; Lee

et al. 1978). 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. 1979, 1985). 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-l mice at 898 mg/kg/day (Ellis et al. 1979; Hong et al. 1985; Lee et al.

1978,1985).

The experimental data are more limited for 2,6-DNT than for 2,4-DNT. After administration of 2,6-DNT,

LD50s have been reported to range from 180 to 795 mg/kg in rats and from 621 to 807 mg/kg in mice (Ellis et

al. 1978; Lee et al. 1975; Vernot et al. 1977). 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).

Intermediate-duration studies have also 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 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 (Lee et al.

1976). No treatment-related deaths were reported when rats were fed up to 155 mg/kg/day 2,6-DNT for the

same duration (Lee et al. 1976). Two of 8 dogs treated with 20 mg/kg 2,6-DNT by capsule died in a 13-week

study (Lee et al. 1976). Thus, dogs seem to be the most sensitive of the three species to intermediateduration

oral 2,6-DNT exposure.
2,4- and 2,6-DINITROTOLUENE 19

2. HEALTH EFFECTS

Administration of up to 150 mg/kg/day Tg-DNT for 14 days was lethal to 6 of 13 pregnant Fischer-344 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).

For 2,4-DNT, all LOAEL values from each reliable study for death in each species and duration category are

recorded in Table 2-l and plotted in Figure 2-l. For 2,6-DNT, all LOAEL values from each reliable study

for death in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2.

2.2.2.2 Systemic Effects

No studies were located regarding musculoskeletal effects in humans or animals after oral exposure to

2,4-DNT or 2,6-DNT. The systemic effects observed after oral exposure are discussed below.

For 2,4-DNT, the highest NOAEL values and all LOAEL values from each reliable study for systemic effects

in each species and duration category are recorded in Table 2-l and plotted in Figure 2-l. For 2,6-DNT, all

LOAEL values from each reliable study for systemic effects in each species and duration category are

recorded in Table 2-2 and plotted in Figure 2-2.

Respiratory Effects. No studies were located regarding respiratory effects in humans after oral exposure

to 2,4-DNT or 2,6-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).
























2,4- and 2,6-DINITROTOLUENE 44

2. HEALTH EFFECTS

Cardiovascular Effects. No studies were located regarding cardiovascular effects in humans after oral

exposure to 2,4-DNT or 2,6-DNT.

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).

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 2,4-DNT or 2,6-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) 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).

Hematological Effects. No studies were located regarding hematological effects in humans after oral

exposure to 2,4-DNT or 2,6-DNT.

Hematological effects have been noted in virtually all animal studies of oral exposure to 2,4-DNT, 2,6-DNT,

and Tg-DNT in which circulating blood has been examined. The most common findings are

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

[RBCs]) and is seen as a compensatory mechanism in anemia resulting from exposure to 2,4- and 2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 45

2. HEALTH EFFECTS

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 (Ellis et al. 1979; Smith 1996).

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). Kozuka et al.

(1979) found methemoglobin concentrations increased to 7 times those of controls in the blood of rats fed

347 mg/kg/day 2,4-DNT in the diet for 6 months. Anemia was observed in a 3-week feeding study in which

male and female CD rats were fed 145 m/kg/day 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. 1978, 1985). 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-l mice administered 413 mg/kg/day and 468 mg/kg/day, 2,4-DNT, respectively in the diet for

13 weeks (Hong et al. 1985; Lee et al 1978). 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; Lee et al. 1978).

Subchronic administration of 2,6-DNT to dogs, rats, and mice resulted in hematological effects in dogs and

rats at concentrations of 20 and 35 mg/kg/day, respectively (Lee et al. 1976). Dogs were found to be anemic

and showed signs of compensatory reticulocytosis at this concentration, and rats showed hemosiderosis and

extramedullary hematopoiesis. Statistically significant hematological effects were not observed in mice at

levels up to 289 mg/kg/day 2,6-DNT but were observed in individual animals (Lee et al. 1976). Dogs

administered 4 mg/kg/day 2,6-DNT by capsule for 13 weeks were found to have extramedullary

erythropoiesis and lymphoid depletion of the spleen (Lee et al. 1976). An intermediate-duration oral MRL of

0.004 mg/kg/day for 2,6-DNT was derived from this LOAEL as described in the footnote in Table 2-2. The

2,6-DNT isomer was not tested for hematological endpoints in studies of chronic duration.

Chronic studies of animals administered 2,4-DNT fortify the weight-of-evidence supporting hematological

effects. In 24-month studies, hematological effects were observed, but the animals often exhibited

“compensated anemia,” an adaptive response to 2,4-DNT exposure (Ellis et al. 1979). Methemoglobinemia

and the presence of Heinz bodies were observed in dogs administered 1.5 mg/kg/day in capsules; no effect

was observed at 0.2 mg/kg/day (Ellis et al. 1985; Lee et al. 1978). The NOAEL of 0.2 mg/kg/day in this dog

study was used as the basis for a chronic oral MRL of 0.002 mg/kg/day for 2,4-DNT. In a 2-year study (with
2,4- and 2,6-DINITROTOLUENE 46

2. HEALTH EFFECTS

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 REK count were found in mid-dose males

compared to controls, and anemia was found in high-dose animals after 1 year (Ellis et al. 1979; Lee et al.

1978, 1985). No changes in methemoglobin or Heinz bodies were found, however. 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 at the

high concentration, with compensatory increases in reticulocytes (Ellis et al. 1979; Hong et al. 1985).

Hematological changes consistent with those observed in anemia were found in pregnant Fischer-344 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 REXs along with hemosiderosis and extramedullary hematopoiesis in males, and increases in mean cell

volume (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 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

DNT indicates that the blood is a primary target of DNT toxicity.
2,4- and 2,6-DINITROTOLUENE 47

2. HEALTH EFFECTS

Musculoskeletal Effects. No studies were located regarding musculoskeletal effects in humans or

animals after oral exposure to 2,4-DNT, 2,6-DNT, or Tg-DNT.

Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to 2,4- or

2,6-DNT.

The hepatotoxic effects of DNT 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 DNT include liver discoloration and inflammation, alteration

of hepatocytes, proliferation of bile duct epithelium, and hyperplastic foci.

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.

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; Lee et al. 1978).

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; Lee et al. 1978).

The most severe hepatotoxicity was found in rats. Concentrations of 2,4-DNT as low as 0.6 mg/kg/day

caused foci of altered hepatocytes or hyperplastic nodules when fed to male rats for 1 year (Ellis et al. 1979;

Lee et al. 1978, 1985). Hepatocellular degeneration and vacuolation accompanied by acidophilic foci and

occasional basophilic foci of cellular alteration were found in Fischer-344 rats fed 27 mg/kg/day 2,4-DNT for

52 weeks (Leonard et al. 1987). The incidences of focal areas of 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 dietary concentrations

of 347-395 mg/kg/day 2,4-DNT 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
2,4- and 2,6-DINITROTOLUENE 48

2. HEALTH EFFECTS

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 (Ellis et al. 1979; Hong et al. 1985). Administration of 1.5 mg/kg/day 2,4-DNT

for 24 months resulted in biliary hyperplasia in dogs; this effect was not seen in dogs administered

0.2 mg/kg/day (Ellis et al. 1979, 1985).

Six weeks of dietary consumption of 7 mg/kg/day 2,6-DNT caused a 380% increase in epoxide hydrolase

(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 and dogs with 2,6-DNT for 13 weeks resulted in liver toxicity (Lee et al. 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 (Lee et

al. 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 (Lee et al. 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 Fischer-344 rats (Leonard et al. 1987).

Irregular liver surfaces were found in male Fischer-344 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

Fischer-344 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).

Renal Effects. No studies were located regarding renal effects in humans after oral exposure to 2,4- or

2,6-DNT.

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. Oral administration of 2,4-DNT to mice
2,4- and 2,6-DINITROTOLUENE 49

2. HEALTH EFFECTS

(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; Lee et al. 1978). 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 (Ellis et al.

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.

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 (Lee et al. 1976). No treatment-related effects on the kidney were found when

rats were fed 2,6-DNT for 13 weeks (Lee et al. 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 (Lee et

al. 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, blood urea nitrogen (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).

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-l mice fed 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.

Endocrine Effects. No studies were located regarding endocrine effects in humans after oral exposure to

2,4- or 2,6-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 Sprague-Dawley 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
2,4- and 2,6-DINITROTOLUENE 50

2. HEALTH EFFECTS

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).

Dermal Effects. No studies were located regarding dermal effects in humans after oral exposure to 2,4- or

2,6-DNT.

Concentrations of up to 261 mgkg/day 2,4-DNT for males or 273 mgkg/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).

Ocular Effects. No studies were located regarding ocular effects in humans after oral exposure to 2,4- or

2,6-DNT.

The eyes of male and female Sprague-Dawley rats administered up to 261 mg/kg/day 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).

Body Weight Effects. No studies were located regarding body weight effects in humans after oral

exposure to 2,4- or 2,6-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. 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 inpalatability.
2,4- and 2,6-DINITROTOLUENE 51

2. HEALTH EFFECTS

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; Ellis et al. 1979,

1985; Hazleton Laboratories 1982; Hong et al. 1985; Kozuka et al. 1979; Lane et al. 1985; Lee et al. 1978,

1985; Leonard et al. 1987; McGown et al. 1983; NCI 1978). In an acute study, rats dosed by gavage with

240 mg/kg 2,4-DNT for 5 days lost weight (Lane et al. 1985). 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 (Ellis et al. 1979; Lee et al. 1978, 1985). This reduction in body

weight gain tended to become more pronounced when 2,4-DNT was continued for periods of l-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- and chronic-duration exposure, but the concentrations of the test article

needed to evoke this effect were considerably higher than in rats (Hong et al. 1985; Lee et al. 1978; NCI

1978). 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).

Administration of 2,6-DNT also caused decreased body weight gain or body weight loss in rats, mice, and

dogs at concentrations ranging from 14 to 145 mg/kg/day in intermediate-duration studies (Lee et al. 1976).

Treatment with 7 mg/kg/day 2,6-DNT decreased body weight in rats at 52 weeks by 18% (Leonard et al.

1987).

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).

2.2.2.3 Immunological and Lymphoreticular Effects

No studies were located regarding immunological and lymphoreticular effects in humans after oral exposure

to 2,4- or 2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 52

2. HEALTH EFFECTS

Testing for immunological effects of DNT is limited. 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. 1978, 1985). 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 (Lee et al. 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-DNT 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 (Lee et al. 1976).

For 2,4-DNT, the highest NOAEL values and all LOAEL values from each reliable study for immunological/

lymphoreticular effects in each species and duration category are recorded in Table 2-l and plotted

in Figure 2- 1. For 2,6-DNT, the highest NOAEL values and all LOAEL values from each reliable study for

immunological/lymphoreticular effects in each species and duration category are recorded in Table 2-2 and

plotted in Figure 2-2.

2.2.2.4 Neurological Effects

No studies were located regarding neurological effects in humans after oral exposure to 2,4- or 2,6-DNT.

Severe clinical signs of neurotoxicity, including incoordination and stiffness that led to an abnormal gait were

observed in dogs given 25 mg/kg/day 2,4-DNT in capsules for 12 days (Ellis et al. 1985; Lee et al. 1978).

These effects progressed with time and this dose was lethal after 22 days. The NOAEL for the neurotoxicity

observed after 12 days was 5 mglkglday. An acute-duration oral MRL of 0.05 mg/kg/day was derived based

on this NOAEL as described in the footnote in Table 2-l. 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.
2,4- and 2,6-DINITROTOLUENE 53

2. HEALTH EFFECTS

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. 1978, 1985). After 3 months of being

fed 2,4-DNT in the diet, 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; Lee et al. 1978). No neurological signs

were observed in mice fed 4 13 mgIkg/day in males or 468 mg/kg/day in females 2,4-DNT in the diet for

13 weeks (Hong et al. 1985; Lee et al. 1978).

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 mg/kg/day or 45.3 mg/kg/day 2,4-DNT to

male and female rats, respectively, for up to 2 years (Ellis et al. 1979; Lee et al. 1978, 1985). This stifflegged

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. 1979,1985). Dogs dosed at 1.5 mg/kg/day 2,4-DNT

in a 2-year study showed loss of hindquarter control (Ellis et al. 1979, 1985). 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. 1979, 1985).

Dogs dosed with 20 or 100 mg/kg/day of 2,6-DNT for 13 weeks exhibited dose-dependent neurotoxic

symptoms that included muscular incoordination, weakness, tremors, and paralysis (Lee et al. 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 (Lee et al. 1976).

Administration of 150 mg/kg/day Tg-DNT to Fischer-344 dams during gestation days 7-20 caused hindlimb

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).

Neurotoxicity appears to be a characteristic syndrome of 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.
2,4- and 2,6-DINITROTOLUENE 54

2. HEALTH EFFECTS

For 2,4-DNT, the highest NOAEL values and all LOAEL values from each reliable study for neurological

effects in each species and duration category are recorded in Table 2-l and plotted in Figure 2-l. For

2,6-DNT, the highest NOAEL values and from each reliable study for neurological effects in each species and

duration category are recorded in Table 2-2 and plotted in Figure 2-2.

2.2.2.5 Reproductive Effects

No studies were located regarding reproductive effects in humans after oral exposure to 2,4- or 2,6-DNT.

Studies in laboratory animals have shown that oral exposure to 2,4-DNT can result in adverse effects on

reproduction. This is evidenced by decreased fertility as well as by 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; Ellis et al. 1979; Kozuka et al. 1979; Lane et al. 1985; Lee et al.

1976, 1978; McGown et al. 1983). In the female reproductive system, ovarian atrophy and dysfunction were

observed (Ellis et al. 1979).

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). Sprague-Dawley rats administered 104, 165, or 261 mg/kg/day

2,4-DNT in the diet for 14 days exhibited oligospermia with degenerative changes, such as syncytial cell

formation and focal spermatic granuloma, in a dose-dependent manner (McGown et al. 1983). A

concentration 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 3 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
2,4- and 2,6-DINITROTOLUENE 55

2. HEALTH EFFECTS

decreased fertility, although no dominant lethal effect was observed (Ellis et al. 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. 1978, 1985). 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 4 13 mg/kg/day 2,4-DNT for 13 weeks

(Hong et al. 1985; Lee et al. 1978) and rats fed 347-395 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 (Lee et al. 1978). The decreased

fertility was not observed in mice fed 295 mg/kg/day (Lee et al. 1978). 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; Lee et al. 1978). No testicular effects were found at 5 mg/kg in the study.

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

effects. Rats that received 0.6 mg/kg/day 2,4-DNT in the diet for up to 2 years had an increased incidence of

seminiferous tubule atrophy (29%) compared to controls (16%) (Ellis et al. 1979; Lee et al. 1978, 1985).

This was the lowest concentration used in this study in which dose-related changes were found. At the

highest dose, 35 mg/kg/day, an 8 1% increase in seminiferous tubule atrophy and spermatogenesis was

reported. Similar changes were found in male CD-l mice fed 14 mg/kg/day 2,4-DNT for 24 months (Hong et

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

follicles and, therefore, a lack of corpora lutea (Hong et al. 1985). The NOAEL for these effects was

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

24 months (Ellis et al. 1979,1985).

Histopathological examination of the testes after treatment with 2,4-DNT has revealed changes which

suggest specific causes for the male infertility observed in animal studies. Dose-dependent 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 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).
2,4- and 2,6-DINITROTOLUENE 56

2. HEALTH EFFECTS

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 (Ellis et al. 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. Decreases in the number of fetal implants were attributed to the adverse impact of 2,4-DNT on

sperm production.

In studies of rats, mice, and dogs dosed with 2,6-DNT for 13 weeks (Lee et al. 1976), decreased

spermatogenesis was observed in male mice administered 5 1 mg/kg/day, but normal spermatogenesis was

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 (Lee et al. 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 (Lee et al. 1976).

Based upon the testicular effects observed after administration of 2,4-DNT or 2,6-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).

The highest NOAEL values and all reliable LOAEL values for reproductive effects in each species and

duration category are recorded in Tables 2-l and 2-2 and plotted in Figures 2-l and 2-2.

2.2.2.6 Developmental Effects

No studies were located regarding developmental effects in humans after oral exposure to 2,4- or 2,6-DNT.

However, developmental toxicity from DNT could potentially occur because exposure to any substance that

depletes the amount of oxygen available to developing fetal tissues can have adverse consequences. The

hematological (and potential oxygen depleting) effects of DNT are reported in Section 2.2.1.2.
2,4- and 2,6-DINITROTOLUENE 57

2. HEALTH EFFECTS

Ellis et al. (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 (Ellis et al. 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.

The highest NOAEL values and all reliable LOAEL values for developmental effects in each species and

duration category are recorded in Tables 2-l and 2-3 and plotted in Figures 2-l and 2-3.

2.2.2.7 Genotoxic Effects

Studies of the effects of various DNT isomers on sperm morphology (Soares and Lock 1980), spermatocyte

DNA repair (Working and Butterworth 1984), and dominant lethal mutations (Ellis et al. 1979; Hodgson et

al. 1976; Soares and Locke 1980) were generally negative for these specific endpoints.

Other genotoxicity studies are discussed in Section 2.5.

2.2.2.8 Cancer

No studies were located regarding cancer in humans after oral exposure to 2,4- or 2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 58

2. HEALTH EFFECTS

The carcinogenic activity of DNT 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 (Ellis et al. 1979; Lee et al. 1978, 1985).

2,4-DNT (98% 2,4-DNT, 2% 2,6-DNT) produced renal tumors (76%) in male CD-l mice fed 95 mg/kg/day

for 2 years (Ellis et al. 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 Ellis et al. (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 (Ellis et al. 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

Fischer-344 strain, lower doses, and shorter exposure durations than did Ellis et al. (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. Ellis et al. (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 Fischer-344 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).
2,4- and 2,6-DINITROTOLUENE 59

2. 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 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 the 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 AR, which is

AlN-76A supplemented with 5% pectin. The number and size of γ-glutamyl transpeptidase-staining foci in

the liver increased in a dose- and time-dependent manner in animals given 2,6-DNT in the NM-07 diet.

Hepatocellular carcinomas and neoplastic nodules were observed only in rats fed NM-07 containing

2,6-DNT. No tumor was observed in rats receiving the control diets or 2,6-DNT in the AlN-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.
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2. 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

initiationpromotion 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.

2.2.3 Dermal Exposure

There are data on occupational exposure of humans to 2,4-DNT and Tg-DNT (see Section 2.2.1) 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 and may account for the quantity of

excreted urinary metabolites that exceeded the amount of inhaled DNT in the operators and loaders.

2.2.3.1 Death

One study was located that examined death among humans exposed to DNT. 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). 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.

No studies were located regarding death in animals after dermal exposure to 2,4- or 2,6-DNT.

2.2.3.2 Systemic Effects

No studies were located regarding respiratory, cardiovascular, renal, body weight, or endocrine effects in

humans or animals after dermal exposure to 2,4-DNT or 2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 61

2. HEALTH EFFECTS

Gastrointestinal Effects. Gastrointestinal complaints of munitions workers exposed to either 2,4-DNT

or Tg-DNT included nausea and vomiting (McGee et al. 1942, 1947). These workers also presumably

inhaled DNT in the occupational setting.

No studies were located regarding gastrointestinal effects in animals after dermal exposure to 2,4-DNT or

2,6-DNT.

Hematological Effects. Hematological effects, such as anemia and cyanosis, have been found in men

employed at munitions factories (McGee et al. 1942, 1947; Perkins 1919). These workers were exposed to

either 2,4-DNT or Tg-DNT. Because these studies lacked worker histories, exposure data, and 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 2,4-DNT or

2,6-DNT.

Musculoskeletal Effects. 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. 1942; 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 2,4-DNT or

2,6-DNT.

Hepatic Effects. In a follow-up 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 DNT via inhalation.
2,4- and 2,6-DINITROTOLUENE 62

2. HEALTH EFFECTS

No studies were located regarding hepatic effects in animals after dermal exposure to 2,4- or 2,6-DNT.

Dermal Effects. The only human studies that noted dermal effects upon topical exposure to 2,4-DNT were

of workers employed by a munitions factory during World War II. The first study found 6 of 154 workers

who complained of dermatitis, which the authors attributed to DNT exposure (McGee et al. 1942). A followup

study, conducted after changes in the manufacturing process designed to reduce DNT exposure were

implemented , reported that 32 of 714 workers complained of dermatitis (McGee et al. 1947). Exposure

levels were not quantified in either of these studies.

Both 2,4- and 2,6-DNT were shown to be mild primary dermal irritants in rabbits (Ellis et al. 1978; Lee et al.

1975).

Ocular Effects. No studies were located regarding ocular effects in humans after dermal exposure to 2,4-

or 2,6-DNT.

No ocular irritation was found in rabbits in a primary eye irritation test using unspecified concentrations of

2,4- or 2,6-DNT (Ellis et al. 1978; Lee et al. 1975). However mild eye irritations were reported in rabbits

treated with 2,4- and Tg-DNT (Ford 1981; Henry 1982).

2.2.3.3 Immunological and Lymphoreticular Effects

No studies were located regarding immunological and lymphoreticular effects in humans after dermal

exposure to 2,4-DNT or 2,6-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 (Ellis et al. 1978; Lee et al. 1975).

2.2.3.4 Neurological Effects

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. 1942, 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.
2,4- and 2,6 DINITROTOLUENE 63

2. HEALTH EFFECTS

No studies were located regarding neurological effects in animals after dermal exposure to 2,4- or 2,6-DNT.

No studies were located regarding the following health effects in humans or animals after dermal exposure to

2,4- or 2,6-DNT:

2.2.3.5 Reproductive Effects

2.2.3.6 Developmental Effects

2.2.3.7 Genotoxic Effects

Genotoxicity studies are discussed in Section 2.5.

2.2.3.8 Cancer

No studies were located regarding cancer in humans or animals after dermal exposure to 2,4- or 2,6-DNT.

2.3 TOXICOKINETICS

2.3.1 Absorption

No information regarding absorption of DNT in children has been located.

2.3.1.l Inhalation Exposure

There are no available studies in which the absorption rates of inhaled 2,4- or 2,6-DNT in humans or laboratory

animals have been evaluated. However, based on analyses of the urinary metabolites of workers in DNT

manufacturing plants(Levine et al. 1985b; Turner 1986; Woollen et al. 1985), it is apparent that dermal

absorption of these two compounds does occur under occupational settings.
2,4- and 2,6-DINITROTOLUENE 64

2. HEALTH EFFECTS

2.3.1.2 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 DNT is 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 DNT in the urine, primarily

within the first 24 hours (Lee et al. 1978; Long and Rickert 1982; Rickert and Long 1981). 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% in the urine (Lee et al. 1978). Increased fecal excretion could be due to reduced

absorption or to greater excretion via the bile.

2.3.1.3 Dermal Exposure

There were no data available specifically on the absorption of 2,4- or 2,6-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 these isomers 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).

2.3.2 Distribution

No information regarding distribution of DNT in children has been located.

2.3.2.1 Inhalation Exposure

No studies were located regarding distribution in humans or animals following inhalation exposure to 2,4- or

2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 65

2. HEALTH EFFECTS

2.3.2.2 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 per

kilogram. When distribution is studied solely by detecting a radioisotope label, it is the labeled atom(s)

which 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 2 stages,

with the first peak occurring l-2 hours and a second peak occurring 8-12 hours after an oral 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 (Lee et al.

1978). 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 (Lee et al.

1975, 1978).
2,4- and 2,6-DINITROTOLUENE 66

2. HEALTH EFFECTS

2.3.2.3 Dermal Exposure

No studies were located regarding distribution in humans or animals following dermal exposure to 2,4- or

2,6-DNT.

2.3.3 Metabolism

The metabolism of DNT 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-amino-2-nitro-, and 2-amino-6-nitrobenzoic acids, and 4-(Nacetyl)

amino-2-nitrobenzoic acid. Trace levels of DNT 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-amino4-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- 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 (less than 1% of

urinary metabolites) of 2-(N-acetyl)amino-4nitrobenzoic acid (Levine et al. 1985b).

Studies in rats have identified a complex pathway for the metabolism of 2,4-DNT (Figures 2-3 and 2-4) and

2,6-DNT (Figure 2-5). Metabolism occurs in the liver and also in the intestine by microflora (Long and



2,4- and 2,6-DINITROTOLUENE 70

2. HEALTH EFFECTS

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-2nitrobenzoic acid (Rickert et al. 1981).

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, DNT appears to be first metabolized by the liver with

themetabolites 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 2,4dinitrobenzyl

alcohol, 2,4-dinitrobenzaldehyde and 2,4-dinitrobenzoic acid (0.09-o. 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.

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 DNT. 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 hepatocarcinogenesis by 2,6-DNT. Metabolism by
2,4- and 2,6-DINITROTOLUENE 71

2. HEALTH EFFECTS

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-diarninotoluene 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’-dimethyl-3,3’-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 glucuromde 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 (Ellis et al. 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-diaminotoluene, 2,4-aminobenzyl
2,4- and 2,6-DINITROTOLUENE 72

2. HEALTH EFFECTS

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 (Lee et al. 1978).

Another study using rats dosed with either 2,4- or 2,6-DNT also demonstrated that the primary urinary

conjugate was the respective dinitrobenzyl 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-amino2-nitrobenzoic acid (0.52%), 4-acetylamino-2-nitrobenzoic acid (3.9%), 4-amino-2-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 2,6dinitrobenzoic acid

(0.17%), 2-amino-6-nitrotoluene (0.44%), and 2,6-dinitrobenzyl alcohol (0.53%) (Mori et al. 1996).

The urinary metabolites of DNT 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 DNT 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 P-450 isoforms responsible for

DNT metabolism have not been identified, cytochromes CYP2E1, CYP2B l/2, and CYP2Cl l/6 are known to

contribute to the side-chain oxidation of toluene by the rat liver, and multiple cytochrome P-450 isoforms

may contribute to the side-chain oxidation of DNT (Chapman et al. 1993). In humans, CYP2El 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 quantitively different from the

expression observed in adults (Komori et al. 1990; Leeder and Kearns 1997). In rats, while sulfotransferase

(the enzyme which catalyzes sulfation) activity is almost at adult levels at birth, UDP-glucuronosyltransferase

(the enzyme which 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 Keams 1997). While the activity of some isoforms of sulfotransferase

may exceed those seen in adults during infancy and early childhood, the activity of UDP-lucuronosyltransferase
2,4- and 2,6-DINITROTOLUENE 73

2. HEALTH EFFECTS

depends on the specific isoforms of the enzyme, and adult levels are generally attained by 6-18 months

(Leeder and Kearns 1997). Since DNT undergoes bioactivation in the liver and by the intestinal microflora,

the toxicity of DNT 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 DNT.

2.3.4 Elimination and Excretion

Information regarding excretion of DNT in children was not located.

2.3.4.1 Inhalation Exposure

In occupational settings, in addition to inhalation, some oral and dermal exposure can occur. The elimination

of DNT 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 DNT. 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.

2.3.4.2 Oral Exposure

No studies were located regarding excretion in humans following oral exposure to 2,4- or 2,6-DNT.

Schut et al. (1983) reported that in mice, urine was the main route of elimination of 3

H-labeled 2,6-DNT, with

about 50% excreted after 8 hours. Lee et al. (1978) observed that most of the radioactivity from 14C-labeled
2,4- and 2,6-DINITROTOLUENE 74

2. HEALTH EFFECTS

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.

2.3.4.3 Dermal Exposure

There are no kinetic data in humans in which the route of exposure was specifically dermal.

Occupationalexposure 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 2.3.4.1.

No studies were located regarding excretion in animals following dermal exposure to 2,4- or 2,6-DNT.
2,4- and 2,6-DINITROTOLUENE 75

2. HEALTH EFFECTS

2.3.5 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 externaVexposure concentration and target tissue dose of

the toxic moiety, and (2) the target tissue dose and observed responses (Andersen et al. 1987; Andersen and

Ktishnan 1994). 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 parametrization, (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 substance-specific

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.

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) is adequately described,

however, this simplification is desirable because data are often unavailable for many biological processes. A
2,4- and 2,6-DINITROTOLUENE 76

2. HEALTH EFFECTS

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 2-6 shows a

conceptualized representation of a PBPK model.

If PBPK models for 2,4- and 2,6-dinitrotoluene 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 2,4- or 2,6-DNT.

2.4 MECHANISMS OF ACTION

The mechanisms of action of DNT is not known to be different in children.

2.4.1 Pharmacokinetic Mechanisms

No information was located regarding the mechanism of absorption of 2,4- or 2,6-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 (Lee et al.

1978; Long and Rickert 1982; Rickert and Long 1981). 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 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,

2,4- and 2,6-DINITROTOLUENE 78

2. HEALTH EFFECTS

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).

2.4.2 Mechanisms of Toxicity

Effects of Metabolism on Toxicity. The primary mechanism of toxicity for DNT involves bioactivation to

form reactive intermediates (Kedderis et al. 1984; Sayama et al. 1989). Detailed information on the

biotransformationof DNT is presented in Section 2.3.3. Briefly, metabolism of DNT 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

Nhydroxylated 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 DNT is described by Ellis

et al. (1979). The effect of DNT 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 reticulocytes, in the blood. If the toxic dose is not

too severe, these compensatory mechanisms suffice. Thus, “compensated anemia,” normal erythrocyte levels
2,4- and 2,6-DINITROTOLUENE 79

2. HEALTH EFFECTS

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 (Ellis et al. 1979).

Carcinogenesis. In hepatic tumor initiation-promotion experiments, Tg-DNT was found to have tumor

promoting and tumor-initiating activity (Leonard et al. 1983, 1986; Mirsalis and Butterworth 1982).

2,6 DNT was indicated to be a complete hepatocarcinogen and is primarily responsible for the carcinogenic

activity of Tg-DNT. Hepatic DNA adducts have been detected by 32P-postlabeling technique in 2,6-DNTtreated

B6C3F, mice and Fischer 344 rats (George et al. 1996). 2,6-Dinitrobenzaldehyde, one of the

metabolites of 2,6-DNT, was found to be a direct-acting mutagen in the Salmonella typhimurium strain

TA98 and Tal00 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. 1983; Sayama et al.

1989). Kedderis et al. (1984) proposed a bioactivation mechanism relating to the genotoxicity of 2,6-DNT in

male Fischer 344 rats. They showed that the active metabolite of 2,6-DNT in the male Fischer 344 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.

2.4.3 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. 1979,1985; Hong et al. 1985; Lane et al. 1985; Lee et al.

1978, 1985; McGee et al. 1942,1947). Other effects for 2,4- and 2,6-DNT, such as reproductive, hepatic,

renal, and cancer have been noted in animals (Ellis et al. 1979, 1985; Hong et al. 1985; Lee et al. 1976,1978,

1985; Leonard et al. 1983, 1986; McGown et al. 1983; Stoner et al. 1984), 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-

and 2,6-DNT than are mice (Ellis et al. 1978; Hong et al. 1985; Lane et al. 1985; Lee et al. 1975, 1978;

Vemot et al. 1977), dogs appear to be the most sensitive of the three species (Ellis et al. 1979, 1985; Lee et

al. 1976, 1978). However, limited intermediate-duration data using 2,6-DNT have shown mice to be more

sensitive than rats (Lee et al. 1976). It should be noted that dogs were fed DNT by capsule in experimental

studies, whereas the rodents received the test chemical in feed (Ellis et al. 1979, 1985; Hong et al. 1985; Lee

et al. 1975, 1976, 1978, 1985).
2,4- and 2,6-DINITROTOLUENE 80

2. HEALTH EFFECTS

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.

2.5 RELEVANCE TO PUBLIC HEALTH

Issues relevant to children are explicitly discussed in 2.6 Children’s Susceptibility and 5.6 Exposures of

Children.

Overview.

The major route of exposure to DNT for humans living near hazardous waste sites is via ingestion of

contaminated water. Because of its low octanol-water partition coefficient, DNT is not expected to

accumulate in homegrown fruits and vegetables. Dermal exposure to DNT could also occur when washing or

bathing with contaminated water. The low vapor pressure of DNT makes inhalation unlikely, although it

could possibly be present in particulate matter in ambient air from leaks in storage containers or from

contaminated soil.

Data for humans exposed to DNT are derived from occupational studies in which exposure concentrations

via the inhalation route were measured. Some dermal exposure may be expected to occur in the workplace

although this should be minimized with modem industrial hygiene practices. No studies were located

regarding health effects in animals following inhalation exposure to DNT. No studies were located regarding

health effects in humans following oral exposure to DNT. Available animal data on the toxic effects of DNT

were derived from oral exposure studies. Discussions of health effects observed in animals and humans are

thus complicated by the need to compare toxicity across different exposure routes. Thus, the effects observed

from inhalation exposure studies in humans may not accurately reflect the effects that might be predicted

after oral exposure to DNT near hazardous waste sites. However, there does appear to be some suggestive

evidence of common target organs following exposure of animals and humans to DNT by different routes.

Possible effects of exposure of humans to primarily Tg-DNT include heart disease, hematological effects, and

neurological effects.

In animal species, 2,4-DNT caused adverse effects in a variety of organs and tissues including the blood,

nervous system, liver, kidney, and gonads. Subcutaneous and mammary gland carcinomas were also
2,4- and 2,6-DINITROTOLUENE 81

2. HEALTH EFFECTS

observed. 2,6-DNT affected the blood in all animal species tested. Effects on the liver or bile duct were

generally observed, and males of all species exhibited adverse effects on the reproductive system. Two toxic

effects, neurotoxicity and renal toxicity, were observed in dogs orally administered 2,6-DNT that were not

observed in other animal species.

Minimal Risk Levels for 2,4- and 2,6-Dinitrotoluene

Inhalation MRLS.

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

2,4-DNT or 2,6-DNT. No suitable NOAEL or LOAEL was found for any duration category.

Oral MRLS.

Studies in humans did not provide sufficient data regarding exposure levels and their correlation with

observed effects. Therefore, animal studies were used for the derivation of the oral MBLs for both 2,4- and

2,6-DNT.

2.4-DNT

• An acute-duration oral MRL of 0.05 mg/kg/day was derived for 2,4-DNT from a NOAEL value of

5 mg/kg/day for neurotoxicity observed in dogs (Ellis et al. 1985; Lee et al. 1978). Male and female

beagle dogs were dosed by capsule with 0, 1,5, or 25 mg/kg/day 2,4-DNT in intermediate- and

chronic-duration studies. However, after 12 days of treatment, the first clinical signs of neurotoxicity

were evident. Minimal signs of neurotoxicity were incoordination and stiffness, giving the animals an

abnormal gait. No clinical signs of neurotoxicity were observed at 5 mg/kg/day. The NOAEL of

5 mg/lcg/day was divided by an uncertainty factor (UF) of 100 (10 for animal-to-human extrapolation,

and 10 for human variability).

• A chronic-duration oral MRL of 0.002 mg/kg/day was derived for 2,4-DNT from a NOAEL value of

0.2 mg/kg in dogs (Ellis et al. 1979, 1985). Beagle dogs were administered 0,0.2, 1.5, or 10 mgkg

2,4-DNT in capsules for up to 24 months. Methemoglobinemia and Heinz bodies were observed in

dogs fed 1.5 mg/kg. Biliary hyperplasia and neurotoxicity (paralysis and cerebellar lesions) were also

noted at this dose. No testicular degeneration was observed up to 10 mgkg 2,4-DNT. The NOAEL of

0.2 mgkg for hematological and neurological effects and biliary hyperplasia was divided by an

uncertainty factor of 100 (10 for animal-to-human extrapolation and 10 for human variability).
2,4- and 2,6-DINITROTOLUENE 82

2. HEALTH EFFECTS

2,6-DNT

Data were insufficient for the derivation of acute-duration oral MRLs for 2,6-DNT. No suitable NOAEL or

LOAEL was found.

• An intermediate-duration oral MRL of 0.004 mg/kg/day for 2,6-DNT was derived from a LOAEL

value of 4 mg/kg, at which extramedullary erythropoiesis in the spleen and lymphoid depletion were

observed in dogs (Lee et al. 1976). Beagle dogs were administered 0,4,20, or 100 mg/kg 2,6-DNT in

capsules for up to 13 weeks. Treatment-related mortality occurred at 20 and 100 mg&g 2,6-DNT. No

neurological effects were found in the 4-mg/kg group, but at 20 mg/kg, listlessness, incoordination, and

lack of balance were found; effects became more severe at 100 mg/kg and progressed to paralysis,

occasional tremors, and inability to eat. Body weight loss correlated with food consumption at 20 and

100 mg/kg. Anemia and compensatory reticulocytosis were also found at 20 and 100 mg!kg. Other

treatment-related effects observed at mid and/or high dose were thymic involution, bile duct

hyperplasia, testicular degeneration, hepatic inflammation, and dilated renal tubules. None of these

effects were observed in animals treated with 4 mg/kg. However, after 13 weeks, mild extramedullary

erythropoiesis in the spleen and lymphoid depletion were observed at 4 mg!kg. The LOAEL of

4 mgkg for hematological effects was divided by an uncertainty factor of 1,000 (10 for use of a

LOAEL, 10 for animal-to-human extrapolation, and 10 for human variability).

Data were insufficient for the derivation of chronic-duration oral MBLs for 2,6-DNT. No suitable NOAEL

or LOAEL was found.

Death.

The limited data available on inhalation exposure to 2,4-DNT or Tg-DNT suggest that there may be an

increase in death due to ischemic heart disease and diseases of the circulatory system (Levine et al. 1986a).

There was no increase found in death due to cancer in this study. Although no data are available regarding

death in animals after inhalation exposure to 2,4- or 2,6-DNT, and only one study reported an increased

incidence of death after occupational exposure to 2,4- or Tg-DNT, it appears unlikely that death would occur

in people living near hazardous waste sites and exposed to low levels of 2,4- or 2,6-DNT. No data are

available on death in humans from oral exposure to 2,4- or 2,6-DNT, but from animal data it can be

reasonably expected that consumption of these compounds may be fatal. Insufficient data are available to

predict whether dermal exposure to 2,4- or 2,6-DNT would cause death in humans.
2,4- and 2,6-DINITROTOLUENE 83

2. HEALTH EFFECTS

Systemic Effects.

Respiratory Effects. Because no studies are available in humans or animals examining the respiratory

endpoints after inhalation or dermal exposure to 2,4- or 2,6-DNT, it is not possible to predict whether

exposure to these compounds via these routes would cause respiratory effects in humans living near

hazardous waste sites. Human data are not available on respiratory effects after oral exposure to 2,4- or

2,6-DNT, but no effects on the respiratory system were found when rats were fed Tg-DNT for up to 2 years

(Hazleton Laboratories 1982).

Cardiovascular Effects. Excessive rates of mortality from ischemic heart disease and residual diseases of

the circulatory system were observed in workers exposed to Tg-DNT and 2,4-DNT at two separate facilities

(Levine et al. 1986a). However, this finding was unusual. The heart disease mortality risk of workers is

commonly lower than that of the general population because of the “healthy worker” effect. It is unlikely that

a chance aggregation of risk factors independent of DNT exposure could explain the observed excess of heart

disease mortality.

Although treatment-related cardiovascular lesions have not been reported in rat or mouse bioassays, these

species are generally resistant to naturally-occurring or experimentally-induced atherosclerosis (Jokinen et al.

1985). Interim sacrifices in a 2-year bioassay in rats did show an exacerbation of spontaneous myocarditis,

although it was not seen at study termination (Hazleton Laboratories 1982). Thus, insufficient data are

available to determine whether cardiovascular effects might result from living near hazardous waste sites.

Gastrointestinal Effects. Some gastrointestinal symptoms, such as nausea and vomiting, have been reported

after workers were exposed to 2,4-DNT, presumably via inhalation and dermal exposure (McGee et al. 1942,

1947). No animal studies are available regarding gastrointestinal effects after inhalation exposure to 2,4-

and 2,6-DNT. The only data available after oral exposure are from a study in which no histopathological

effects on the gastrointestinal tract were observed in rats fed Tg-DNT for up to 2 years (Hazleton

Laboratories 1982). Thus, insufficient data are available to predict whether low-level exposure to 2,4- or

2,6-DNT might result in gastrointestinal effects in persons living near hazardous waste sites.

Hematological Effects. Early observations of workers exposed to 2,4-DNT (levels of exposure not

described) report symptoms of cyanosis (McGee et al. 1942, 1947; Perkins 1919). McGee et al. (1942,

1947) also reported anemia. Occupational conditions at the time of these reports (pre-1950) undoubtedly
2,4- and 2,6-DINITROTOLUENE 84

2. HEALTH EFFECTS

resulted in higher levels of human exposure than would occur in modem manufacturing facilities.

Engineering, ventilation, and industrial hygiene improvements have reduced the levels of modem

occupational exposure to DNT. Unfortunately, recent studies of exposed workers (Ahrenholz 1980;

Ahrenholz and Meyer 1982; Hamill et al. 1982; Levine et al. 1985a) did not monitor hematological profiles.

In animal studies of intermediate duration, methemoglobinemia and its sequelae (Heinz bodies, anemia,

reticulocytosis), hemosiderosis, extramedullary hematopoiesis, and slight cyanosis (Lane et al. 1985) were

observed when 2,4-, 2,6-, and Tg-DNT were administered (Hazleton Laboratories 1977, 1982; Lee et al.

1976, 1978). Extramedullary erythropoiesis in the spleen and lymphoid depletion have been observed after

intermediate-duration treatment of dogs with 2,6-DNT (Lee et al. 1976). In animal studies of chronic

duration, anemia was observed (Ellis et al. 1979; Hazleton Laboratories 1982), but the animals often

appeared to adapt, as indicated by a decrease in the hematological effects in the second year of exposure.

Based on these findings in humans and animals, it appears that hematological effects may occur in persons

exposed to 2,4-DNT at low levels near hazardous waste sites.

Musculoskeletal Effects. Workers at munitions plants that produced 2,4- or Tg-DNT have complained of

muscle weakness, joint pain, and other incapacitating symptoms (McGee et al. 1942; Perkins 1919). Because

these workers were most likely exposed to high levels of DNT without protective equipment, these effects

may not necessarily be observed in persons exposed to low levels. No human or animal data are available on

musculoskeletal effects of 2,4-, 2,6-, or Tg-DNT after oral exposure.

Hepatic Effects. Medical surveys of workers exposed to Tg-DNT performed during the 1970s and 1980s

revealed no significant differences in hepatic blood chemistry profiles (Ahrenholz 1980; Ahrenholz and

Meyer 1982). In earlier surveys of workers exposed presumably to 2,4-DNT, 2 of 154 workers (McGee et al.

1942) and 29 of 714 workers (McGee et al. 1947) indicated symptoms of liver tenderness. These reports are

probably not significant. The incidence of liver tenderness reported by McGee et al. (1947) was increased

over earlier reports by McGee et al. (1942), despite improvements in engineering, ventilation, and industrial

hygiene in the intervening period that were likely to decrease the magnitude of worker exposures to DNT. In

addition, alcohol consumption, which may also have been a cause of liver tenderness, was not taken into

account.

A consistent finding in several studies of rats, mice, and dogs fed high doses of 2,4-, 2,6-, and Tg-DNT has

been the occurrence of adverse effects on the liver or biliary tract (Ellis et al. 1979; Hazleton Laboratories
2,4- and 2,6-DINITROTOLUENE 85

2. HEALTH EFFECTS

1982; Hong et al. 1985; Lee et al. 1976, 1978,1985). Lesions in rats were dose-dependent and may have

progressed from foci of altered hepatocytes to proliferative nodules to hepatocellular carcinoma (Ellis et al.

1979; Hazleton Laboratories 1982; Leonard et al. 1987).

The significance of hepatotoxicity observed in animal studies to potential adverse liver effects in humans

exposed to DNT isomers is uncertain. The negative findings in humans cannot be considered conclusive.

Studies of DNT exposure are limited by the small groups of workers studied and by the lack of individual

exposure monitoring. The consistent observation of hepatotoxicity in animals indicates that a potential exists

for hepatotoxicity in humans.

Endocrine Effects. Although no histopathological effects on adrenal, pituitary, or thyroid glands have been

observed in several bioassays, after oral exposure to 2,4- or Tg-DNT (Hazleton Laboratories 1985; McGown

et al. 1983), an increase in the incidence and severity of parathyroid hyperplasia, and fatty metamorphoses

and vascular ectasia were found in rats fed Tg-DNT in a chronic study (Hazleton Laboratories 1982). No

data are available on endocrine effects in humans after exposure to 2,4-, 2,6-, or Tg-DNT. Therefore, the

likelihood that exposure to 2,4- or 2,6-DNT will cause deleterious effects on the endocrine system in people

cannot be determined.

Renal Effects. Medical surveys of workers exposed to Tg-DNT revealed no significant differences in renal

blood chemistry profiles (Ahrenholz 1980; Ahrenholz and Meyer 1982). The limitations of these studies

were discussed previously. Adverse effects in the kidney have been observed in laboratory animals exposed

to 2,4- and 2,6-DNT, but the observations have not been as consistent as reports of hematological,

reproductive, and hepatic effects in exposed animals.

Renal dysplasia was observed in male, but not female, mice exposed chronically (up to 2 years) to 2,4-DNT

(Ellis et al. 1979). Cystic degeneration, atypical epithelium lining the cysts, and a variety of tumors were

reported by Ellis et al. (1979). Subchronic administration of 2,6-DNT to dogs, but not mice or rats, resulted

in less severe renal toxicity that included kidney degeneration (Lee et al. 1976).

The potential for renal toxicity in humans exposed to DNT is uncertain; 2,4-DNT has elicited severe renal

effects only in the male mouse. Renal effects were observed in dogs administered 2,6-DNT but not when

2,4-DNT was administered.
2,4- and 2,6-DINITROTOLUENE 86

2. HEALTH EFFECTS

Dermal Effects. Dermatitis was attributed to dermal exposure to 2,4-DNT in a small percentage of

munitions workers exposed topically to DNT (McGee et al. 1942, 1947). Both 2,4- and 2,6-DNT are mildly

irritating to rabbit skin (Ellis et al. 1978; Lee et al. 1975). Mild sensitization has been observed in guinea

pigs after 2,6-DNT exposure but was not observed after exposure to 2,4-DNT (Ellis et al. 1978; Lee et al.

1975). Therefore, although limited data are available, it appears that direct contact with low levels of 2,4- or

2,6-DNT may cause dermatitis and/or dermal sensitization in humans; however, insufficient data are

available to state this with certainty.

Ocular Effects. Only animal data are available on ocular effects after exposure to 2,4- or 2,6-DNT.

Histopathological examination of the eyes of rats fed Tg-DNT for up to 2 years showed no abnormalities

(Hazleton Laboratories 1982). Neither 2,4- nor 2,6-DNT was found to cause eye irritation in rabbits (Ellis et

al. 1978; Lee et al. 1975). There was no mention of ocular effects in studies of munitions workers exposed to

2,4- or Tg-DNT (McGee 1942, 1947; Perkins 1919). Therefore, it appears unlikely that low-level exposure

to 2,4- or 2,6-DNT would cause ocular effects in people living near hazardous waste sites.

Body Weight Effects. Effects on body weight have not been reported in munitions workers (McGee et al.

1942, 1947; Perkins 1919), but numerous studies have shown body weight loss or decreased body weight

gain in rats, mice, and dogs after oral treatment with 2,4-, 2,6-, or Tg-DNT (Bloch et al. 1988; Ellis et al.

1979, 1985; Hazleton Laboratories 1982; Hong et al. 1985; Kozuka et al. 1979; Lane et al. 1985; Lee et al.

1978,1985; Leonard et al. 1987; NCI 1978). However, in most of these studies there was a concurrent

decrease in food consumption. Therefore, it is not known whether exposure to low levels of 2,4- or 2,6-DNT

found near hazardous waste sites would cause deleterious effects on body weight in humans.

Immunological and Lymphoreticular Effects. Although no data are available regarding

immunological or lymphoreticular effects in humans, some data on these end points are available in animals.

No effects on serum concentrations of IgE, the antibody associated with allergic or hypersensitive reactions,

were reported in rats or dogs exposed to 2,4- or 2,6-DNT (Ellis et al. 1985; Lee et al. 1976, 1978, 1985).

Mild sensitization was observed in guinea pigs after dermal exposure to 2,6-DNT, but not after exposure to

2,4-DNT (Ellis et al. 1978; Lee et al. 1975). Therefore, it may be expected that human sensitizing potential

to 2,4-DNT would be low but may occur after exposure to 2,6-DNT. Lymphoreticular effects have been

reported in rats, mice, and dogs administered 2,6-DNT (Lee et al. 1976) and rats administered Tg-DNT

(Hazleton Laboratories 1977, 1982). Splenic effects included alterations in appearance such as discoloration,

enlargement, and surface irregularities; thymic involution was also seen in dogs (Hazleton Laboratories 1977,
2,4- and 2,6-DINITROTOLUENE 87

2. HEALTH EFFECTS

1982; Lee et al. 1976). Based on these data in several species, it is possible that immunological or

lymphoreticular effects may occur in people living near hazardous waste sites exposed to 2,4- or 2,6-DNT,

but insufficient data are available to state this with certainty.

Neurological Effects. Early reports of workers occupationally exposed presumably to 2,4-DNT refer to

symptoms indicative of neurotoxicity in humans. Perkins (1919) reported headaches, sleepiness, pain in the

joints, and dizziness in exposed workers. McGee et al. (1942) reported that occupationally exposed workers

complained of nausea, insomnia, headaches, dizziness, and tingling pains in the extremities. More recent

occupational studies (Ahrenholz 1980; Ahrenholz and Meyer 1982; Hamill et al. 1982; Levine et al. 1985a)

failed to examine workers for symptoms of neurotoxicity.

In animals, the nervous system has been observed to be a major target of 2,4- and 2,6-DNT toxicity (Ellis et

al. 1979, 1985; Kozuka et al. 1979; Lee et al. 1978, 1985). Clinical signs have been most pronounced in

dogs, and less severe symptoms have been observed in rats and mice (Ellis et al. 1979; Lee et al. 1976,

1978). Initial symptoms in dogs included incoordination and stiffness, especially of the hind legs, resulting in

an abnormal gait. More seriously affected dogs had paralysis of the hind legs which progressed to the

forelimbs and eventually the neck. CNS lesions, including cerebellar vacuolization, hypertrophy and focal

gliosis, and some cerebellar and brain stem perivascular hemorrhage were seen in dogs fed 2,4-DNT (Ellis et

al. 1979, 1985). In the mouse, there were signs of depression and hyperexcitability. Neurotoxic symptoms in

rats administered 2,4-DNT were less common, but some rats administered 2,6-DNT had neuromuscular

symptoms of incoordination and abnormal gait.

There are no data on the biochemical events involved in the toxicity of the nervous system. Based on the

available human and animal data, it appears that neurological effects may occur in persons exposed to low

levels of 2,4- or 2,6-DNT.

Reproductive Effects. A consistent finding in several studies in rats, mice, and dogs dosed with 2,4- or

2,6-DNT has been the impairment of the male reproductive system. Testicular atrophy, degeneration of the

seminal vesicles, and decreased sperm production have been observed in the test animals (Bloch et al. 1988;

Ellis et al. 1979; Lee et al. 1976, 1978).

Sertoli cells play a major role in maintaining spermatogenesis in the testis. Bloch et al. (1988) reported that

fine structural alterations of Sertoli cells accompanied the diminished sperm count in rats acutely exposed to
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2. HEALTH EFFECTS

2,4-DNT. Other deleterious effects included frayed basal lamina and distortion of peritubular tissue. Sertoli

cell cultures prepared from testes of Wistar rats and treated with up to 100 pM 2,4- or 2,6-DNT remained

intact, although at 50 FM of 2,4- or 2,6-DNT, some damage was observed as many of the germ cells were

absent and some Sertoli cells contained cytoplasmic vacuoles (Reader and Foster 1990). Germ cell

detachment from Sertoli germ cell cocultures was significantly increased (p<0.05) compared to controls at

10 FM 2,4- or 2,6-DNT (Reader and Foster 1990). Further evidence of disruption of Sertoli cell function

was observed as increased production of lactate and pyruvate, although increases in pyruvate production were

minimal with 2,6-DNT (Reader and Foster 1990). Thus, it appears that structural changes in the Sertoli cells

may be precipitating events responsible for spermatogenic disruption noted in 2,4-DNT exposed rats. Bloch

et al. (1988) also reported increased serum levels of FSH. Increased FSH levels have been associated with

Sertoli cell misfunction.

In rodent studies measuring the fertility of test animals dosed with 2,4-DNT, there have been marked and

significant decreases in the number of fetal implants, which have been attributed to the adverse impacts of

2,4-DNT on sperm production (Ellis et al. 1979; Lane et al. 1985; Lee et al. 1978).

Based on the adverse effects on sperm production and fertility seen in oral studies in animals, assessments of

the reproductive effects of 2,4- and 2,6-DNT on occupationally exposed workers have been conducted. As

described previously, inhalation is assumed to be the main route of exposure, with probable concurrent

dermal and oral exposure. Three of these studies, Hamill et al. (1982), Ahrenholz and Meyer (1982), and

Levine et al. (1985a), reported no detectable differences in sperm levels or fertility rates as a result of

occupational exposure.

In an earlier, study Ahrenholz (1980) reported a significant reduction in the sperm counts of exposed male

workers as well as an increase of marginal statistical significance in the number of spontaneous abortions in

the wives of exposed workers.

The small exposure populations and lack of historical individual exposure monitoring limit the effectiveness

of the occupational studies to detect adverse effects on reproduction. Although the findings reported by

Ahrenholz (1980) are suggestive of a reproductive problem in exposed workers (especially with regard to the

findings of reproductive toxicity in animal studies), the human results cannot be considered conclusive,

however, neither can they be dismissed as insignificant. Therefore, insufficient data are available to
2,4- and 2,6-DINITROTOLUENE 89

2. HEALTH EFFECTS

determine whether people exposed to low levels of 2,4- or 2,6-DNT near hazardous waste sites may have

reproductive effects.

Developmental Effects. No defects were found in rat pups after dams were fed 2,4-DNT in the diet

during gestation (Ellis 1979). Decreased pup viability in this study was believed to result from a high

incidence of maternal death during parturition and maternal neglect.

Tg-DNT was orally administered to pregnant rats during gestation and adverse effects upon blood elements

and organ weights were observed in the dams and the fetuses (Jones-Price et al. 1982). The findings of fetal

hematotoxicity imply that oxygen supply to developing tissues may be impaired by exposure of mothers to

DNT. Exposure to any substance that depletes the amount of oxygen available to developing fetal tissues can

have adverse consequences. However, insufficient data are available to predict whether low-level exposure to

2,4- or 2,6-DNT may cause developmental effects in humans.

Genotoxic Effects. Some results of in vitro genotoxicity assays are presented in Tables 2-3 (2,4-DNT),

2-4 (2,6-DNT), and 2-5 (Tg-DNT). Results of in vivo genotoxicity assays are presented in Table 2-6. DNT

causes gene mutations in the reverse mutation assay using S. typhimurium. However, the test system has

given variable results because of the need for metabolic activation and the sensitivity of the tester strains.

Unscheduled DNA synthesis (UDS) and S-phase synthesis (SPS) were induced in vitro in the hepatocytes of

Fischer-344 rats treated with 2,4-DNT in vivo (Mirsalis et al. 1989). The genotoxicity of Tg-DNT is

believed to be due to the potent genotoxicity of the 2,6-DNT component, as was evidenced in an in vivo-in

vitro hepatocyte UDS system (Mirsalis and Butterworth 1982). 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-amino6-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. 1989b). The putative metabolite

2,6-dinitrobenzaldehyde was a direct acting mutagen, that is, it did not require activation (Sayama et al.

1989b). Urine from Fischer-344 rats administered 75 mg/kg 2,6-DNT by gavage tested positive for

mutagenicity using S. typhimurium TA 98 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).






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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, three

DNA adducts were found in the liver of Fischer-344 rats (La and Froines 1992). Four DNA adducts, which

were not identified, were found in the liver of rats treated with 75 mg/kg 2,6-DNT by gavage (Chadwick et al.

1993). The formation of 4 DNA adducts was also observed after intraperitoneal administration of 219 mg/kg

2,6-DNT to Fischer-344 rats in a study by La and Froines (1992, 1993). One adduct accounts for the

majority of the radioactivity measured; about 85% of the total was 1 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 lo-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-hydroxylamino6-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 more than 40% in the

2 weeks after exposure (La and Froines 1992).

Cancer. 2,4-DNT administered in the diet of mice caused cancer of the kidneys (Ellis et al. 1979); 2,4-,

2,6-, and Tg-DNT caused hepatocellular carcinoma in rats (Ellis et al. 1979; Hazleton Laboratories 1982;

Leonard et al. 1987). 2,4-DNT was shown to be a tumor promoter, but not a tumor initiator, using in vivo

hepatic initiation-promotion protocols (Leonard et al. 1983, 1986; Mirsalis and Butter-worth 1982). 2,6-DNT

and Tg-DNT were shown to be complete carcinogens using the same protocols (Leonard et al. 1983,1986).

The results of Leonard et al. (1983, 1986, 1987) and Mirsalis and Butterworth (1982) suggest or show that

2,6-DNT is the isomer most responsible for the carcinogenic response observed in animal studies with

Tg-DNT. No increase in morphological transformation was observed in Syrian hamster embryo cells (SHE)

compared to controls when SHE cells were exposed to 2,4-, 2,6-, or Tg-DNT, or to the DNT metabolites

2,4-diaminotoluene, 2-amino-4-nitrotoluene, 2-amino-6-nitrotoluene, or 2,4-dinitrobenzoic acid (Holen et al.

1990). In addition, none of these isomers had initiator-like or promotor-like effects in this assay when tested

with O-tetradecanoyl phorbol 13-acetate or benzo[a]pyrene, respectively.

In order to evaluate the possibility that DNT might also cause cancer in humans, Levine et al. (1986b)

performed a retrospective cohort mortality study at two army ammunition plants that used Tg-DNT and/or

2,4-DNT. No significant increases in mortality from malignant neoplasms as a whole or from particular

cancers (liver, lung, gallbladder, kidney, and connective tissues) were observed. The study was limited by

small cohort size, and could have detected only an 8-fold or greater increase in liver or gallbladder cancer. As
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2. HEALTH EFFECTS

a result, the negative findings of Levine et al. (1986b) do not refute the animal cancer findings with regard to

the potential for cancer in humans. The cancer classification for 2,4- and 2,6-DNT mixture is B2: probable

human carcinogen (EPA 1992). An upperbound q1* for oral exposure to 2,4- and 2,6-DNT mixture was

estimated by EPA (1986d, 1998) to be 0.68 (mg/kg/day) -1 based on the combined tumor incidence of liver

and mammary gland tumors in rats. Doses associated with excess cancer risks of 10-4, 10-5, 10-6, and 10-7 by

the oral routes are plotted in Figure 2-1.

2.6 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 due to 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 5.6 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 pre-natal and post-natal 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 (Morselh 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 (Widdowson and Dickerson

1964; Foman et al 1982; Owen and Brozek 1966; Altman and Dittmer 1974; Foman 1966). The infant also

has an immature blood-brain barrier (Adinolfi 1985; Johanson 1980) and probably an immature blood-testis
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2. HEALTH EFFECTS

barrier (Setchell, BP and Waites GMH 1975). Many xenobiotic metabolizing enzymes have distinctive

developmental patterns and 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 (Leeder and Keams 1997; Komori 1990; Vieira et al 1996; NRC 1993). Whether differences in

xenobiotic metabolism make the child more or less susceptible also depend 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 the newborn who has a low glomerular filtration rate and has not

developed efficient tubular secretion and resorption capacities (Altman and Dittmer 1974; West et al 1948;

NRC 1993). Children and adults may differ in their capacity to repair damage from chemical insults.

Children also have a longer 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 while others may

decrease susceptibility to the same chemical. For example, the fact that infants breathe more air per kilogram

of body weight than adults may be somewhat counterbalanced by their alveoli being less developed, so there

is a disproportionately smaller surface area for 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 DNT 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 DNT does not cause dominant lethal mutation or

increases in the proportion of nonviable conception (Ellis et al. 1979).
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2. HEALTH EFFECTS

DNT has 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 Salmonella typhimurium

strains TA98 and TAlOO 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 DNT is not shown to bind to DNA and cause mutations in most of the short-term 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, DNT did not cause dominant

lethal mutation or increases in the proportion of nonviable conceptions following exposure of male animals

(Ellis et al. 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 DNT and its metabolites will accumulate in maternal tissues because of its low octanolwater

partition coefficient. No studies are available that demonstrate DNT or its metabolites cross the

placenta or get into breast milk. Thus, it is unlikely that the developing fetus or nursing infant would be

exposed to DNT as a consequence of maternal exposure prior to gestation. However, developmental toxicity

from DNT 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 hematoxicity of DNT 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 DNT 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-l 8 months of age (Leeder and Keams 1997).

Thus, the toxicity of DNT may be different in children.

2.7 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/NFX 1989).

Due to a nascent understanding of the use and interpretation of biomarkers, implementation of biomarkers as

tools of exposure in the general population is very limited. A biomarker of exposure is a xenobiotic
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2. HEALTH EFFECTS

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 (NASLNRC 1989). The

preferred biomarkers of exposure are generally the substance itself or 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., 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 2,4- and 2,6-dinitrotoluene are discussed in Section 2.7.1.

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 2,4- and 2,6-dinitrotoluene are

discussed in Section 2.7.2.

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 2.9, Populations That Are Unusually Susceptible.

There are no biomarkers of exposure effects that have been validated in children or in adults exposed as

children.
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2.7.1 Biomarkers Used to Identify or Quantify Exposure to 2,4- and 2,6-Dinitrotoluene

Spectrophotometric analysis of complexes of primary arylamines in urine, resulting from the reduction of

DNT and its metabolites, has been used to biomonitor DNT workers (Smith et al. 1995). The method is

specific for primary arylarnines and eliminates interferences from other classes of amines that might be

present in the urine. Metabolic intermediates and conjugates of DNT can also be detected if they are present

as primary arylamines. Although this method cannot identify or quantify individual metabolites, it is simple

and accurate and yields results in about 1 hour.

Workers exposed to DNT 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 DNT 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 DNT ranged from 0.5 to

4.9 mg/day, less than the total excreted. These results indicate that dermal and oral exposure contributed to

the body burden of DNT.

Woollen et al. (1985) determined that the urinary concentration of a Tg-DNT metabolite, 2,4-dinitrobenzoic

acid, was less than 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.

2.7.2 Biomarkers Used to Characterize Effects Caused by 2,4- and 2,6-Dinitrotoluene

For more information on biomarkers for renal and hepatic effects of chemicals see ATSDRDC

Subcommittee Report on Biological Indicators of Organ Damage (1990) and for information on biomarkers

for neurological effects see OTA (1990).

After exposure to DNT, methemoglobin levels in the blood may be elevated (Elleuhorn 1997). The

methemoglobinemia present may be quite profound and its onset is often delayed by up to 4 hours (Ellenhorn
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2. HEALTH EFFECTS

1997). Another hematological change that might be present in individuals who have undergone repeated or

prolonged exposure to DNT 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 (Block et al. 1988; Ellis et al.

1979). However, decrease in sperm counts in workers exposed to DNT has been reported in only one study

(CDC 1981).

2.8 INTERACTIONS WITH OTHER CHEMICALS

Reduced tolerance to alcohol was observed in some workers presumably exposed to 2,4-DNT and other

workers reported that alcohol ingestion intensified the symptoms of 2,4-DNT exposure (McGee et al. 1942).

Perkins (19 19) 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 Fischer-344 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.

Interaction of DNT with other chemicals has not been observed in children.
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2.9 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE

A susceptible population will exhibit a different or enhanced response to 2,4- and 2,6-dinitrotoluene than will

most persons exposed to the same level of 2,4- and 2,6-dinitrotoluene 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 may result in reduced detoxification or excretion of 2,4- and

2,6-dinitrotoluene, or compromised function of target organs affected by 2,4- and 2,6-dinitrotoluene.

Populations that are at greater risk due to their unusually high exposure to 2,4- and 2,6dinitrotoluene are

discussed in Section 5.7, Populations with Potentially High Exposure.

Humans sensitive to DNT 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 DNT 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 19 19), the extent of this compromise has not been determined.

The susceptibility of children to the health effects of DNT may be different from that of adults, as discussed

in Section 2.6.

2.10 METHODS FOR REDUCING TOXIC EFFECTS

This section will describe clinical practice and research concerning methods for reducing toxic effects of

exposure to 2,4- and 2,6-dinitrotoluene. 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 2,4- and

2,6-dinitrotoluene. 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 2,4- and 2,6-dinitrotoluene: Bronstein and Currance 1994; Ellenhom 1997.
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2. HEALTH EFFECTS

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.

2.10.1 Reducing Peak Absorption Following Exposure

Limited information from humans indicates that DNT is absorbed after inhalation exposure, while animal

data suggest that DNT is rapidly and completely absorbed after oral exposure. Efforts to reduce absorption

following acute exposure to DNT 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 (Bronstein and Currance 1994). It is suggested that eyes exposed to

DNT be copiously irrigated with water and normal saline (Bronstein and Currance 1994). If ingestion of

DNT occurs, it is suggested that the mouth be rinsed and water can be administered for dilution if the patient

can swallow, has a good gag reflex, and is not drooling (Bronstein and Currance 1994). In addition, the use

of activated charcoal has been suggested (Bronstein and Currance 1994). Induction of emesis is

contraindicated (Bronstein and Currance 1994). In patients who present within 2 to 4 hours of DNT

ingestion, gastric lavage may be helpful in decreasing peak absorption following exposure (Ellenhom 1997).

There may also be some benefit in administering activated charcoal and cathartics after lavage (Ellenhorn

1997).

2.10.2 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 (Ellenhorn 1997).

2.10.3 Interfering with the Mechanism of Action for Toxic Effects

Exposure to DNT can cause profound methemoglobinernia with its sequelae (cyanosis and Heinz body

formation), anoxia, and death (Ellenhorn 1997). The antidote used for serious methemoglobinemia is

methylene blue (tetramethylthionine chloride), but treatment with methylene blue is not indicated for all

patients (Ellenhorn 1997). Treatment with methylene blue is believed to be effective because it acts as a

cofactor to increase the erythrocyte reduction of methemoglobin in the presence of NADPH (Ellenhorn 1997).

Methylene blue is oxidized and the resulting molecule becomes an electron donor for the nonenzymatic
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reduction of methemoglobin to oxyhemoglobin (Ellenhorn 1997). Exchange transfusion and/or packed RBC

transfusion may be useful for patients who do not respond to methylene blue or for patients with G6PD- or

NADPH-methemoglobin reductase deficiencies (Ellenhorn 1997).

2.11 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 2,4- and 2,6-DNT 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 2,4- and 2,6-dinitrotoluene.

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.

2.11.1 Existing Information on Health Effects of 2,4- and 2,6-Dinitrotoluene

The existing data on health effects of inhalation, oral, and dermal exposure of humans and animals to 2,4-,

2,6-, and Tg-DNT are summarized in Figures 2-7,2-8, and 2-9, respectively. The purpose of these figures is

to illustrate the existing information concerning the health effects of 2,4-, 2,6, 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 (ATSDR 1989), is

substancespecific 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.
2,4- and 2,6-DINITROTOLUENE 106

2. HEALTH EFFECTS

2.11.2 Identification of Data Needs

As shown in Figures 2-7 (2,4-DNT), 2-8 (2,6-DNT), and 2-9 (Tg-DNT), there are limited data on health

effects in humans, primarily for Tg-DNT, following inhalation exposure.

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-life-time studies.

Acute-Duration Exposure. Although there are no human data available from acute-duration oral

exposure to 2,4- or 2,6-DNT, the data currently available from animal studies using single-dose exposure to

2,4- and 2,6-DNT are appropriate for evaluation of oral toxicity (Ellis et al. 1978; Lee et al. 1975; Vernot et

al. 1977). The LD50s 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 (Ellis et al. 1978; Lee et al. 1975; Vernot et al. 1977). After oral

administration of 2,6-DNT, LD50s ranged from 180 to 795 mg/kg in rats and from 621 to 807 mg/kg in mice

(Ellis et al. 1978; Lee et al. 1975; Vemot et al. 1977). Ataxia was observed in these animals before death.

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). 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). An acute-duration oral MRL has been derived for

2,4-DNT from a NOAEL for neurotoxicity in dogs (Ellis et al. 1985; Lee et al. 1978). Data were insufficient

to derive an acute-duration oral MRL for 2,6-DNT.

There were no acute-duration inhalation or dermal studies in humans available for evaluation. Both 2,4- and

2,6-DNT were shown to be mild primary dermal irritants in rabbits (Ellis et al. 1978; Lee et al. 1975). Acute

inhalation and dermal studies would be useful for determination of route-specific toxicity.



2,4- and 2,6-DINITROTOLUENE 110

2. HEALTH EFFECTS

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. 1979, 1985; Hazleton

Laboratories 1977, 1982; Hong et al. 1985; Jones-Price et al. 1982; Lee et al. 1976,1978,1985; McGown et

al. 1983; Smith et al. 1996). 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; Lee et al. 1976,1978). Mild

hepatocellular dysplasia was observed in mice fed 2,4-DNT in the diet for 13 weeks (Hong et al. 1985; Lee et

al. 1978), but no hepatotoxicity was observed after 2,4-DNT administration to rats or dogs for the same

duration (Ellis et al. 1985; Lee et al. 1978). However, treatment with 2,6-DNT did cause bile duct

hyperplasia in rats and mice (Lee et al. 1976). This lesion, as well as hepatic degeneration, was observed in

dogs dosed with 2,6-DNT (Lee et al. 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; Lee et al. 1978). Administration of 2,6-DNT to dogs for 13 weeks caused

renal inflammation and degeneration, which were not observed in rats or mice (Lee et al. 1976). Decreased

body weight gain or weight loss was observed in rats and mice after administration of 2,4-DNT (Ellis et al.

1979; Hong et al. 1985; Lee et al. 1978, 1985; Leonard et al. 1987; NCI 1978) and in rats, mice, and dogs

after administration of 2,6-DNT (Lee et al. 1976). An intermediate-duration oral MRL has been derived for

2,6-DNT based on a LOAEL for hematological effects in dogs (Lee et al. 1976). Subchronic inhalation and

dermal studies 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 2,4- or 2,6-DNT. A retrospective cohort mortality study performed using data from

workers at ammunition plants that used 2,4- 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 (Ellis et al. 1979; Leonard et al. 1987). Renal cancer was observed in mice after

administration of 2,4-DNT in the diet (Ellis et al. 1979). An upperbound qi* has been derived for oral

exposure to 2,4-DNT/2,6-DNT mixture (EPA 1986d, 1998).
2,4- and 2,6-DINITROTOLUENE 111

2. HEALTH EFFECTS

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. Studies performed in nonhuman primates

to investigate further the potential cardiovascular effects of both chemicals would help to elucidate the

mechanism of heart disease observed.

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. 1979, 1985; Hazleton

Laboratories 1982; Lee et al. 1978, 1985; Leonard et al. 1978; NCI 1978). 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. 1979, 1985; Hong et al. 1985; Lee et al,.

1978). A chronic-duration oral MRL has been derived for 2,4-DNT based on a NOAEL of 0.2 mg/kg for

hematological and neurological effects and biliary hyperplasia in dogs (Ellis et al. 1979, 1985). 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. 1979, 1985; Hong et al. 1985; Leonard et

al. 1987). Renal cystic dysplasia was observed in mice, but not rats or dogs, treated orally with 2,4-DNT for

chronic-duration periods (Ellis et al. 1979; Hong et al. 1985). 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. 1979, 1985; Hazleton Laboratories 1982; Hong et al. 1985; Leonard et al. 1987; NCI

1978).

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.
2,4- and 2,6-DINITROTOLUENE 112

2. HEALTH EFFECTS

Genotoxicity. Both 2,4- and 2,6-DNT cause gene mutations in the reverse mutation assay using S.

typhimurium. (Couch et al. 198 1; Dellarco and Prival1989; Ellis et al. 1978; Spanggord et al. 1982b;

Tokiwa et al. 198 1). 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 unscheduled

DNA synthesis 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 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; Ellis et al. 1979; Lee et al. 1976, 1978). 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 (Ahrenholz and Meyer 1982;

Hammill et al. 1982; Levine et al. 1985a). 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 (Ahrenholz 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.
2,4- and 2,6-DINITROTOLUENE 113

2. HEALTH EFFECTS

Developmental Toxicity. No data are available regarding developmental effects in humans after oral

exposure to DNT, 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 endpoints are available in animals. The currently available information on the

potential immunotoxic effects of 2,4- and 2,6-DNT is sufficient to describe the sensitizing potential of DNT.

Mild sensitization has been reported in guinea pigs after dermal exposure to 2,6-DNT, but not 2,4-DNT

(Ellis et al. 1978; Lee et al. 1975). 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. 1976, 1978, 1985). Studies have not been performed that would describe effects on

immunocompetence following exposure to DNT. 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. 1979, 1985; Kozuka et al. 1979; Lee et al. 1979, 1985). Clinical signs in dogs have

included incoordination and stiffness of the hind legs leading to complete paralysis; cerebellar vacuolation,

hypertrophy, and focal gliosis; are cerebellar and brain stem hemorrhage. In mice, depression and

hyperexcitability were observed, while some rats administered 2,6-DNT showed neuromuscular symptoms.

More systematic examination of the neurological effects of these compounds in laboratory animals would be

useful to assess fully behavioral abnormalities and morphological damage to the nervous system. The

biochemical mechanism of dinitrotoluene 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. 1942; Perkins 19 19).

However, the more recent occupational studies performed failed to examine workers for symptoms of
2,4- and 2,6-DINITROTOLUENE 114

2. HEALTH EFFECTS

neurotoxicity (Ahrenholz 1980; Ahrenholz and Meyer 1982; Hammill et al. 1982; Levine et al. 1985a).

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 DNT

suggest a potential for heart disease in exposed populations (Levine et al. 1986a). Doses of DNT associated

with heart disease in humans have not been determined. Further studies with historical cohort monitoring

data would be useful to verify these findings.

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 DNT. 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

endpoints in humans would be useful.

Biomarkers of Exposure and Effect

Exposure. Recently a rapid, accurate method for determining exposure to DNT has been developed using

spectrophotometric analysis of complexes of primary arylamines, which result from the reduction of DNT

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
2,4- and 2,6-DINITROTOLUENE 115

2. HEALTH EFFECTS

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 DNT 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 DNT 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 would be valuable in

determining the significance of findings to 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 DNT 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

DNT 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 DNT. Methylene blue treatment is used with patients

presenting with serious methemoglobinemia (Ellenhorn 1997). No additional studies are considered

necessary at this time to examine further methods for reducing body burden of DNT. 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. No data are available on the health effects of DNT on exposed children. Few

usable data are available on whether the developmental process is altered by exposure to DNT; this

information is summarized above under the Developmental Toxicity heading.
2,4- and 2,6-DINITROTOLUENE 116

2. HEALTH EFFECTS

There is inadequate experimental evidence to evaluate if the pharmacokinetics of DNT are different in

children. There are no studies on whether DNT or its 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 DNT and its metabolites are stored in maternal

tissues and thus might be later mobilized during gestation or lactation; however, DNT and its 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 DNT or its mechanisms of action

are different in children. As discussed in section 2.6, newborns are highly sensitive to the

methemoglobingenerating effect of DNT because of their deficiency in methemoglobin reductase (Gruener

1976), which reduces methemoglobin back to hemoglobin. In addition, newborns have a high 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 DNT. It will also be helpful to have data on the metabolism and

mechanism of action of DNT on children to determine if children are more vulnerable than adults to health

effects from exposure to DNT, 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

DNT is shown to be genotoxic, it is not known if parental exposure to DNT may affect children via parental

germ cells, or if DNT may indirectly affect the fetus during maternal exposure.

Child health data needs relating to exposure are discussed in 5.8.1 Data Needs: Exposures of Children.

2.11.3 Ongoing Studies

No information was located regarding ongoing research related to the potential health effects of 2,4- or

Tg-DNT.

A project to develop and apply biomarkers of exposure associated with 2,6-DNT is being conducted by

S. Rappaport at the University of North Carolina at Chapel Hill (FEDRIP 1997). The project will ultimately

perform epidemiological studies of exposed populations in order to provide exposure-biomarker relationship.

It is anticipated that in vivo and in vitro measurements of the protein adducts will provide data on the

disposition of reactive intermediates and mechanisms of toxicity.
2,4- and 2.6-DINITROTOLUENE 117

3. CHEMICAL AND PHYSICAL INFORMATION

3.1 CHEMICAL IDENTITY

Information regarding the chemical identity of 2,4- and 2,6-DNT is located in Table 3-1.

3.2 PHYSICAL AND CHEMICAL PROPERTIES

Information regarding the physical and chemical properties of 2,4- and 2,6-DNT is located in Table 3-2.

Data regarding specific isomers of DNT have been provided whenever possible. However, DNT is generally

produced as a technical-grade mixture of the two isomers, with approximately 5% other substances. This 5%

contains predominantly the other isomers of DNT, which are not discussed in this profile. Where information

pertains to Tg-DNT, it has been so noted.



2,4- and 2,6-DINITROTOLUENE 121

4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

4.1 PRODUCTION

Tables 4-l and 4-2 list the facilities in each state that manufacture or process 2,4-DNT and 2,6-DNT,

respectively; the intended use, and the range of maximum amounts of 2,4- and 2,6-DNT that are stored on

site. There are currently 3 facilities that produce or process 2,4-DNT in the United States: First Chemical

Corporation in Pascagoula, MS; U.S. Army Radford Army in Radford, VA; and Bayer Corporation in New

Martinsville, WV. There is one facility that produces or processes 2,6-DNT in the United States: Bayer

Corporation in New Martinsville, WV. The data listed in Tables 4-l and 4-2 are derived from the Toxics

Release Inventory (TR196 1998). Only certain types of facilities were required to report. Therefore, this is

not an exhaustive list.

The chemicals 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 1998). This mixture is commercially

prepared by the nitration of toluene with concentrated sulfuric and nitric acid (Etnier 1987).

4.2 IMPORT/EXPORT

No data describing import or export activities for 2,4- or 2,6-DNT were located. Imports of nitrobenzenes

and nitrotoluenes into the United States for 1994 totaled 1,676,582 kg (NTDB 1996).

4.3 USE

The most commercially important use of DNT is as a chemical intermediate in the production of toluene

diisocyanate, a precursor to polyurethane polymers (HSDB 1998). It has been estimated that 99% of all

DNT produced is used for this purpose (CMR 1983). Additionally, DNT is used in the production of TNT,

and as a waterproofing, plasticizing, and gelatinizing agent in explosives (HSDB 1998). DNT is also used as

an intermediate in the production of dyes and as a modifier for smokeless powders in the munitions industry

(HSDB 1998). 2,4-DNT is used in the air bags of automobiles (Ellenhorn 1997).


2,4- and 2,6-DINITROTOLUENE 124

4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

4.4 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 2,4-DNT and 2,6-DNT is controlled by a number

of federal regulations (see Chapter 7).

Only limited information is available regarding the appropriate disposal of DNT. NTOSH recommends small

quantities be swept onto paper or other suitable 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 1998). DNT has 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 1998).
2,4- and 2,6-DINITROTOLUENE 125

5. POTENTIAL FOR HUMAN EXPOSURE

5.1 OVERVIEW

2,4-DNT and 2,6-DNT have been identified in at least 69 and 53 of the 1,467 current or former EPA

National Priorities List (NPL) hazardous waste sites, respectively (HazDat 1998). However, the number of

sites evaluated for 2,4-DNT and 2,6-DNT is not known. The frequency of these sites within the United

States can be seen in Figures 5-l and 5-2.

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 2,4- and 2,6-DNT have been obtained primarily from the workplace.

DNT has been found in waste water and groundwater in and around munitions sites (Jenkins et al. 1986), and

4-nitrotoluene and dinitrobenzene, structural analogues of DNT, are taken up by plants (McFarlane et al.

1987). However, predictions of environmental exposure pathways based on measurements of structural

analogues of DNT are severely limited by the complex abiotic reactions of DNT in the environment and by

the different pathways, rates, and products of biological reduction and/or oxidation of 2,4-DNT and

2,6-DNT.

The relatively low log octanol-water partition coefficients of the DNT isomers (1.98 and 1.72 for 2,4-DNT

and 2,6-DNT, respectively) predict that DNT released to the environment would not bioaccumulate.

However, the log octanol-water partition coefficients are sufficiently large to indicate adsorption to soil

organic matter. The bioavailability and potential toxicity of soil- and sediment-bound products are also

unknown. DNT is degraded by oxidation, photolysis, and biotransformation in water or soil, but a variety of

degradation products, about which very little is known, is formed. DNT may bind to clays and clay colloids

and facilitate its oxidation. It is otherwise expected to be stable in water because it is not hydrolyzed. The

relatively low volatility and high solubility of DNT indicates that it will tend to remain in water for long

periods of time, unless acted upon by light, oxygen, or biota. As a result, DNT can be transported to

groundwater or surface waters.


2,4- and 2,6-DINITROTOLUENE 128

5. POTENTIAL FOR HUMAN EXPOSURE

Studies of occupational exposures to DNT indicate that inhalation and dermal contact can result in absorption

of DNT into the body. However, inhalation and dermal contact are less likely to be critical environmental

exposure pathways for the general population. Individuals exposed outside the workplace would be more

likely to ingest DNT via contaminated drinking water and food.

5.2 RELEASES TO THE ENVIRONMENT

According to the Toxics Release Inventory (TRI), in 1996, a total of 6,844 pounds (5,080 kg) of 2,4-DNT

from 3 large processing facilities and 1,315 pounds (592 kg) 2,6-DNT from 2 large processing facilities were

released to the environment (TR196 1998). Tables 5-l and 5-2 list amounts released from these facilities. In

addition, an estimated 1,866 pounds (840 kg) of 2,4-DNT and 58 pounds (26 kg) 2,6-DNT were transferred

offsite (TR196 1998). The TRI data should be used with caution because only certain types of facilities are

required to report. This is not an exhaustive list.

2,4-DNT and 2,6-DNT have been identified in a variety of environmental media (air, surface water,

groundwater, soil, and sediment) collected at 69 and 53 of the 1,467 NPL hazardous waste sites, respectively

(HazDat 1998).

5.2.1 Air

According to the Toxics Release Inventory, in 1996, the estimated releases of 4,202 pounds (1,891 kg)

2,4-DNT from 3 large processing facilities and 1,048 pounds (472 kg) 2,6-DNT from 1 large processing

facility to air accounted for about 77% of total environmental releases (TR196 1998). Tables 5-l and 5-2 list

amounts released from these facilities. The TRI data should be used with caution, however, since only certain

types of facilities are required to report. This is not an exhaustive list.

2,4-DNT has been identified in air samples colleced at 1 of the 69 NPL hazardous waste sites, and 2,6-DNT

has been identified in air samples colleced at 1 of the 53 NPL hazardous waste sites where they were detected

in some environmental media (HazDat 1998).

The DNT isomers have relatively low vapor pressures (2,4-DNT, 5.1x10-3 torr [20°C]; 2,6-DNT, 0.018 torr

[20°C]) (Maksimov 1968) and quite low Henry’s Law constants (2,4-DNT, 4.5x10-6 atm-m3

 mol-1 [calc.,

20°C]; 2,6-DNT, 7.9x10-6 atm-m3 mol-1 [calc., 20°C]) (Mabey et al. 1982). As a result, DNT is not


2,4- and 2,6-DINITROTOLUENE 131

5. POTENTIAL FOR HUMAN EXPOSURE

expected to volatilize from water. However, DNT may be released to or transported in the air as dusts or

aerosols or adsorbed to other suspended particles. Exposure to airborne DNT may occur in the workplace.

Environmental exposure to DNT in the air may occur if contaminated surface soils are eroded and reentrained.

Minute amounts of nitrotoluene are formed by the photochemical reaction of toluene, nitrogen oxides, and

sunlight (Atkinson et al. 1980). Although DNT could be formed subsequently, it would be subject to

photolysis and would not be likely to accumulate enough to contribute significantly to human exposure.

5.2.2 Water

According to the Toxics Release Inventory, in 1996, the estimated releases of 2,4-DNT and 2,6-DNT of

776 pounds (349 kg) from 2 large processing facilities and 209 pounds (94 kg) from 1 large processing

facility, respectively, to water accounted for about 12% of total environmental releases (TRI96 1998).

Tables 5-l and 5-2 list amounts released from these facilities. The TRI data should be used with caution,

however, since only certain types of facilities are required to report. This is not an exhaustive list.

2,4-DNT has been identified in surface water at 19 of 69 NPL hazardous waste sites, and in groundwater at

54 of 69 NPL hazardous waste sites where it was detected in some environmental media (HazDat 1998).

2,6-DNT has been identified in surface water at 18 of 53 NPL hazardous waste sites, and in groundwater at

32 of 53 NPL hazardous waste sites where it was detected in some environmental media (HazDat 1998).

The occurrence of DNT 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). DNT occurs 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 DNT in waste waters from other manufacturing uses (e.g., polyurethane forms) has not been

reported. The frequency of detection of DNT 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 DNT, and the

median of positive samples was less than 10 μg/L. The presence of DNT was not detected in samples of

sediment or biota.
2,4- and 2,6-DINITROTOLUENE 132

5. POTENTIAL FOR HUMAN EXPOSURE

The water solubility of 2,4- and 2,6-DNT (270 ppm and 180 ppm, respectively) (Mabey et al. 1982) is

sufficient to permit transport in surface or groundwaters. Thus, releases to water are very important sources

of potential human exposure. Prior to introduction of discharge controls, washings from munitions

manufacturing and demilitarization facilities could produce thousands of gallons of contaminated effluent per

day at a single facility. Moreover, water contamination may persist. For example, an old effluent lagoon

contained detectable quantities of DNT about 12 years after use had been discontinued (Jenkins et al. 1986).

Hashimoto et al. (1982) reported the release of as much as 150 kg/day of DNT isomers in the effluent from a

single coastal industrial drain in Dokai Bay, Japan, with a daily average release of 76 kg/day over 7 months.

However, DNT concentrations in the bay decreased with distance from the site more rapidly than predicted by

simple dilution and tidal action. The salting-out effect of sea water on nitroaromatic compounds was

probably responsible for this effect (Hashimoto et al. 1984).

5.2.3 Soil

According to the Toxics Release Inventory, in 1996, there were no reported releases of 2,4-DNT or 2,6-DNT

to soil (TR196 1998). The TRI data should be used with caution, however, since only certain types of

facilities are required to report. This is not an exhaustive list

2,4-DNT has been identified in soil at 35 of 69 NPL hazardous waste sites, and in sediment at 16 of 69 NPL

hazardous waste sites where it was detected in some environmental media (HazDat 1998). 2,6-DNT has been

identified in soil at 20 of 53 NPL hazardous waste sites, and in sediment at 9 of 53 NPL hazardous waste

sites where it was detected in some environmental media (HazDat 1998).

No information on releases of DNT to soil were located. The extensive use of DNT as an intermediate in the

synthesis of toluenediisocyanate and polyurethane foam is not a reported source of releases to soil.

5.3 ENVIRONMENTAL FATE

5.3.1 Transport and Partitioning

The water solubilities of DNT are moderate (Callahan et al. 1979), and the octanol-water partition

coefficients are low (Mabey et al. 1982). As a result, there is a potential for transport of DNT by surface or
2,4- and 2,6-DINITROTOLUENE 133

5. POTENTIAL FOR HUMAN EXPOSURE

groundwater. Spanggord et al. (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. Mabey et al. (1982)

calculated sediment-water partitioning coefficients of 45 and 92 for 2,4-DNT and 2,6-DNT, respectively.

Depending on the nature of the sediment load, the total concentration of DNT carried in the soil and water

column could be high. DNT in buried munitions wastes could potentially be released to groundwater or

transported as contaminated soil and sediment.

The expected bioaccumulation of DNT in animal tissues is negligible. The low octanol-water partition

coefficients do not indicate a concern for biological lipid accumulation (Trabalka and Garten 1982). Values

of Kow lower than 2.5 for nonionized organic chemicals predict bioconcentration solely dependent on the

magnitude of exposure. The log Kow values for 2,4-DNT and 2,6-DNT are 1.98-2.01 and 2.05-2.28,

respectively (Callahan et al. 1979; Mabey et al. 1982), indicating that bioaccumulation is not likely to occur.

Direct measurement of plant uptake of DNT 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; Nolt 1988) suggests that 2,4- and 2,6-DNT would be readily taken up by plants.

However, plant uptake of related nitroaromatic compounds such as 2,4,6-TNT and its byproduct 4-amino2,6-DNT has 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 (Cataldo et al. 1989).

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 long-term

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 DNT is

so dependent on environmental conditions and the presence of effective microorganisms, the protective view

that DNT is of heavy concern for persistence in water, plant uptake, and leaching to graundwater may be

warranted. The lack of concern for bioaccumulation, multimedia/multispecies action, and atmospheric action

also appears to be justified.
2,4- and 2,6-DINITROTOLUENE 134

5. POTENTIAL FOR HUMAN EXPOSURE

5.3.2 Transformation and Degradation

5.3.2.1 Air

Based on its rapid photolysis in water, DNT is presumed to be subject to oxidation of its methyl group,

decarboxylation, ring oxidation, and/or nitroreduction in air and sunlight. Indirect photolysis must be

considered as well. Although the literature implies that these routes lead to destruction of DNT, no

experimental measurements were located in the available studies.

5.3.2.2 Water

DNT 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

(Andrews and Osmon 1977; Bausum et al. 1992; Bradley et al. 1995; Freedman et al. 1996; Ho 1986;

Noguera and Freedman 1996; Roth and Murphy 1979). Ho (1986) studied photooxidation of 2,4-DNT in

aqueous solution in the presence of hydrogen peroxide and suggested that the degradation pathway of

2,4-DNT was, although it includes other intermediates, as follows: 2,4-DNT -- 1,3-dinitrobenzene -

hydroxynitrobenzene derivatives + carboxylic acids – CO2 H2,O, and HNO3. Oxidation of aqueous DNT

with hydrogen peroxide or UV irradiation alone was very slow, and elimination was not complete. Dillert et

al. (1995) reported that degradations of DNT and several other nitroaromatics were accelerated in irradiated

TiO2, suspensions and that the degradation rates were dependent on time, solution pH, and light intensity. At

given temperature, pH, and photo intensity, degradation rates were shown in the order of 2-nitrotoluene >

nitrobenzene > DNT > 1,3-dinitrobenzene > TNT > trinitrobenzene, and the degradations followed first order

kinetics. There was very little influence on degradation rates by changes in pH (Dillert et al. 1995; Kumar

and Davis 1997).

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 DNT in waste water have spurred considerable study of the abiotic and

biotic fate of 2,4- and 2,6-DNT. Spanggord et al. (1980) reported that the half-lives of DNT in 3 sunlit

natural waters were 3-10 hours, whereas the photolysis half-life in distilled water was 43 hours. Simmons
2,4- and 2,6-DINITROTOLUENE 135

5. POTENTIAL FOR HUMAN EXPOSURE

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. Since the calculated rates

of oxidation are less by a factor of about 10-5, photolysis is probably the major route of degradation of DNT

in oxygenated waters.

DNT may be also degraded by ozonation and chlorination. Lee and Hunter (1985) reported that both ozone

and chlorine produced less than 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.

In the absence of sunlight and oxygen, any losses of DNT would apparently be dependent on biodegradation.

Spanggord et al. (1980) observed biodegradation in an aerobic environment, with a half-life of less than

1 hour. Under anaerobic conditions, the half-life of 2,6-DNT in non-acclimated sewage is found to be

28 days, with no loss of the compound under aerobic conditions during the same period (Hallas and

Alexander 1983). Similarly, Liu et al. (1984) reported no loss of 2,4-DNT in aerobic municipal activated

sludge to which benzene was added as a carbon source for bacterial growth, but complete biotransformation

of 2,4-DNT within 14 days under anaerobiosis. 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 DNT from microorganisms taken from areas that are

frequently exposed to DNT (Bausum et al. 1992; Bradley et al. 1995; 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 DNT was shown to be

converted to CO2 with 2,4-DNT conversion occurring at a greater rate than that of 2,6-DNT; concentrations
2,4- and 2,6-DINITROTOLUENE 136

5. POTENTIAL FOR HUMAN EXPOSURE

ranged from 0.004 to 10.0 ppm (Bausum et al. 1992). The rate of mineralization to CO2 was concentration

dependant and increased with increasing concentration.

In the Bradley et al. (1995) study, a culture of microorganisms taken from aquifer sediments at an

explosivescontaminated site was observed to be capable of removing added 2,4- and 2,6-DNT. The removal rate

of 2,4-DNT after 70 days was greater than 99% after application of 100 μM of 2,4-DNT. Removal of 2,6-DNT

at the same concentration was less efficient, with 60% of the compound being removed after 70 days.

Breakdown products from 2,4-DNT degradation included 4-amino-2-nitrotoluene and 2-amino4-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 mg/L and 142 mg/L,

respectively. Ethanol and ether were chosen because they are often found in munitions manufacturing

wastewater 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 DNT was mineralized to a significant degree.

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 (DAT) (Cheng et al.

1996). During the biotransformation, two intermediates were formed: 2-amino-4-nitrotoluene and 4-amino2-nitrotoluene. 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 DNT, the other with activated

sludge that was not routinely exposed to nitroaromatic compounds. The acclimated culture consumed DNT

twice as fast as the unacclimated culture, with reduction of DNT to aminonitrtoluenes as the initial pathway.

The principal metabolites identified in the acclimated culture were 6-nitroindazole, 2-nitrotoluene,

4 nitrotoluene, as well as products from acetylation at the pm-u position (4-acetamide-2-nitrotoluene and

4 acetamidetoluene). Reduction of aminonitrotoluenes to 2,4-diaminotoluene also occurred, and its

subsequent disappearance results in accumulation of significant amount of nonfilterable material in both

cultures. The soluble metabolites formed from the unacclimated culture were more hydrophilic. Initial
2,4- and 2,6-DINITROTOLUENE 137

5. POTENTIAL FOR HUMAN EXPOSURE

characterization of the highly hydrophilic metabolites indicated approximately equal amounts of

negativelycharged and neutral compounds.

Biotransformation of DNT by a Pseudomonas aeruginosa strain, which was isolated from a propellant

wastewater treatment plant, was observed under both aerobic and anoxic conditions (Noguera and Freedman

1996). The biotransformation was mainly reductive under both of these conditions. The primary breakdown

products were 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, Zacetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and

2,4-diacetamidetoluene.

5.3.2.3 Sediment and Soil

Little information was located regarding transformation of DNT in 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).

5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT

Reliable evaluation of the potential for human exposure to 2,4-DNT and 2,6-DNT depends in part on the

reliability of supporting analytical data from environmental samples and biological specimens. In reviewing

data on 2,4-DNT and 2,6-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.

5.4.1 Air

Measurements of DNT in ambient air were not located. In an occupational environment, Ahrenholz (1980)

measured breathing zone air concentrations of Tg-DNT that ranged from undetected to 23 μg/m3

 (timeweighted

average [TWA]). Tg-DNT concentrations in the area air samples ranged from undetected to 420
2,4- and 2,6-DINITROTOLUENE 138

5. POTENTIAL FOR HUMAN EXPOSURE

μg/m3

 (TWA). Ahrenholz and Meyer (1982) reported that area air samples in a manufacturing facility

contained TWA concentrations of Tg-DNT that ranged from undetected to 890 μg/m3

. Levine et al. (1985b)

also measured personal air samples of 2,4-DNT at 10-440 μg/m3

 and 2,4- and 2,6-DNT at 50-590 μg/m3 in

the workplace.

5.4.2 Water

As priority pollutants, 2,4- and 2,6-DNT are monitored routinely in U.S. waters, but both the number of

positive samples (1.1 - 1.6%) and the mean value (<10 μg/L) of those samples indicate that release of the

compounds to water is neither widespread nor very great. No residues of 2,4- or 2,6-DNT were reported in

sediments or biota (Staples et al. 1985). Residues of 2,4-DNT were detected in the groundwater of 1.5% of

800-900 hazardous waste sites (CLPSD 1988). The geometric mean concentration in groundwater was 58

μg/L. In surface waters, 2,4-DNT was detected at 1.0% of hazardous waste sites at a geometric mean

concentration of 104 μg/L. In surface waters of the river Elbe in Germany, concentrations of 2,4-DNT

ranged from 0.1 to 1.3 μg/L, while concentrations of 2,6-DNT ranged from 0.08 to 0.5 μg/L (Feltes et al.

1990). Sohr et al. (1995) reported 2,4-DNT and 2,6-DNT concentrations of 0.7 μg/L and 3.1 μg/L,

respectively, at contaminated warfare sites in Germany. Other less contaminated sites contained 28 ng/L

2,4-DNT and 19 ng/L 2,6-DNT (Sohr et al. 1995).

Residues of 2,6-DNT were detected in the groundwater of 1.0% of waste sites at a geometric mean

concentration of 18 μg/L. 2,6-DNT was detected in the surface water of only 0.3% of the waste sites

(CLPSD 1988).

5.4.3 Sediment and Soil

Hoke et al. (1993) reported that only low concentrations of 2,4-DNT and 2,6-DNT were detected in sediment

of the Great Calumet River-Indian Harbor. Concentrations of 2,4-DNT in sediments ranged from the

detection limit of 0.01 μg/L to 0.07 μg/L. 2,4-DNT concentrations in sediment pore water ranged from

0.1 μg/L to 1.7 μg/L. 2,6-DNT was not detected in sediment samples (LOD = 0.01 l.μg/L) and subsequently

was not analyzed for in sediment pore water.
2,4- and 2,6-DINITROTOLUENE 139

5. POTENTIAL FOR HUMAN EXPOSURE

Concentrations ranging from less than 0.1 mg/kg (detection limit) to 117 mg/kg of 2,4-DNT were found at

the Joliet Army Ammunition Plant, in Joliet, IL, an NPL site. 2,6-DNT was detected on this site at

concentrations ranging from less than 0.1 mg/kg to 8 mg/kg (Simini et al. 1995).

5.4.4 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). DNT was not detected in fish from Great Lakes harbors and

tributaries in Ohio and Wisconsin (DeVault 1985).

5.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE

Sources of exposure to DNT for the general population include processing facilities that manufacture or

process DNT, as well as hazardous waste sites that release this chemical. There are 3 large processing

facilities that release a total of 3,080 kg of 2,4- and 2,6-DNT to the environment annually (TR196 1998), and

2,4- and 2,6-DNT has been found in at least 69 and 53 waste sites, respectively (HazDat 1998). The general

population may be exposed to DNT via inhalation, dermal contact, and incidental ingestion pathways.

Occupational exposure to DNT 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 pathways will also involve inhalation, dermal contact, and incidental

ingestion.

Studies on occupational exposure to DNT are limited. Levine et al. (1985b) evaluated the 7-hour TimeWeighted Average (TWA) personal exposure of workers to DNT technical grade (Tg-DNT) and measured

urinary metabolites of DNT 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

. Air exposure concentrations of loaders, who load

storage tanks, collect samples, and perform cleaning tasks, averaged 0.32 mg/m3

. Exposure of maintenance

mechanics averaged 0.12 mg/m3

, and the exposure of acid-stripper operators was 0.06 mg/m3. 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 DNT in loaders and operators exceeded

those that would have resulted from the inhaled concentrations, although the workers wore gloves for

operations that might have led to dermal exposure. In another study of occupational absorption in an
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5. POTENTIAL FOR HUMAN EXPOSURE

explosives manufacturing facility (Woollen et al. 1985), personal airborne exposure to DNT ranged from

undetectable to 0.1 mg/m3

. Area samples collected near dusty parts of the process ranged from 0.02 to

2.68 mg/m3

. However, air concentrations could not account for the observed excretion levels of the

metabolite 2,4-dinitrobenzoic acid, indicating probable dermal uptake.

Two major studies of occupational exposure to DNT (Levine et al. 1985b; Woollen et al. 1985) reported the

following: maximum excretion of metabolites occurred at or after the end of a working shift; excretion was

practically complete by the start of the next shift; and rates of 2,4-dimtrobenzoic acid excretion in the urine

made a plausible marker of exposure, provided that sampling patterns were adequate to compensate for the

wide variation over time, both between individuals and for the same person. In both studies, difficulties in

detecting metabolites and frequent occurrence of samples with values near the limits of detection hampered

interpretation of actual exposure and disposition. Higher exposures in the study by Levine et al. (1985b)

permitted more extensive analysis. The results of both studies indicated that dermal contact and inadvertent

ingestion were contributing routes of exposure for male and female workers.

The Occupational Safety and Health Administration (OSHA) established an 8-hour Time-Weighted Average

(TWA) Permissible Exposure Limit (PEL) for dinitrotoluene as 1.5 mg/m3

, with skin designation to indicate

the potential significant contribution to the overall exposure by the cutaneous route (OSHA 1998). The

American Conference of Governmental Industrial Hygienist (ACGIH)‘s Threshold Limit Value (TLV) for

dinitrotoluene is 0.2 mg/m3

, with skin notation (ACGIH 1998). 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 dinitrotoluene as 1.5 mg/m3

,

with skin designation (NIOSH 1997).

5.6 EXPOSURES OF CHILDREN

This section focuses on exposures from conception to maturity at 18 years in humans and briefly considers

potential pre-conception exposure to germ cells. Differences from adults in susceptibility to hazardous

substances are discussed in 2.6 Children’s Susceptibility.

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, and breathe more air per kilogram of body weight, and have a
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5. POTENTIAL FOR HUMAN EXPOSURE

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; they put things in their mouths; they may

ingest inappropriate things such as dirt or paint chips; they spend more time outdoors. Children also are

closer to the ground, and they do not have the judgement of adults in avoiding hazards (NRC 1993).

No studies are available that monitor the level of exposure of children to DNT. No measurements have been

made of DNT or its metabolite levels in aminiotic fluid, meconium, cord blood, or neonatal blood to test for

prenatal exposure, nor have measurements been made of DNT or metabolite levels in breast milk. However,

because of the low octanol-water partition coefficient of DNT and excretion in the urine, it is not expected to

accumulate in maternal tissues.

Although DNT can degrade in water by several mechanisms, it has the potential to be transported in surface

water or groundwater due to its moderate water solubility. Therefore, children playing in DNT contaminated

surface water 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 or municipal water contaminated with DNT 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 DNT is 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 parents’ work clothes, skin, hair,

tools, or other objects removed from the workplace. No information is available concerning exposure from

consumer products because DNT is used mainly for military and industrial purposes.

Although DNT is genotoxic in in vivo test systems, it is found to be negative in dominant lethal mutations

(Ellis et al. 1979) and spermatocyte DNA repair (Working and Butterworth 1984). There is no evidence that

exposure of parental germ cells to the active form of DNT could plausibly occur since DNT does not

accumulate in tissue.
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5. POTENTIAL FOR HUMAN EXPOSURE

5.7 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES

In addition to individuals who are occupationally exposed to 2,4-DNT and 2,6-DNT (see Section 5.5), there

are several groups within the general population that have potentially high exposures (higher than

background levels) to 2,4-DNT and 2,6-DNT. These populations include individuals living in proximity to

sites where 2,4-DNT and 2,6-DNT were produced or sites where 2,4-DNT and 2,6-DNT were disposed, and

individuals living near one of the NPL hazardous waste sites where 2,4-DNT and 2,6-DNT have been

detected in some environmental media (HazDat 1998).

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. 2,4-DNT and 2,6-DNT do not

appear to be widespread in the environment, and they were not frequently detected at hazardous waste sites.

5.8 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 2,4- and 2,6-DNT is available. Where adequate information is not

available, ATSDR, in conjunction with the 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 2,4- and 2,6-DNT.

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.
2,4- and 2,6-DINITROTOLUENE 143

5. POTENTIAL FOR HUMAN EXPOSURE

5.8.1 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 DNT is presented in Chapter 3 and the data appear to be adequate

(HSDB 1998; Lide 1993). The isomers of DNT have many similar traits, including identical molecular

weights), but 2,4-DNT has higher melting and boiling points than 2,6-DNT and a greater solubility in water

(HSDB 1998). DNT is generally produced as a technical-grade mixture comprised of 95% 2,4- and

2,6-DNT, and 5% other substances. The other substances are predominantly isomers of DNT not discussed

in this profile.

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

chemical release and off-site transfer information to the EPA. The Toxics Release Inventory (TRI), which

contains this information for 1996, became available in May of 1998. This database is updated yearly and

should provide a list of industrial production facilities and emissions. The TRI reported that DNT was

produced at 3 sites within the United States (TR196 1998).

USITC statistics on synthetic organic chemical production (USITC 1987) do not describe DNT production.

Uses of DNT appear to be well characterized (CMR 1983; HSDB 1998). The most commercially important

use of DNT is as a chemical intermediate in the production of toluene diisocyanate, a precursor to

polyurethane polymers (HSDB 1998). It has been estimated that 99% of all DNT produced is used for this

purpose (CMR 1983). DNT is recognized as a potentially hazardous chemical and is subject to a variety of

regulations (see Chapter 7), but disposal practices and restrictions are not adequately documented.

Environmental Fate. The low octanol-water partition coefficients of the DNT isomers predict that DNT

released to the environment would not bioaccumulate and would be weakly bound to soil organic matter. The

relatively low volatility and high solubility of DNT indicate that it 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). DNT has been found in wastewater and groundwater in

and around munitions sites (Jenkins et al. 1986; Spanggord and Suta 1982). The occurrence of DNT in

wastewater from other manufacturing uses such as polyurethane forms has not been reported.
2,4- and 2,6-DINITROTOLUENE 144

5. POTENTIAL FOR HUMAN EXPOSURE

Given the importance of information about the behavior of DNT in the water column, and the extensive range

of available information relative to that topic (Gillett 1983; Hashimoto et al. 1982, 1984; Jenkins et al. 1986;

Mabey et al. 1982; Spanggord et al. 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 DNT

in soil, 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 DNT in environmental media or 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). DNT was not detected in fish from Great Lakes

harbors and tributaries in Ohio and Wisconsin (DeVault 1985).

Food Chain Bioaccumulation. Limited information indicates that DNT is not widely distributed in the

environment. Residues of DNT have not been observed in fish samples and 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; Mabey et al. 1982; Trabalka and Garten 1982). The expected bioaccumulation of

DNT is animal tissues in negligible. The log Kow, values for 2,4- and 2,6-DNT are 1.98-2.01 and 2.05-2.28,

respectively (Callahan et al. 1979; Mabey et al. 1982), indicating that bioaccumulation is not likely to occur.

The bioavailability and potential toxicity of soil- and sediment-bound products are unknown. Degradation of

DNT forms a variety of products, about which very little is known. Additional information would help to

confirm or refute indications of low potential for bioaccumulation of DNT in foods.

Exposure Levels in Environmental Media. Reliable monitoring data for the levels of 2,4- and

2,6-DNT in contaminated media at hazardous waste sites are needed so that the information obtained on

levels of 2,4- and 2,6-DNT in the environment can be used in combination with the known body burdens of

2,4- and 2,6-DNT 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 DNT has not occurred (Staples et al.

1985). Monitoring for priority pollutants in wastewater, drinking water, and biota has failed to detect
2,4- and 2,6-DINITROTOLUENE 145

5. POTENTIAL FOR HUMAN EXPOSURE

residues in sediments or biota (Staples et al. 1985). DNT was detected at very low frequencies in drinking

water, the number of positive samples ranging from 1.1 - 1.6% and the mean value of those samples reported

at less than 10 μg/L. 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). Residues of

2,4-DNT were detected in the groundwater of 1.5% of 800-900 hazardous waste cites (CLPSD 1988). The

geometric mean concentration in groundwater was 58 μg/L. In surface waters, 2,4-DNT was detected at

1.0% of hazardous waste sites at a geometric mean concentration of 104 g/L.

Exposure Levels in Humans. This information is necessary for assessing the need to conduct health

studies on these populations. 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 DNT and its metabolite levels in blood

and urine will be useful to provide an estimate of internal dose of exposure.

Exposures of Children. No exposure and body burden studies have been conducted on children;

consequently, it is not known if children differ from adults in their weight-adjusted intake of DNT, or if

unique exposure pathways for children exist. Since DNT is 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 DNT has 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 DNT and are occupationally exposed to

significant quantities of DNT might potentially be exposed to DNT 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.

Exposure Registries. No exposure registries for DNT (2,4- or 2,6-DNT) were located. Neither of these

substances is currently one of the compounds for which a subregistry has been established in the National

Exposure Registry. The substance will be considered in the future when chemical selection is made for

subregistries to be established. The information that is amassed in the National Exposure Registry facilitates
2,4- and 2,6-DINITROTOLUENE 146

5. POTENTIAL FOR HUMAN EXPOSURE

the epidemiological research needed to assess adverse health outcomes that may be related to exposure to this

substance.

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.

5.8.2 Ongoing Studies

Photochemical reactions of 2,4-DNT in aqueous solutions containing cationic surfactant, nonionic surfactant,

a hydrogen donor, or a base are being investigated by C.A. Diehl et al. (1995) at Purdue University.

A project to develop and apply biomarkers of exposure associated with 2,6-DNT is being conducted by

Dr. Steve Rappaport at University of North Carolina at Chapel Hill (FEDRIP 1998).

A project to develop an anaerobic biodegradative process for the biodegradation of DNT in aqueous streams

is being conducted by D. Munnecke at Environmental Biotechnologies, Inc. in Montara, CA (FEDRIP 1997).
2,4- and 2,6-DINITROTOLUENE 147

6. ANALYTICAL METHODS

The purpose of this chapter is to describe the analytical methods that are available for detecting, and/or

measuring, and/or monitoring 2,4-DNT and 2,6-DNT, its metabolites, and other biomarkers of exposure and

effect to 2,4-DNT and 2,6-DNT. 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.

6.1 BIOLOGICAL SAMPLES

The need to determine DNT 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 DNT in biological samples is available in the literature. DNT has been determined in ocean

floor fauna using thin layer chromatography (TLC) (Hoffsommer et al. 1972). Procedures have been

described for the examination of swabs for traces of explosives, including 2,6-DNT using high-performance

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. DNT

and its 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 DNT

and its metabolites can also be performed after reduction to primary arylamines and subsequent coupling of

diazo compounds to produce a colored complex (Smith et al. 1995).
2,4- and 2,6-DINITROTOLUENE 148

6. ANALYTICAL METHODS

Dichloromethane is the solvent of choice for extracting DNT from water samples (EPA 1982a) and from

wastes (EPA 1986a). Reversed-phase high-performance liquid chromatography (RP-HPLC) is attractive for

the determination of DNT 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 DNT from low-lipid biological samples and for

subsequent HPLC determination after sample cleanup.

Methods for the determination of the DNT in biological samples are given in Table 6-l.

6.2 ENVIRONMENTAL SAMPLES

The basic method for collecting DNT 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. New instrumentation for the detection of 2,4-DNT and other explosives has recently been developed

that 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; the detection limit for 2,4-DNT is 20 ppt

(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).

DNT is 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 DNT 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). Supercritical fluid

extraction (SFE) has been used with GC to reduce the preparation time of DNT in solids, such as soil

(Francis et al. 1995). A sonic extraction-liquid chromatographic method has also 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).

The analysis of DNT is normally done by GC with a variety of detectors, including flame ionization detector

(FIJI), electron capture detector (ECD), Hall electrolytic conductivity detector (HECD), thermionic specific


2,4- and 2,6-DINITROTOLUENE 151

6. ANALYTICAL METHODS

detector (TSD), fourier transform infrared (FT-IR), 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

structurespecific 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 GUMS 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).

A sensitive and selective technique that has been used to identify trace amounts of 2,4-DNT and other

explosive vapors is negative ion mobility spectrometry (Clark et al. 1995). This technique makes use of a

tunable laser ionization source to produce characteristic negative ions, which can be used to identify the

chemicals present (Clark et al. 1995). 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 DNT 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 for 2,4-DNT and 0.003 μg/L for 2,6-DNT; these detection limits are sensitive enough to meet the

suggested requirements for EPA health advisories and water quality criteria.

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 with a standard deviation of 3.4 μg/L for concentrations up to 250

μg/L. HPLC with photodiode array detection was used by Bouvier et al. (1995) to separate and quantitate

2,4- and 2,6-DNT in water samples. Recoveries ranged from 95 to 100%, and minimum detection limits

were 0.041- 0.160 μg/L (Bouvier et al. 1995). A convenient method for analysis of 2,4- and 2,6-DNT in
2,4- and 2,6-DINITROTOLUENE 152

6. ANALYTICAL METHODS

contaminated soils used high performance liquid chromatography with minimal sample preparation (Preslan

et al. 1993).

Methods for the determination of DNT in environmental samples are summarized in Table 6-2.

6.3 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 2,4-DNT and 2,6-DNT is available. Where adequate information is not

available, ATSDR, in conjunction with the 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 2,4-DNT and 2,6-DNT.

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.

6.3.1 Identification of Data Needs

Methods for Determining Biomarkers of Exposure and Effect. The available methods for the

determination of DNT and its metabolites in biological samples are inadequate. Although one method exists

for determination of DNT and its 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 modem

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). 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.







2,4- and 2,6-DINITROTOLUENE 160

6. ANALYTICAL METHODS

Methods for Determining Parent Compounds and Degradation Products in Environmental

Media. DNT 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; NIOSH 1977; 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, 1 μg/L in surface waters, and 10 μg/L in effluent waters. Analytes well include

numerous semivolatile compounds and some compounds that are only “semi-soluble” in water, as well as

volatile compounds (boiling point <150°C). A comprehensive review of the literature leading up to these

efforts has been published (Pellizzari et al. 1985). 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.

6.3.2 Ongoing Studies

No ongoing studies were identified on analytical methods for DNT and its metabolites.
2,4- and 2,6-DINITROTOLUENE 161

7. REGULATIONS AND ADVISORIES

The international, national, and state regulations and guidelines regarding 2,4-DNT and 2,6-DNT in air,

water, and other media are summarized in Table 7-l.

ATSDR has derived two MRL values for 2,4-DNT and one MRL value for 2,6-DNT. An acute-duration oral

MRL of 0.05 mg/kg/day was derived for 2,4-DNT based on neurotoxicity in dogs (Ellis et al. 1985; Lee et al.

1978). A chronic-duration oral MRL of 0.002 mg/kg/day was derived for 2,4-DNT based on a NOAEL of

0.2 mg/kg/day for neurotoxicity, Heinz bodies, and biliary tract hyperplasia in dogs (Ellis et al. 1979, 1985).

For 2,6-DNT, an intermediate-duration oral MRL of 0.004 mg/kg/day was derived based on hematological

effects of splenic extramedullary erythropoiesis and lymphoid depletion in dogs (Lee et al. 1976).

The International Agency for Research on Cancer (IARC) classifies 2,4-DNT and 2,6-DNT as Group 2B

carcinogens (possibly carcinogenic to humans) (IARC 1996). The U.S. EPA assigns Class 2B (human

carcinogen) (EPA 1996).

2,4-DNT and 2,6-DNT are on the list of chemicals in “The Emergency Planning and Community Right-toKnow Act of 1986” (EPA 1989a, 1988a). Section 313 of Title III of the Super-fund Amendments and

Reauthorization Act (SARA) requires owners and operators of certain facilities that manufacture, import,

process, or otherwise use the chemicals on this list to report annually any release of those chemicals to any

environmental media over a specified threshold level.

OSHA requires employers of workers who are occupationally exposed to 2,4-DNT and 2,6-DNT to institute

engineering controls and work practices to reduce and maintain employee exposure at or below permissible

exposure limits (PEL). If the employer can document that 2,4-DNT and 2,6-DNT are used in the workplace

less than 30 days per year, the employer can use any combination of engineering controls, work practice

controls, or respirators to reduce employee exposure to or below the (PEL) of 1.5 mg/m3

. PELs are 8-hour

time-weighted averages (TWA). Respirators must be provided and used during the time period necessary to

install or implement feasible engineering and work practice controls, or where controls are not yet sufficient.

Respirators are also required when the employer determines that compliance with the PEL is not feasible with

engineering or work practice controls, such as maintenance and repair activities, vessel cleaning, or other

operations where exposures are intermittent and limited in duration, and in emergencies (OSHA 1987).



2,4- and 2,6-DINITROTOLUENE 165

7. REGULATIONS AND ADVISORIES

2,4-DNT and 2,6-DNT are regulated by the Clean Water Effluent Guidelines in 40 CFR Part 401. For each

point source category, 2,4-DNT and 2,6-DNT may be regulated as part of a group of chemicals controlled as

total toxic organics, or may have a specific regulatory limitation. The point source categories for which

2,4-DNT and 2,6-DNT are controlled as total toxic organics include electroplating and metal finishing (EPA

1998f). The point source category for which 2,4-DNT and 2,6-DNT have a specific regulatory limitation is

organic chemicals, plastics, and synthetic fibers (EPA 1998g).

EPA has developed oral reference dose (RfD) of 0.002 mg/kg/day and 0.001 mg/kg/day for 2,4- and

2,6-DNT, respectively (EPA 1998d).

An oral slope factor (q1*) of 6.8x10-l (mg/kg/day)-1 has been derived for the 2,4-/2,6-DNT mixture and is

used for the cancer risk assessment of both 2,4- and 2,6-DNT (EPA 1998).

2,4- and 2,6-DINITROTOLUENE 167

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2,4- and 2,6-DINITROTOLUENE 193

9. GLOSSARY

Acute Exposure-Exposure to a chemical for a duration of 14 days or less, as specified in the Toxicological

Profiles.

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 a 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.

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.

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.

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.

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.

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 occurred. The terms, as used here, include malformations and variations, altered growth, and in utero

death.

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.

Immediately Dangerous to Life or Health (IDLH)-The maximum environmental concentration of a

contaminant from which one could escape within 30 min without any escape-impairing symptoms or

irreversible health effects.

Intermediate Exposure-Exposure to a chemical for a duration of 15-364 days, as specified in the

Toxicological Profiles.
2,4- and 2,6-DINITROTOLUENE 194

9. GLOSSARY

Immunologic Toxicity-The occurrence of adverse effects on the immune system that may result from

exposure to environmental agents such as chemicals.

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 which 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 is

expected to have caused death in humans or animals.

Lethal Dose(50) (LD50)-The dose of a chemical which 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 dose 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.

Malformations-Permanent structural changes that may adversely affect survival, development, or function.

Minimal Risk Level-An estimate of daily human exposure to a dose of a chemical that is likely to be

without an appreciable risk of adverse noncancerous effects over a specified duration of exposure.

Mutagen-A substance that causes mutations. A mutation is a change in the genetic material in a body cell,

Mutations can lead to birth defects, miscarriages, or cancer.

Neurotoxicity--The occurrence of adverse effects on the nervous system following exposure to chemical,

No-Observed-Adverse-Effect Level (NOAEL)-The dose of 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.

Permissible Exposure Limit (PEL)-An allowable exposure level in workplace air averaged over an 8-hour

shift.
2,4- and 2,6-DINITROTOLUENE 195

9. GLOSSARY

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).

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 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

CERCLA. Reportable quantities are (1) 1 pound or greater or (2) for selected substances, an amount

established by regulation either under CERCLA or under Sect. 3 11 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.

Short-Term Exposure Limit (STEL)-The maximum concentration to which workers can be exposed for

up to 15 min continually. No more than four excursions are allowed per day, and there must be at least 60

min between exposure periods. The daily TLV-TWA may not be exceeded.

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)-A concentration of a substance to which most workers can be exposed

without adverse effect. The TLV may be expressed as a TWA, as a STEL, or as a CL.

Time-Weighted Average (TWA)-An allowable exposure concentration averaged over a normal 8-hour

workday or 40-hour workweek.

Toxic Dose (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.

Uncertainty Factor (UF)-A factor used in operationally deriving the RfD 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 LOAEL data rather

than NOAEL data. Usually each of these factors is set equal to 10.

2,4- and 2,6-DINITROTOLUENE 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 Super-fund Amendments and Reauthorization Act (SARA) [Pub. L. 99-4991,

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.
2,4- and 2,6-DINITROTOLUENE A-2

APPENDIX A

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 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 a hundredfold 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, expert panel peer reviews, and agencywide 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, h4RLs in the

most recent toxicological profiles supersede previously published levels. For additional information

regarding MRLs, please contact the Division of Toxicology, Agency for Toxic Substances and Disease

Registry, 1600 Clifton Road, Mailstop E-29, Atlanta, Georgia 30333.






2,4- and 2,6-DINITROTOLUENE B-l

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

Tables and Figures for Levels of Significant Exposure (LSE)

Tables (2-l) 2-2, and 2-3) and figures (2-l and 2-2) 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, minimal risk levels (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 No-Observed-Adverse- Effect Levels (NOAELs),

Lowest-Observed-Adverse-Effect Levels (LOAELs), or Cancer Effect Levels (CELs).

The legends presented below demonstrate the application of these tables and figures. Representative

examples of LSE Table 2-l and Figure 2-l are shown. The numbers in the left column of the legends

correspond to the numbers in the example table and figure.

LEGEND

See LSE Table 2-1

(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. When sufficient data
2,4-and 2,6-DINITROTOLUENE B-2

APPENDIX B

exists, 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 Table 2-1,

2-2, and 2-3, respectively). LSE figures are limited to the inhalation (LSE Figure 2-l) and oral (LSE

Figure 2-2) 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 2 “18r” data

points in Figure 2-l).

(5) Species The test species, whether animal or human, are identified in this column. Section 2.5,

“Relevance to Public Health,” covers the relevance of animal data to human toxicity and Section 2.3,

“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 regimen

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 1,1,2,2-tetrachloroethane via inhalation for

6 hours per day, 5 days per week, for 3 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,l systemic effect (respiratory) was investigated.

(8) NOAEL A No-Observed-Adverse-Effect Level (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
2,4- and 2,6-DINITROTOLUENE B – 3

APPENDIX B

the respiratory system which was used to derive an intermediate exposure, inhalation MRL of 0.005

ppm (see footnote “b”).

(9) LOAEL A Lowest-Observed-Adverse-Effect Level (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

endpoint 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. Ml&s are not derived from

Serious LOAELs.

(10) Reference The complete reference citation is given in chapter 8 of the profile.

(11) CEL A Cancer Effect Level (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

ootnotes. Footnote “b” indicates the NOAEL of 3 ppm in key number 18 was used to derive an MFU

of 0.005 ppm.

LEGEND

See Figure 2-1

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 intermediate and chronic 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, 18r NOAEL is the critical endpoint for which an intermediate inhalation

exposure MRL is based. As you can see from the LSE figure key, the open-circle symbol indicates to a
2,4- and 2,6-DINITROTOLUENE B-4 

APPENDIX B

NOAEL for the test species-rat. 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 38r is 1 of 3 studies for which Cancer Effect Levels were derived. The diamond

symbol refers to a Cancer Effect Level for the test species-mouse. The number 38 corresponds to the

entry in the LSE table.

(18) Estimated Upper-Bound Human Cancer Risk Levels This is the range associated with the upper-bound

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.

The Relevance to Public Health section provides a health effects summary based on evaluations of existing

toxicologic, epidemiologic, and toxicokinetic information. This summary is designed to present interpretive,

weight-of-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 section covers end points in the same order they appear within the Discussion of Health Effects by Route

of Exposure section, by route (inhalation, oral, 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 section. If

data are located in the scientific literature, a table of genotoxicity information is included.

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 Data Needs section.


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APPENDIX B

Chapter 2 (Section 2.5)

Relevance to Public Health

Interpretation of Minimal Risk Levels

Where sufficient toxicologic information is available, we have derived minimal risk levels (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. They 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 2.5,

“Relevance to Public Health,” contains basic information known about the substance. Other sections such as

2.8, “Interactions with Other Substances,” and 2.9, “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 the Environmental Protection Agency (EPA) provides (Barnes

and Dourson 1988) to determine reference doses for lifetime exposure (RfDs).

To derive an MRL, ATSDR generally selects the most sensitive endpoint 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 endpoint are available, ATSDR derives an MRL using the most sensitive species (when

information from multiple species is available) with the highest 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 (UP) 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 LSE Tables.