TOXICOLOGICAL PROFILE FOR 

3,3’-DICHLOROBENZIDINE 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
 

Public Health Service
 

Agency for Toxic Substances and Disease Registry
 

December 1998 
3,3’-DICHLOROBENZIDINE 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. 
3.3’-DICHLOROBENZIDINE iii 

UPDATE STATEMENT
 

A Toxicological Profile for 3,3’-Dichlorobenzidine 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
 


*Legislative Background vi 

The toxicological profiles are developed in response to the Superfund 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 FR 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. 
___________________________________________________________________________ 

___________________________________________________________________________ 

3.3’-DICHLOROBENZIDINE 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 1-888-42-ATSDR in 1999) 

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

E-mail: atsdric@,cdc.nov Internet: http://atsdrl.atsdr.cdc.nov: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. 
________________________________________________________________________ 

________________________________________________________________________ 

3,3’-DICHLOROBENZIDINE 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 IIIMedicalManagement 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 Centerfor Environmental Health (NCEH) 

focuses on preventing or controlling disease, injury, and disability related to the 

interactions between people and their environment outside the workplace. 

Contact: NCEH, Mailstop F-29,4770 Buford Highway, NE, Atlanta, GA 30341-3724 •

Phone: 770-488-7000 • FAX: 770-488-7015. 

The National Institute for Occupational Safety and Health (NIOSH) 

conducts research on occupational diseases and injuries, responds to requests for 

assistance by investigating problems of health and safety in the workplace, recommends 

standards to the Occupational Safety and Health Administration (OSHA) and the Mine 

Safety and Health Administration (MSHA), and trains professionals in occupational 

safety and health. Contact: NIOSH, 200 Independence Avenue, SW, Washington, DC 

2020 1 • Phone: 800-356-4674 or NIOSH Technical Information Branch, Robert A. Taft 

Laboratory, Mailstop C-19,4676 Columbia Parkway, Cincinnati, OH 45226-1998 •

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 •

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, 10 10 Vermont Avenue, NW, #5 

13, Washington, DC 20005 l Phone: 202-347-4976 l FAX: 202- 347-4950 •

e-mail: aoec@,dgs.dnsys.com • 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 • Phone: 847-228-6850 • FAX: 847-228-1856. 
3,3’-DICHLOROBENZIDINE ix 

CONTRIBUTORS
 

CHEMICAL MANAGER(S)/AUTHORS(S):
 

Lori L. Miller, M.P.H.
 

ATSDR Division of Toxicology, Atlanta, GA
 

Cassandra Smith-Simon, M.S.
 

ATSDR, Division of Toxicology, Atlanta, GA
 

Fernando Llados, Ph.D.
 

Research Triangle Institute, Research Triangle Park, NC
 

Steve Kueberuwa, M.S.
 

Research Triangle Institute, Research Triangle Park, NC
 

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. 

3.3’-DICHLOROBENZIDINE xi 

PEER REVIEW
 

A peer review panel was assembled for 3,3’-dichlorobenzidine. The panel consisted of the 

following members : 

1. Dr. Herbert Cornish, Private Consultant, 830 W. Clark Rd., Ypsilanti, MI 48 198; 

2. Dr. Arthur Gregory, Private Consultant, 1 Gregory Lane, Luray, VA 22835; 

3. Dr. Philip Leber, Private Consultant, 1344 Jefferson Ave., Akron, OH 443 13; and 

4. Dr. Robert Rubin, Johns Hopkins School of Public Health, Environmental Health Sciences,

 Baltimore, MD 21205. 

These experts collectively have knowledge of 3,3’-dichlorobenzidine’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(1)( 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. 









3,3’-DICHLOROBENZIDINE 1 

1. PUBLIC HEALTH STATEMENT
 

This public health statement tells you about 3,3’-dichlorobenzidine 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. 3,3’-Dichlorobenzidine has been found in at least 32 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 3,3’-dichlorobenzidine 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 3,3’-dichlorobenzidine, 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 .l WHAT IS 3,3’-DICHLOROBENZIDINE? 

3,3’-Dichlorobenzidine is a gray-to-purple colored crystalline solid. It changes from a solid to a 

gas very slowly. 3,3’-Dichlorobenzidine salt, the major form in actual use, is a stable, off-white 

colored crystalline solid that does not evaporate. Neither 3,3’-dichlorobenzidine nor its salt occur 

naturally in the environment. They are manufactured for use in the production of pigments for 

printing inks, textiles, plastics and enamels, paint, leather, and rubber. Whether 3,3’­

dichlorobenzidine or the salt is present as such depends on the acidity of the soil or water as well 

as other 
3,3’-DICHLOROBENZIDINE 2 

1. PUBLIC HEALTH STATEMENT
 

factors. In most environmental samples, such as water and soils, 3,3’-dichlorobenzidine would be 

expected to exist in the free amino form, not as the salt. For more information, see Chapters 3 

and 4. 

1.2 WHAT HAPPENS TO 3,3’-DICHLOROBENZIDINE WHEN IT ENTERS THE 

ENVIRONMENT? 

3,3’-Dichlorobenzidine breaks down rapidly when exposed to natural sunlight. In air and 

sunshine, it is estimated that half of the chemical breaks down within 9.7 hours. In water exposed 

to natural sunlight, 3,3’-dichlorobenzidine is expected to break down rapidly, with half being 

removed in approximately 90 seconds. In soil, where no sunlight is present, the compound may 

last for several months. Under certain conditions, 3,3’-dichlorobenzidine can break down in soil 

to form another compound, benzidine, which is toxic. For more information, see Chapter 5. 

1.3 HOW MIGHT I BE EXPOSED TO 3,3’-DICHLOROBENZIDINE? 

3,3’-Dichlorobenzidine is used to make pigments (substances used to give color to something, for 

example, paint). You are most likely to be exposed to 3,3’-dichlorobenzidine if you work inside 

plants where the chemical is manufactured or used. However, employers have limited workers’ 

exposure to the chemical by using closed systems for processing as well as other methods for 

reducing its concentration in the air to very low levels and by requiring workers to wear 

protective clothing and use special equipment. If you were exposed in such a workplace, it would 

probably be by breathing in the dust or by getting the chemical on your skin. Careless handling or 

accidental spillage of the chemical could result in exposure to potentially hazardous levels of 

3,3’-dichlorobenzidine. People may be exposed to the chemical if they live or work near land 

where plant wastes have been stored or buried, or close to lakes, streams, or rivers near where 

plants discharge process water or store wastes. Most people do not live near a source of the 

chemical. The Canadian government has published calculations that show that exposure of the 

Canadian general population to 3,3’-dichlorobenzidine in air, soil, or water is extremely low. If 

you do live in areas near a source of the chemical (such as a hazardous waste site that contains 
3,3’-DICHLOROBENZIDINE 3 

1. PUBLIC HEALTH STATEMENT
 

dye or pigment manufacturing wastes), some exposure could occur if you or a child accidentally 

or purposely ingested small amounts of contaminated soil, drank contaminated water, or ate fish 

caught in waters near the source. However, studies of water and fish taken from locations near 

dye-manufacturing plants did not find the chemical. 

3,3’-Dichlorobenzidine has no agricultural or food chemical uses, so exposure to it by eating 

contaminated food is not likely. More information about the presence of 3,3’-dichlorobenzidine in 

our environment and how it disappears by being broken down by other chemicals and processes 

can be found in Chapter 5. 

1.4 HOW CAN 3,3’-DICHLOROBENZIDINE ENTER AND LEAVE MY BODY? 

In the workplace, 3,3’-dichlorobenzidine may enter the body when workers breathe dust 

contaminated by 3,3’-dichlorobenzidine and through skin contact. You are not likely to be 

exposed to 3,3’-dichlorobenzidine unless you drink water or eat dirt contaminated with 

3,3’-dichlorobenzidine in the vicinity of a hazardous waste site where 3,3’dichlorobenzidine has 

been stored and leakage has occurred. When 3,3’-dichlorobenzidine does enter the body, very 

little of it leaves the body unchanged. Most of it (over 90%) is changed to related chemical 

substances called metabolites, which leave the body, mainly in urine and to a lesser extent in 

feces, within 72 hours after exposure. More information can be found in Chapter 2. 

1.5 HOW CAN 3,3’-DICHLOROBENZIDINE AFFECT MY HEALTH? 

Some workers exposed to the salt form of 3,3’-dichlorobenzidine complained of sore throat, 

respiratory infections, stomach upset, headache, dizziness, caustic burns, and dermatitis (an 

inflammation of the skin). However, with the exception of dermatitis, it is not certain that 

3,3’-dichlorobenzidine causes these health effects because the workers were also exposed to other 

chemicals at the same time. There is no evidence that 3,3’-dichlorobenzidine affects the nervous 

system, the ability to fight disease, or the ability of people to have children. 
3,3’-DICHLOROBENZIDINE 4 

1. PUBLIC HEALTH STATEMENT
 

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. 

Death has occurred in laboratory animals that ate very high levels of 3,3’-dichlorobenzidine 

mixed in their food for short periods of time. Laboratory animals exposed to moderate levels of 

3,3’-dichlorobenzidine mixed with food for a long time suffered mild injury to the liver. 

Studies show that 3,3’-dichlorobenzidine caused cancer of the liver, skin, breast, bladder, and 

tissues that form blood (leukemia), and other sites in laboratory animals that ate 3,3’­

dichlorobenzidine in their food. There is no evidence that 3,3’-dichlorobenzidine has caused 

cancer in people who worked with it or who were exposed to it unknowingly or by accident for a 

short or long time. However, because of the many types of cancer that 3,3’-dichlorobenzidine has 

caused in different tissues of many types of laboratory animals, 3,3’-dichlorobenzidine should be 

thought of as probably capable of causing human cancer if exposure to the chemical is 

sufficiently high. 

The Environmental Protection Agency (EPA) has determined that 3,3’-dichlorobenzidine is a 

“probable human carcinogen.” The U.S. Department of Health and Human Services (DHHS) has 

determined that 3,3’-dichlorobenzidine and its salt may reasonably be expected to be 

cancercausing substances (carcinogens). The International Agency for Research on Cancer 

(IARC) has determined that 3,3’-dichlorobenzidine is possibly carcinogenic to humans. More 

information can be found in Chapter 2. 
3,3’-DICHLOROBENZIDINE 5 

1. PUBLIC HEALTH STATEMENT
 

1.6 HOW CAN 3,3’-DICHLOROBENZIDINE 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. 

Children might be exposed to 3,3’-dichlorobenzidine if they eat small amounts of soil 

contaminated with 3,3’-dichlorobenzidine. However, studies suggest that it is very difficult to 

release 3,3’-dichlorobenzidine once it becomes attached to soil. Exposure via contaminated soil 

may occur if they live in an area near a source of the chemical (such as a hazardous waste site that 

contains 3,3’-dichlorobenzidine). Children can also be exposed if the parents work at chemical 

facilities where 3,3’-dichlorobenzidine is handled and bring home contaminated clothing or tools 

or if they do not shower before coming home. There are no known unique exposure pathways for 

children. 

There have been no studies of health effects in children exposed to 3,3’-dichlorobenzidine. We 

have no information on whether 3,3’-dichlorobenzidine causes birth defects in children. It is 

unknown whether birth defects would occur in the offspring of pregnant animals that breathed or 

eaten 3,3’-dichlorobenzidine, or had it on their skin. In studies in which pregnant mice were 

injected with high amounts of 3,3’-dichlorobenzidine under the skin, the kidneys of their babies 

did not develop properly and some babies developed renal tumors. However, it is highly unlikely 

that humans will encounter such exposure conditions. 

There is no information to determine whether children are different in their sensitivity to the 

health effects of 3,3’-dichlorobenzidine from adults. There is indirect evidence that 3,3’­

dichlorobenzidine or its breakdown products can cross the placenta, but we do not know for 

certain whether it can be transferred to the young via the mother’s breast milk. Sometimes when 

children have been exposed to chemicals before they are born, the chemical or its breakdown 

products can be found in amniotic fluid, meconium, cord blood, or neonatal blood; however, no 

information 
3,3’-DICHLOROBENZIDINE 6 

1. PUBLIC HEALTH STATEMENT
 

about such measurements was found for 3,3’-dichlorobenzidine. More information regarding 

children’s health and 3,3’-dichlorobenzidine can be found in Section 2.6. 

1.7 	HOW CAN FAMILIES REDUCE THE RISK OF EXPOSURE TO

 3,3’-DICHLOROBENZIDINE? 

If your doctor finds that you have been exposed to significant amounts of 3,3’-dichlorobenzidine, 

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

your state Department of Public Health to investigate. 

3,3’-Dichlorobenzidine has no agricultural or food chemical uses, so exposure to it by eating 

contaminated food is not likely. It is sometimes possible to carry 3,3’-dichlorobenzidine from 

work on your clothing, skin, hair, tools, or other objects removed from the workplace. This has 

happened in factories that produce 3,3’-dichlorobenzidine. In this way, you may contaminate 

your car, home, or other locations outside work where children might be exposed to 3,3’­

dichlorobenzidine. You should know about this possibility if you work with 3,3’­

dichlorobenzidine. 

Your occupational health and safety officer at work can and should tell you whether chemicals 

you work with are dangerous and likely to be carried home’on your clothes, body, or tools. Ask 

if you should shower and change clothes before you leave work, store your street clothes in a 

separate area of the workplace, or launder your work clothes at home separately from other 

clothes. The Occupational Safety and Health Administration (OSHA) requires Material Safety 

Data Sheets (MSDSs) for many chemicals used at your place of work. MSDS information should 

include chemical names and hazardous ingredients, and important information such as fire and 

explosion data, potential health effects, how you get the chemical(s) in your body, how to 

properly handle the materials, and what to do in the case of emergencies. Your employer is 

legally responsible for providing a safe workplace and should freely answer your questions about 

hazardous chemicals. U.S. OSHA or your state OSHA-approved occupational safety and health 

program can answer any further questions and help your employer identify and correct problems 

with hazardous substances. OSHA or your state OSHA-approved occupational safety and health 

program will listen to your formal complaints about workplace health hazards and inspect your 
3,3’-DICHLOROBENZIDINE 7 

1. PUBLIC HEALTH STATEMENT
 

workplace when necessary. Employees have a right to seek safety and health on the job without 

fear of punishment. More information regarding exposure to 3,3’-dichlorobenzidine can be found 

in Sections 5.5, 5.6, and 5.7. 

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

 EXPOSED TO 3,3’-DICHLOROBENZIDINE? 

Exposure to 3,3’-dichlorobenzidine can be determined by finding the chemical or its metabolites 

in urine. The test is not commonly available to the general population, but it is available to 

workers who may be exposed to potentially hazardous levels of the chemical in the workplace 

(for example, by careless handling or accidental spills). The test is accurate and provides evidence 

that exposure has occurred. However, since 3,3’-dichlorobenzidine does not remain long in the 

body, the test must be performed very soon after the possible exposure. Also, measured urine 

levels of 3,3’-dichlorobenzidine or its metabolites do not tell you whether it will affect your 

health. More information can be found in Chapter 6. 

1.9 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO

 PROTECT HUMAN HEALTH? 

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. 
3,3’-DICHLOROBENZIDINE 8 

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 3,3’-dichlorobenzidine include the 

following: 

EPA has determined that 3,3’-dichlorobenzidine is a “probable human carcinogen” and has 

placed several limits on the chemical in the environment to protect human health. Under the 

Clean Water Act of 1977, EPA controls discharges of 3,3’-dichlorobenzidine to industrial waste 

waters. The agency has listed 3,3’-dichlorobenzidine as a hazardous waste and requires that any 

spill of one pound or more be reported to the National Response Center. 

Although the FDA has classified 3,3’-dichlorobenzidine as a carcinogen, no regulatory guidelines 

have been enacted. The FDA has concluded that the food supply is not in danger from 

3,3’-dichlorobenzidine. 

3,3’-Dichlorobenzidine is one of a number of compounds regulated by OSHA. To control 

exposures to 3,3’-dichlorobenzidine in workplace air and to protect the health of workers, 

OSHA’s regulatory standards provide strict guidelines for handling, using, and storing the 

compound. They also include the requirements for personal protective equipment, training, 

labeling, and posting and engineering controls. OSHA also requires that initial medical screening 

and regular medical examinations be made available to any employee who is exposed to 3,3’-

dichlorobenzidine at potentially hazardous levels. 

NIOSH considers 3,3’-dichlorobenzidine a “potential occupational carcinogen” and recommends 

workplace practices and controls to reduce exposures to the lowest possible level. NIOSH 

defines potential occupational carcinogens as substances which may cause an increased incidence 

of benign and/or malignant neoplasm, or a substantial decrease in the latency period between 

exposure and onset of neoplasms in humans. 
3,3’-DICHLOROBENZIDINE 9 

1. PUBLIC HEALTH STATEMENT
 

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. 

* To order toxicological profiles, contact: 

National Technical Information Service
 

5285 Port Royal Road
 

Springfield, VA 22 16 1
 

Phone: (800) 553-6847 or (703) 487-4650
 

3,3’-DICHLOROBENZIDINE 11 

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 3,3’­

dichlorobenzidine. 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 exposureinhalation, 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-observedadverse-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 
3,3’-DICHLOROBENZIDINE 12 

2. HEALTH EFFECTS
 

distinction between “less serious” effects and “serious” effects is considered to be important 

because it helps the users of the profiles to identify levels of exposure at which major health 

effects start to appear. LOAELs or NOAELs should also help in determining whether or not 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 3,3’­

dichlorobenzidine are indicated in Table 2- 1 and Figure 2- 1. Because cancer effects could occur 

at lower exposure levels, Figure 2-1 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. 

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. 

2.2.1 Inhalation Exposure 

3,3’-Dichlorobenzidine is not a volatile chemical. In the air, it may exist as dust particles or 

bound to particulate matter. The absorption of 3,3’-dichlorobenzidine from such respirable 

particles into the body depends, in part, on the size of the particle. Large particles tend to deposit 

in the upper airways and are subsequently cleared by ciliary action with little absorption across 

lung tissues. However, the ciliary action transports the particles to the epiglottis where they are 

often swallowed, leading to gastrointestinal absorption. Smaller particles can penetrate more 

deeply into the respiratory tree, where 3,3’-dichlorobenzidine absorption may be significant. 
3,3’-DICHLOROBENZIDINE 13 

2. HEALTH EFFECTS
 

2.2.1.1 Death 

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

3,3’-dichlorobenzidine. No fatalities were observed in rats observed for 14 days following a 

l-hour exposure to an unspecified concentration of 3,3’-dichlorobenzidine dihydrochloride dust 

(Gerarde and Gerarde 1974). No deaths were reported in male rats exposed to 23,700 mg/m3 

3,3’-dichlorobenzidine base (dust) for 2 hours per day for 7 days (Gerarde and Gerarde 1974). 

2.2.1.2 Systemic Effects 

No studies were located regarding cardiovascular, hematological, musculoskeletal, hepatic, renal, 

endocrine, dermal, ocular, body weight, or metabolic effects in humans or animals after 

inhalation exposure to 3.3’-dichlorobenzidine. 

Respiratory Effects. Upper respiratory infection and sore throat were listed among several 

principal reasons for visits to a company’s medical clinic by workers handling 

3,3’-dichlorobenzidine dihydrochloride (Gerarde and Gerarde 1974). However, there is no 

conclusive evidence that these effects were due to inhalation of 3,3’-dichlorobenzidine 

dihydrochloride. 

No adverse health effects were observed in male rats exposed by inhalation to 

3,3’- dichlorobenzidine free base (23,700 mg/m3

) 2 hours per day for 7 days (Gerarde and 

Gerarde 1974). In another study, 10 rats were exposed to an unspecified concentration of 

3,3’-dichlorobenzidine dihydrochloride dust particles for 1 hour and then observed for 14 days. 

Slight-to-moderate pulmonary congestion and one pulmonary abscess were observed upon 

necropsy (Gerarde and Gerarde 1974). The effects observed in the study using the ionized 

(hydrochloride) form of 3,3’-dichlorobenzidine may have been due to the irritative properties of 

hydrochloric acid released from the salt in combination with particulate toxicity. 

Gastrointestinal Effects. Gastrointestinal upset was one of the symptoms reported by employees 

who worked with 3,3’-dichlorobenzidine dihydrochloride (dihydro salt of 3,3’-dichlorobenzidine) 

(Gerarde and Gerarde 1974). However, there is no conclusive evidence that the gastrointestinal 

effects, or other symptoms reported by employees, resulted specifically from inhalation of 

3,3’-dichlorobenzidine dihydrochloride. 
3,3’-DICHLOROBENZIDINE 14 

2. HEALTH EFFECTS
 

No studies were located regarding gastrointestinal effects in animals following inhalation 

exposure to 3,3’-dichlorobenzidine. 

2.2.1.3 Immunological and Lymphoreticular Effects 

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

exposure to 3,3’-dichlorobenzidine. 

2.2.1.4 Neurological Effects 

The only relevant information regarding neurological effects in humans exposed to 

3,3’-dichlorobenzidine was found in an early study which reported that headache and dizziness 

were among several principal reasons why employees working with 3,3’-dichlorobenzidine in a 

chemical manufacturing plant visited the company medical clinic (Gerarde and Gerarde 1974). 

However, there is no conclusive evidence that these symptoms were caused specifically by 

3,3’-dichlorobenzidine since there was exposure to other chemicals as well. No further 

information was provided. 

No studies were located regarding neurological effects in animals after inhalation exposure to 

3,3’-dichlorobenzidine. 

No studies were located regarding the following effects in humans or animals after inhalation 

exposure to 3,3’-dichlorobenzidine: 

2.2.1.5 Reproductive Effects 

2.2.1.6 Developmental Effects 

2.2.1.7 Genotoxic Effects 

Genotoxicity studies are discussed in Section 2.5. 
3,3’-DICHLOROBENZIDINE 15 

2. HEALTH EFFECTS
 

2.2.1.8 Cancer 

Several epidemiological studies have investigated cancer incidences among workers 

occupationally exposed to 3,3’-dichlorobenzidine (Gadian 1975; Gerarde and Gerarde 1974; 

MacIntyre 1975; Myslak et al. 1991). Exposure may have been by both inhalation and dermal 

routes. 

Due, in part, to structure-activity considerations, epidemiological studies of potential cancer 

effects of occupational exposure to 3,3’-dichlorobenzidine have been particularly concerned with 

bladder tumors, since 3,3’-dichlorobenzidine is structurally similar to benzidine, a chemical 

which is known to be a human bladder carcinogen. The possible role of benzidine-based azodyes 

as a carcinogenic risk factor for painters in a major industrial area of Germany was investigated 

by Myslak et al. (1991). The cohort consisted of 403 male patients (case group) treated in the 

period 1984-1987 for urological tumors: 290 had a diagnosis of bladder carcinoma and 113 had a 

diagnosis of bladder papilloma. The mean duration of employment was 29 years (range 2-48 

years). A comparison group (reference group) of 426 patients with benign prostate disease was 

also included in the study. Cases and controls responded to questionnaires regarding employment 

history. Questionnaires were analyzed for occupational categories. A painter was defined as a 

person employed in this occupation for at least 6 months at any time of his working history and 

who had never been employed in another occupation known to be causally associated with 

bladder cancer. Of the bladder tumor patients, 21 were painters; among referents, 8 were painters. 

This difference among the groups was statistically significant; the relative risk of painters to be 

associated with bladder tumor was 2.76 (p<0.01). Occupation as painter (primarily house painter) 

was far more frequent among bladder tumor patients than would be expected from census data. 

The relative risk of bladder tumors for current smokers and ex-smokers was 1.13, which led 

Myslak et al. (1991) to suggest that the risk of smoking for bladder tumors was less than the 

occupational risk for the painters. The authors noted that a large number of benzidine-based 

azodyes were manufactured in Germany in the past. During that time it was usual for painters to 

prepare the paints themselves, allowing for possible exposure to dyes and pigments derived from 

benzidine, 3,3’-dichlorobenzidine, 3,3’-dimethylbenzidine (o-tolidine), 3,3’-dimethoxybenzidine 

(o-dianisidine), and 2-naphthylamine (Myslak et al. 1991). While the results of this study suggest 

that occupational exposure to benzidine-like chemicals is associated with an increased incidence 

in bladder tumors, the specific role of 3,3’-dichlorobenzidine, if any, is unknown. 
3,3’-DICHLOROBENZIDINE 16 

2. HEALTH EFFECTS
 

No other epidemiological studies have found either bladder tumors or excess tumors at other sites 

that were associated with 3,3’-dichlorobenzidine (Gadian 1975; Gerarde and Gerarde 1974; 

MacIntyre 1975). However, these studies were conducted with workers who were exposed to 

3,3’-dichlorobenzidine for less than 20 years. Since a period of 5 to 50 years may follow the 

exposure to bladder carcinogens and the diagnosis of bladder cancer by a physician (Badalament 

1998), an adequate latency period for 3,3’-dichlorobenzidine-induced tumors may not have 

elapsed for some individuals. Also, the number of workers examined in these studies was 

relatively small, thus limiting the statistical power to detect a significant increase in bladder 

cancer mortality (incidence). Finally, the possibility that 3,3’-dichlorobenzidine is a human 

carcinogen under certain undefined exposure conditions cannot be totally ruled out. 

In one of these reports, no bladder tumors were found in a group of 35 workers who handled only 

3,3’-dichlorobenzidine; in the same dyestuff plant, bladder tumors occurred in 3 out of 14 

workers exposed to both benzidine and 3,3’-dichlorobenzidine. The investigator reported a total 

exposure time of 68,505 hours, equivalent to nearly 140 full-time working years (Gadian 1975). 

No cases of bladder tumors were found in an epidemiology study of 259 workers exposed to dry 

and sernidry 3,3’-dichlorobenzidine base and hydrochloride. Cytological analyses of the urine 

(Papanicolaou tests) were negative. Workers were exposed to an average of less than 16 years 

each to 3,3’-dichlorobenzidine, which means that an adequate exposure duration and/or the latent 

period following exposure may not have been reached for tumor expression (MacIntyre 1975). 

In a retrospective epidemiological study of workers employed in a dye and pigment 

manufacturing plant that used 3,3’-dichlorobenzidine as chemical precursor, no bladder tumors 

were observed in a cohort of 207 workers, most of whom had been exposed for up to 15 years 

(Gerarde and Gerarde 1974). However, in this study there was no evidence that any valid system 

of medical surveillance of workers ever existed during the years that 3,3’-dichlorobenzidine was 

used at the plant. A number of employees had not been followed up for 15 years or more 

(Gerarde and Gerarde 1974). Other limitations of this study included using data from a very small 

and incomplete sample of workers; focusing solely on the occurrence of bladder tumors; and 

using data that may have been misleading and, at times, apparently inaccurate. 
3,3’-DICHLOROBENZIDINE 17 

2. HEALTH EFFECTS 

No studies were located regarding cancer effects in animals after inhalation exposure to 

3,3’-dichlorobenzidine. However, cancer effects have been observed in animal studies where 

3,3’-dichlorobenzidine was administered orally or by other routes. See Sections 2.2.2.8 and 2.5 

for further information. 

2.2.2 Oral Exposure 

Indirect gastrointestinal tract exposure may occur from breathing contaminated airborne dust in 

the workplace. The respiratory deposition pattern of inhaled 3,3’-dichlorobenzidine depends 

primarily on the mass median aerodynamic diameter (MMAD) of the particles. The mucociliary 

clearance mechanism moves most particulates with a MMAD of l-5 μm out of the lower 

respiratory tract, thus allowing their passage into the gastrointestinal tract. Larger particles 

(>5 μm) impacting in the nasopharyngeal region would also be eventually ingested. Oral 

exposure may potentially occur in the general environment by drinking contaminated 

groundwater. Occupational exposure by the oral route is not expected to be significant. Exposure 

through eating food is unlikely since 3,3’-dichlorobenzidine has never had an application as an 

agricultural or food chemical. Children may be exposed to 3,3’-dichlorobenzidine if they 

consume contaminated soil; however, the bioavailability of 3,3’-dichlorobenzidine from soil is 

quite low. All of the available data on the effects of 3,3’-dichlorobenzidine following oral 

exposure are derived from studies in experimental animals. Table 2- 1 and Figure 2- 1 summarize 

available data. 

2.2.2.1 Death 

No studies were located regarding lethal effects in humans after oral exposure to 

3,3’-dichlorobenzidine. 

In rats, the acute-duration oral LD50 (lethal dose, 50% kill) for 3,3’-dichlorobenzidine free base 

administered in pure olive oil was estimated to be 7,070 mg/kg, whereas the LD50 for a 20% 

suspension of the dihydrochloride salt in corn oil was 3,820 mg/kg (Gerarde and Gerarde 1974). 

The cause of death was not discussed. Given this high LD50 acute lethality in humans following 

oral exposure is unlikely. Both oral LD50 values for 3,3’-dichlorobenzidine are shown in Table 

2-1 and plotted in Figure 2-l. 






3,3’-DICHLOROBENZIDINE 24 

2. HEALTH EFFECTS
 

2.2.2.2 Systemic Effects 

No studies were located regarding respiratory, hematological, hepatic, renal, or body weight 

effects in humans after oral exposure to 3,3’-dichlorobenzidine. No studies were located 

regarding cardiovascular, gastrointestinal, musculoskeletal, endocrine, dermal, ocular, or 

metabolic effects in humans or animals after oral exposure to 3,3’-dichlorobenzidine. 

The highest NOAEL values and all LOAEL values for oral exposure from each reliable study for 

systemic effects in each species and duration category for 3,3’-dichlorobenzidine are shown in 

Table 2-1 and plotted in Figure 2- 1. 

Respiratory Effects. Dyspnea was observed in 1 of 6 female dogs exposed to 10.4 mg/kg/day 

3,3’-dichlorobenzidine for 6.6 years, which probably resulted as a secondary effect of liver 

disease, that this dog was experiencing. No respiratory effects were observed in any other dogs, 

including controls (Stula et al. 1978). 

Hematological Effects. Although hematological effects may not be sensitive indicators for 

3,3’-dichlorobenzidine toxicity, hemoglobin adducts have been detected in female Wistar rats 

orally administered single 127 or 253 mg/kg doses of 3,3’-dichlorobenzidine (Birner et al. 1990) 

or with repeated doses between 0.3 and 5.8 mg/kg/day (Joppich-Kuhn et al. 1997). It was 

suggested that metabolically formed nitroso derivatives and the formation of a sulfinic acid amide 

with cysteine residues in hemoglobin may be the mechanism of adduct formation (Birner et al. 

1990). Hydrolysis yielded mainly 3,3’-dichlorobenzidine; N-acetylated 3,3’-dichlorobenzidine 

was also detected. The more recent study found that adduct formation was dose-related (JoppichKuhn et al. 1997). It was further observed that at low doses of 3,3’-dichlorobenzidine, N-acetyl3,3’-dichlorobenzidine adducts and 3,3’-dichlorobenzidine adducts were formed at similar levels, 

but at the highest dose level tested (5.8 mg/kg/day) the dichlorobenzidine adduct was 

predominant, suggesting saturation of the acetylation pathway at high dose (Joppich-Kuhn et al. 

1997). While hemoglobin adduct formation does not imply altered or abnormal hemoglobin 

function, adduct formation may be a suitable biomarker of human exposure to 

3,3’-dichlorobenzidine (see Section 2.7). Hematological variables (erythrocyte count, 

hemoglobin concentration, hematocrit, and leucocyte count) were found to be normal in dogs 

exposed to 10.4 mg/kg/day 3,3’-dichlorobenzidine for 7 years (Stula et al. 1978). 
3,3’-DICHLOROBENZIDINE 25 

2. HEALTH EFFECTS
 

Hepatic Effects. Limited animal evidence suggests that chronic-duration oral exposure to 

3,3’-dichlorobenzidine results in mild-to-moderate liver injury. Six female dogs exposed to 

3,3’-dichlorobenzidine (10.4 mg/kg/day) all had modestly elevated plasma glutamic-pyruvic 

transaminase (GPT) during the first 3 years of a 7-year treatment period (Stula et al. 1978). 

Thereafter, GPT levels returned to normal in three of the experimental animals, two remained 

elevated for the duration of the study. Elevated GPT levels may have been due to the test 

chemical that caused chronic hepatic injury to these dogs that ultimately led to development of 

liver tumors. One of the six dogs, sacrificed after 42 months of the test, showed a marked 

fatty change in the liver. It should be noted that the study is limited by use of one dose level, 

precluding dose-response evaluations. It should be mentioned, however, that none of the six 

control dogs exhibited adverse liver effects. 

Renal Effects. Urinary parameters (blood urea nitrogen, pH, osmolality, volume, protein, sugar, 

and sediment) were normal in female dogs exposed to 3,3’-dichlorobenzidine (10.4 mg/kg/day) 

throughout a 7-year study in which female dogs were exposed to 10.4 mg/kg/day 

3,3’-dichlorobenzidine. At necropsy, no histological effects to the kidneys were reported in any of 

the dogs (Stula et al. 1978). 

Body Weight Effects. In a study in which female dogs were exposed to 10.4 mg/kg/day 

3,3’-dichlorobenzidine for 7 years, there were no significant differences in body weight between 

treated and control dogs during the study period (Stula et al. 1978). 

2.2.2.3 Immunological and Lymphoreticular Effects 

No studies were located regarding immunological and/or lymphoreticular effects in humans or 

animals after oral exposure to 3,3’-dichlorobenzidine. 

2.2.2.4 Neurological Effects 

No studies were located regarding neurological effects in humans after oral exposure to 3,3’-

dichlorobenzidine. 

In a 3,3’-dichlorobenzidine carcinogenicity study, 1 of 6 dogs exhibited convulsions after 21, 28, 

or 42 months of oral treatment with 10.4 mg/kg/day over a period of 3.5 years (Stula et al. 1978). 

Necropsy 
3,3’-DICHLOROBENZIDINE 26 

2. HEALTH EFFECTS
 

at 42 months revealed slight neuronal degeneration; although the specific location was not 

indicated, histological examination was performed on the brain and spinal cord. No neurological 

effects were observed in any other dogs, including controls. This LOAEL value for neurological 

effect for oral exposure to 3,3’-dichlorobenzidine is shown in Table 2- 1 and plotted in 

Figure 2- 1. 

2.2.2.5 Reproductive Effects 

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

to 3,3’-dichlorobenzidine. 

2.2.2.6 Developmental Effects 

No studies were located regarding developmental effects in humans or animals after oral 

exposure to 3,3’-dichlorobenzidine. 

2.2.2.7 Genotoxic Effects 

No studies were located regarding genotoxic effects in humans after oral exposure to 

3,3’-dichlorobenzidine. 

Genotoxic effects have been reported in animals treated with 3,3’-dichlorobenzidine. A single 

dose of 3,3’-dichlorobenzidine (1,000 mg/kg) administered to male and pregnant female mice 

induced micronuclei in polychromatic erythrocytes in the bone marrow of the males and in the 

liver of the fetuses, but not in bone marrow of the dams (Cihak and Vontorkova 1987). A 

micronucleus test is performed to detect a chemical’s ability to induce chromosomal aberrations. 

However, the relevance of micronuclei formation to human health is not known. The reason for 

the lack of effect of 3,3’-dichlorobenzidine on bone marrow micronuclei formation in the mothers 

is unclear, but it may be related to deficiencies in the metabolic activation of 

3,3’-dichlorobenzidine in female mice. The relative importance of pregnancy is unknown since 

the study did not evaluate nonpregnant females. In another study, an increase in unscheduled 

deoxyribonucleic acid synthesis (UDS) was observed in cultured liver cells from male mice 

previously pretreated orally with single doses of ≥ 500 mg/kg 3,3’-dichlorobenzidine; no 

response was observed at a dose of ≤200 mg/kg (Ashby and Mohammed 1988). 
3,3’-DICHLOROBENZIDINE 27 

2. HEALTH EFFECTS
 

3,3’-Dichlorobenzidine was also shown to bind extensively to tissue deoxyribonucleic acid 

(DNA) in rats and mice. Single oral administration of 20 or 100 mg/kg radiolabeled 

3,3’-dichlorobenzidine to male Sprague-Dawley rats and Swiss-Webster mice resulted in 

extensive binding of the compound to tissue (liver, bladder, and intestine) DNA at 12, 24, or 96 

hours, and 9 or 14 days after treatment (Ghosal and Iba 1990). 

The UDS assay is used to measure the repair that follows DNA damage. However, the relevance 

of UDS to human health is not known. While results were positive in two assay in animals, 

sufficient data are not available from more predictive indicator assays to adequately characterize 

the genotoxic potential for 3,3’-dichlorobenzidine in humans. Other genotoxicity studies are 

discussed in Section 2.5. 

2.2.2.8 Cancer 

There are no epidemiological studies linking cancer in humans to oral exposure to 

3,3’-dichlorobenzidine. However, based on the findings of oral studies in animals, 

3,3’-dichlorobenzidine may be regarded as a chemical that would probably induce cancer in 

humans given sufficient exposure to the agent. An IARC review of the existing cancer toxicity 

data for 3,3’-dichlorobenzidine concluded that, although no case report on exposure to 

3,3’-dichlorobenzidine was available, because 3,3’-dichlorobenzidine and benzidine may be made 

in the same plant, it is not possible to exclude 3,3’-dichlorobenzidine’s contribution to the 

incidence of bladder cancer attributed to benzidine (IARC 1982a). Studies in animals 

demonstrated that 3,3’-dichlorobenzidine is carcinogenic in rats, hamsters, mice and dogs (see 

below). 

A statistically significant increased incidence of hepatomas was observed in male ICR/JCL mice 

exposed to 0.1% 3,3’-dichlorobenzidine in the diet (170 mg/kg/day) at 6 months (8 of 8 treated as 

opposed to 0 of 5 controls) and 12 months (18 of 18 treated as opposed to 2 of 2 1 controls) 

(Osanai 1976). Hepatic tumors were observed in 4/l 8 strain D mice exposed to 11.2-l 1.9 mg 

3,3’-dichlorobenzidine/kg/day in the diet for 10 months (Pliss 1959). 

No bladder carcinomas were observed in rats exposed to 0.03% 3,3’-dichlorobenzidine in the diet 

(27 mg/kg/day) for 4 or 40 weeks (Ito et al. 1983), nor were any mammary tumors observed in 

rats administered approximately 49 mg 3,3’-dichlorobenzidine dihydrochloride/kg/day by gavage 

once every 3 days over a 30-day period and sacrificed 8 months later (Griswold et al. 1968). 
3,3’-DICHLOROBENZIDINE 28 

2. HEALTH EFFECTS
 

In a study in which rats were exposed to 10-20 mg 3,3’-dichlorobenzidine per day (120 

mg/kg/day) in feed 6 days per week for 12 months, tumors were observed at a variety of sites, 

including the Zymbal gland (7 of 29 animals), mammary gland (7/29), bladder (3/29), 

hematopoietic system (3/29), skin (3/29), ileum (2/29), connective tissue (2/29), salivary gland 

(2/29), liver (l/29), and thyroid (l/29) (Pliss 1959). No tumors were reported in 130 control 

animals. In a later study, the same investigator reported that oral administration of an unspecified 

dose (in the range of 125-500 mg/kg) of 3,3’-dichlorobenzidine by gavage to rats for 10-13 

months resulted in the development of tumors of the skin, sebaceous and mammary glands, and 

papillomas of the urinary bladder (Pliss 1963). Because the frequency of administration of the 

compound was not provided, a daily dose could not be estimated. 

In another rat study, 3,3’-dichlorobenzidine was administered to 50 male (70 mg/kg/day) and 50 

female (80 mg/kg/day) Sprague-Dawley rats, in a standard diet for up to 16 months (Stula et al. 

1975). In rats fed 3,3’-dichlorobenzidine in the diet for a total of 349 days (females) and 353 days 

(males), histopathological evaluations revealed mammary adenocarcinoma (16% incidence), 

malignant lymphoma (14%) granulocytic leukemia (20%), carcinoma of the Zymbal gland (18%) 

in males, and mammary adenocarcinoma (59%) in females. These tumors were either totally 

absent or occurred statistically less frequently in untreated controls. The authors noted that most 

of these tumors appeared to arise in the bone marrow and hematopoietic foci in the spleen and 

liver with subsequent metastasis to other organs. Only one dose level was used in the study, 

however, and information on the purity of the test substance was not provided. 

In a subsequent study by this investigator, hepatocellular carcinomas (67% incidence) and 

papillary transitional cell carcinomas of the urinary bladder (83%) were observed in female dogs 

fed approximately 10.4 mg/kg/day orally in gelatin capsules over a period of 6.6-7.1 years (Stula 

et al. 1978). These tumors were absent in untreated controls. Although a small number of dogs (6) 

were evaluated, and only one sex and one dose were used, the significant increase in tumor rate in 

this group of dogs demonstrates unequivocally the carcinogenicity of this chemical in this 

species. 

Transitional cell bladder carcinomas and liver cell and cholangiomatous tumors were observed in 

hamsters fed a diet containing 0.3% 3,3’-dichlorobenzidine (300 mg/kg/day) (Sellakumar et al. 

1969). This level was determined to be the maximum tolerated dose. In an earlier study, a diet 

containing 0.1% 3,3’-dichloro­
3,3’-DICHLOROBENZIDINE 29 

2. HEALTH EFFECTS
 

benzidine (59-64 mg/kg/day) fed to Syrian golden hamsters for their lifetimes did not cause 

significant carcinogenic effects or changes in bladder pathology (Saffiotti et al. 1967). 

A synergistic role for 3,3’-dichlorobenzidine in the development of bladder cancer has been 

suggested. This was proposed in a study in which no carcinomas were found in any rats 

administered one of the following: 0.03% 3,3’-dichlorobenzidine in the diet, 0.001% BBN 

(N-butyl-N-(hydroxybutyl)nitrosamine) in drinking water, 0.0005% 2-acetylaminofluorene 

(2-AAF) in the diet, or 0.04% N-[4-(5nitro-2-furyl)-2-thiazolyllformamide (FANFT) in the diet 

for a period of 40 weeks (Ito et al. 1983). However, when BBN plus 3,3’-dichlorobenzidine were 

fed together at the same dose levels as above, there was a marked increase in the presence of 

papillary or nodular hyperplasia in the rat bladder, and the appearance of one papilloma. 

Based on these findings, the authors suggested that 3,3’-dichlorobenzidine had a synergistic effect 

on the carcinogenicity of BBN. In rats sequentially administered BBN (0.0l%), FANFT (0.15%), 

2-AAF (0.025%), and 3,3’-dichlorobenzidine (0.03%) for 4 weeks each, the incidence of bladder 

cancer after administration of the 4 chemicals was no different than after administration of the 

first 3, suggesting no interactive effect of any type for 3,3’-dichlorobenzidine (Ito et al. 1983). 

The Cancer Effect Level (CEL), (i.e., lowest dose that produced a tumorigenic response for each 

species) and the duration category of exposure to 3,3’-dichlorobenzidine are shown in Table 2-l 

and plotted in Figure 2-l. Based on the increased incidence in mammary adenocarcinomas in rats 

reported in the Stula et al. (1975) study, EPA calculated a q1* of 0.45 (mg/kg/day)-1. Doses 

corresponding to risk levels ranging from 10-4 to 10-7 are 2.2x10-4 to 2.2x10-7 mg/kg/day, 

respectively, as indicated in Figure 2-l. 

2.2.3 Dermal Exposure 

Because of large particle size and increased usage of closed systems and protective clothing, 

dermal absorption is expected to be minimal in occupational environments. Conditions of high 

humidity and high temperature are known to enhance dermal absorption of chemicals following 

skin contact. 

2.2.3.1 Death 

No studies were located regarding lethal effects in humans after dermal exposure to 3,3’-

dichlorobenzidine. The minimum dermal lethal dose for 3,3’-dichlorobenzidine (free base) for 

male and female New Zealand 
3,3’-DICHLOROBENZIDINE 30 

2. HEALTH EFFECTS
 

albino rabbits with skin intact was reported to be greater than 8,000 mg/kg (Gerarde and Gerarde 

1974). The cause of death was not discussed. No discernible skin irritation was observed when 

3,3’-dichlorobenzidine dihydrochloride was applied to the intact or abraded skin of rabbits; the 

dose was not provided (Gerarde and Gerarde 1974). This minimum dermal lethal dose in female 

New Zealand albino rabbits is shown in Table 2-2. Dermal exposure is not likely to cause death in 

humans. 

2.2.3.2 Systemic Effects 

No information was located regarding cardiovascular, gastrointestinal, hematological, 

musculoskeletal, hepatic, renal, endocrine, body weight, or metabolic effects in animals or 

humans following dermal exposure to 3,3’-dichlorobenzidine. 

Very limited data were found regarding the effects of dermal exposure to 3,3’-dichlorobenzidine. 

The highest NOAEL value and all LOAEL values for dermal exposure for this study are shown in 

Table 2-2. 

Respiratory Effects. Although no respiratory effects have been reported in humans following 

dermal exposure exclusively to 3,3’-dichlorobenzidine, upper respiratory infection and sore throat 

were among the principal reasons for visits to a company’s medical clinic by workers who 

handled 3,3’-dichlorobenzidine (Gerarde and Gerarde 1974). However, there is no conclusive 

evidence that these effects were due specifically to 3,3’-dichlorobenzidine exposure. Workers 

may have been exposed to this and/or other agents by both inhalation and dermal routes. 

No studies were located regarding respiratory effects in animals after dermal exposure to 

3,3’-dichlorobenzidine. 

Dermal Effects. Dermatitis was cited as the only verified health problem encountered by workers 

in contact with the free base of 3,3’-dichlorobenzidine in a dichlorobenzidine manufacturing plant 

(Gerarde and Gerarde 1974). 

There was no discernable skin irritation when 3,3’-dichlorobenzidine dihydrochloride (at an 

unstipulated dose) was applied to the intact and abraded skin of rabbits (Gerarde and Gerarde 

1974). Similarly, an 

3,3’-DICHLOROBENZIDINE 32 

2. HEALTH EFFECTS
 

aqueous suspension of 3,3’-dichlorobenzidine instilled intradermally into rats at a dose of 700 

mg/kg did not produce adverse effects (Gerarde and Gerarde 1974). 

Ocular Effects. No studies were located regarding the ocular effects of 3,3’-dichlorobenzidine in 

humans. 

No effects were reported in rabbits when 100 mg of dichlorobenzidine (free base) was placed in 

the conjunctival sac of the eye (Gerarde and Gerarde 1974). It should be noted that the authors 

did not report the duration of exposure or the vehicle used. However, 0.1 mL of 

3,3’-dichlorobenzidine dihydrochloride in a 20% corn oil suspension produced erythema, pus, 

and cornea1 opacity, giving a 76% score in the Draize test within an hour when placed in the 

conjunctival sac of the eye of the rabbit (Gerarde and Gerarde 1974). This response is very likely 

associated with the release of hydrochloric acid following the salt’s contact with the moist surface 

of the eye. 

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

exposure to 3.3’-dichlorobenzidine: 

2.2.3.3 Immunological and Lymphoreticular Effects 

2.2.3.4 Neurological Effects 

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 

Several epidemiological studies have investigated cancer incidences among workers 

occupationally exposed to 3,3’-dichlorobenzidine and other arylamines (Gadian 1975; Gerarde 

and Gerarde 1974; MacIntyre 1975; Myslak et al. 1991). Exposure may have been by both 

inhalation and dermal routes. These studies are discussed in greater detail under Section 2.2.1.8 

(inhalation cancer effects). 
3,3’-DICHLOROBENZIDINE 33 

2. HEALTH EFFECTS
 

Due, in part, to structure-activity considerations, epidemiological studies of potential cancer 

effects of occupational exposure to 3,3’-dichlorobenzidine have been particularly concerned with 

bladder tumors since benzidine is a known human carcinogen in which the bladder is the primary 

target. While one studyfound an excess incidence of bladder tumors among German painters who 

may have been exposed to 3,3’-dichlorobenzidine (Myslak et al. 1991), the causality is equivocal 

largely because it had been common for painters to prepare the paints themselves, allowing for 

possible exposure to other carcinogenic dyes and pigments derived from benzidine, 

3,3’-dichlorobenzidine, 3,3’-dimethylbenzidine (o-toluidine), 3,3’-dimethoxybenzidine 

(o-dianisidine), and 2-naphthylamine (Myslak et al. 1991). 

A more recent study found an association between bladder cancer and exposure to arylamines 

(Ouellet-Hellstrom and Rench 1996). This study examined the cancer incidence in a cohort of 

704 workers employed at a Connecticut chemical plant between 1965 and 1989. The plant 

produced a variety of chemicals including arylamines such as 3,3’-dichlorobenzidine, o-tolidine, 

and o-dianisidine, but not benzidine; benzidine production ceased prior to mid-1965. Skin contact 

was found to be the main route of exposure. Only workers never exposed to benzidine were 

selected to participate and only confirmed cancer cases were considered in the analysis. As a 

result of a worker survey, the information on follow-up yielded 8,624 person-years of observation 

for a follow-up rate of 97% for male employees and 1,660 person-years for a follow-up rate of 

97% for female employees. There were a total of 27 cancer cases, 23 in males and 4 in females. 

Three of the 23 male cases were non-melanoma skin cancers and were not included in the 

analysis. There were 7 cases of bladder cancer, all in males; two were diagnosed in workers first 

employed after 1972, four in workers first employed at the age of 40 or older, and five in 

workers who worked at least 5 years or more. All bladder cancers had a follow-up period of 8 

years or more. The standardized incidence ratio (observed/expected, SIR) for bladder cancer was 

8.3 (C.I. 3.3-17.0). In addition, the association between bladder cancer cases and exposure to 

arylamines increased with cumulative exposure. One bladder cancer case was a current smoker 

and the other six were former smokers. The authors (Ouellet-Hellstrom and Rench 1996) 

recognized that the study could not evaluate cancer risks for specific arylamines, but as indicated 

above, the results supported an association between bladder cancer and arylamine exposure. They 

also indicated that although smoking is known to increase the risk of bladder cancer by a factor of 

two, it is unlikely that smoking alone explains the eight-fold increase in bladder cancer risk 

observed in the study. 
3,3’-DICHLOROBENZIDINE 34 

2. HEALTH EFFECTS
 

No studies were located regarding carcinogenicity in animals following dermal exposure to 

3,3’-dichlorobenzidine. 

2.3 TOXICOKINETICS 

Very limited studies exist on the toxicokinetics of 3,3’-dichlorobenzidine in humans. Most of the 

available information is on urinary elimination of the compound following occupational 

exposure. Evidence from animal studies suggest that 3,3’-dichlorobenzidine is rapidly absorbed 

from the gastrointestinal tract. Animals administered a single oral dose of 

[

14C]-3,3’-dichlorobenzidine showed highest concentrations of radioactivity in the liver, kidney, 

lung, spleen, heart, pancreas, and testes. In rats, a major step in the elimination of 

3,3’-dichlorobenzidine is metabolic transformation. N-Acetyl metabolites (N-acetyl3,3’-dichlorobenzidine and N,N-diacetyl-3,3’-dichlorobenzidine) have been detected in urine of 

rats. N-acetyl metabolites are formed in vivo by hepatic N-acetyltransferase(s). In humans, some 

isozyme(s) of N-acetyltransferase show marked polymorphic differences; it is thus possible that 

the proportion of the dose of 3,3’-dichlorobenzidine converted to its N-acetyl metabolites in 

humans may vary widely between individuals. The metabolites undergo rapid excretion primarily 

in urine and to a lesser extent in feces. Unchanged 3,3’-dichlorobenzidine occurs as a minor 

urinary excretion product. 

2.3.1 Absorption 

There is no information regarding absorption of 3,3’-dichlorobenzidine in children by any route 

of exposure. 

2.3.1.1 Inhalation Exposure 

3,3’-Dichlorobenzidine has been detected in the urine of workers in 3,3’-dichlorobenzidinehandling plants under conditions which favored inhalation of 3,3’-dichlorobenzidine-bound 

particulate matter (Handke et al. 1986; London and Boiano 1986; Meigs et al. 1954). Under these 

conditions, it is reasonable to expect that some of the 3,3’-dichlorobenzidine found in the urine 

could have come from inhalation exposure. However, conditions in the plants were also 

conducive to dermal exposure. Therefore, some of the 3,3’-dichlorobenzidine dose found in the 

urine could have come from dermal exposure. In addition, since the mucocilliary clearance 

mechanism moves most of the larger particulates (5-10 μm) out of the lungs into 
3,3’-DICHLOROBENZIDINE 35 

2. HEALTH EFFECTS
 

the gastrointestinal tract, it is reasonable to expect that some gastrointestinal dose was received as 

well. No information was located on absorption in animals following inhalation exposure. 

2.3.1.2 Oral Exposure 

No quantitative data were located on the absorption of 3,3’-dichlorobenzidine following oral 

exposure in humans. However, a study in volunteers found acetylated metabolites in the urine 24 

hours after a single 250 mg oral dose of 3,3’-dichlorobenzidine, which suggested that the 

compound is absorbed (Belman et al. 1968). 

In animals, absorption of 3,3’-dichlorobenzidine from the gastrointestinal tract is rapid. Following 

a dose of 40 mg/kg, the plasma level of unchanged 3,3’-dichlorobenzidine attained a peak 

concentration of 1.25 μg/mL at 4 hours in Sprague Dawley rats. Further, about 90% of the 

administered radioactivity was excreted in feces (via bile) and urine within 72 hours largely as 

metabolites, indicating a high bioavailability, typical of primary arylamines. The elimination is 

biphasic, with half-lives of 6 hours and 14 hours in plasma for the rapid and slow phases, 

respectively (Hsu and Sikka 1982). 

2.3.1.3 Dermal Exposure 

No studies were located regarding absorption of 3,3’-dichlorobenzidine following dermal 

exposure in humans. Because of large particle size and increased usage of closed systems and 

protective clothing, dermal absorption is minimized. In animals, dermally applied 

3,3’-dichlorobenzidine (in acetone) is moderately absorbed. Based on the amount of radioactivity 

remaining at the site of application, the extent of dermal absorption of applied 

[

14C]-3,3’-dichlorobenzidine to the shaved skin of rats at 1, 8, and 24 hours following the 

application was estimated to be 6, 23, and 49%, respectively (Shah and Guthrie 1983). 

2.3.2 Distribution 

There is no information regarding distribution of 3,3’-dichlorobenzidine or metabolites in 

children after exposure by any route. 
3,3’-DICHLOROBENZIDINE 36 

2. HEALTH EFFECTS 

2.3.2.1 Inhalation Exposure 

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

3,3’-dichlorobenzidine. 

2.3.2.2 Oral Exposure 

No studies were located regarding distribution of 3,3’-dichlorobenzidine in humans after oral 

exposure. 

In animals, orally absorbed 3,3’-dichlorobenzidine is widely distributed. In a study in which 

3,3’-dichlorobenzidine was orally administered to female Wistar rats in single doses of 0.25 mL 

3,3’-dichlorobenzidine in propylene glycol at 0.5 or 1 mmol/kg (127 or 253 mg/kg) by gavage, 

hemoglobin adducts of 3,3’-dichlorobenzidine were isolated from the blood of the animals 

(Birner et al. 1990). Similar results were obtained in rats dosed with 0.3-5.8 mg 

3,3’-dichlorobenzidine/kg/day for 4 weeks (Joppich-Kuhn et al. 1997). The distribution of 

radioactivity in rat tissues after the oral administration of [14C]-3,3’-dichlorobenzidine 

has been studied (Hsu and Sikka 1982). Twenty-four hours after a single oral dose, the highest 

levels of radioactivity were found in the liver, followed by the kidney, lung, spleen, heart, 

pancreas, and testes, in that order. This pattern did not depend on dose. After 96 hours, tissues 

that retained 0.02% or more of the administered radioactivity were liver (1.48%), muscle 

(0.37%), kidney (0.19%), and lung (0.02%). Erythrocytes retained more of the radioactivity than 

lung, but attention was not paid to the hematopoietic system in this study (Hsu and Sikka 1982). 

The effect of repetitive 3,3’-dichlorobenzidine administration on tissue levels of radioactivity was 

also studied by Hsu and Sikka (1982). Radioactivity in tissues of animals that received six daily 

doses of 3,3’-dichlorobenzidine was generally three to four times as high as the radioactivity in 

tissues of animals that received a single dose. Similarly, the rate of decline of radioactivity in 

tissues was generally higher in animals that received a single dose than in those treated with 

multiple doses of the compound. The authors concluded that repeated dosing with 

3,3’-dichlorobenzidine did not result in a substantial retention of 14C, and the compound may be 

considered to have a fairly low tendency to accumulate in tissues following repetitive dosing 

(Hsu and Sikka 1982). Overall, bioaccumulation of this chemical in rats is considered to be 

minimal following oral exposure of any duration. 
3,3’-DICHLOROBENZIDINE 37 

2. HEALTH EFFECTS
 

There is indirect evidence that 3,3’-dichlorobenzidine or metabolites can cross the placenta. A 

study that examined the potential genotoxic effects of 3,3’-dichlorobenzidine found that oral 

administration of 3,3’-dichlorobenzidine to pregnant rats induced micronuclei in the liver of 

fetuses (Cihak and Vontorkova 1967). There is no information regarding accumulation of 

3,3’-dichlorobenzidine or metabolites in breast milk or its potential transfer to offspring via breast 

milk. 

2.3.2.3 Dermal Exposure 

No studies were located regarding distribution of 3,3’-dichlorobenzidine in humans following 

dermal exposure. The distribution of [14C]-3,3’-dichlorobenzidine in rat tissues following dermal 

application was studied by Shah and Guthrie (1983). Tissues retaining >0.1% of the administered 

radioactivity 24 hours after application were liver (4.09%), blood (0.75%) and lung (0.45%). The 

level in the lung was the same at the 8- and 24-hour time points. Differences in the tissue 

distribution pattern of total radioactivity between the oral and dermal routes of 

3,3’-dichlorobenzidine administration may be presumed to reflect differences in the rates of 

absorption from these sites. These differences suggest that the target organ in which 

3,3’-dichlorobenzidine exerts an adverse effect may depend on the route of exposure to the 

compound. Organ toxicity can be better evaluated in comparative studies designed to test tissue 

distribution and persistence exposure. 

2.3.3 Metabolism 

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

3,3’-dichlorobenzidine. 

Information from a study in which 4 volunteers ingested a single 250 mg dose of 

3,3’-dichlorobenzidine suggests that this chemical undergoes N-acetylation and that metabolites 

may be excreted in the urine either free or as glucuronides (Belman et al. 1968). N-Acetylation 

appears to be the major path for the metabolism of 3,3’-dichlorobenzidine in mammals (Lazear et 

al. 1979; Reid et al. 1984; Tanaka 1981). Studies in animals also indicate that 

3,3’-dichlorobenzidine is extensively metabolized. Bile and urine of rats given single oral doses 

of [14C]-3,3’-dichlorobenzidine (40 mg/kg/day) contained 5 metabolites of 3,3’-dichlorobenzidine 

in addition to the parent compound. None of the metabolites were identified, but a majority were 

reported to be conjugates (Hsu and Sikka 1982). A 24-hour urine sample of rats given a 
3,3’-DICHLOROBENZIDINE 38 

2. HEALTH EFFECTS 

single oral dose of 3,3’-dichlorobenzidine (50 mg/kg/day) contained unchanged 

3,3’-dichlorobenzidine, N,N’-diacetyl 3,3’-dichlorobenzidine, and N-acetyl 3,3’-dichlorobenzidine in 

a ratio of 1:3:10 (Tanaka 1981). Indirect evidence for the formation of nitroso derivatives was found 

in a study in which 3,3’-dichlorobenzidine was administered to female Wistar rats by gavage (Bimer 

et al. 1990). Hemoglobin adducts were detected by the release of 3,3’-dichlorobenzidine after alkaline 

hydrolysis. The authors stated that the most likely process by which the adducts were formed was a 

reaction between a nitroso derivative of 3,3’-dichlorobenzidine and sulfhydryls in cysteine residues of 

hemoglobin. 

No studies were located regarding the metabolism of 3,3’-dichlorobenzidine in humans following 

dermal exposure. In a 24-hour urine sample of rats given a single dermal application of 

3,3’-dichlorobenzidine (50 mg/kg/day), N,N ‘-diacetyl 3,3’-dichlorobenzidine (but not N-acetyl 3,3’­

dichlorobenzidine or the unchanged chemical) was detected (Tanaka 1981). Since the utagenicity of 

diacetylated product is much less than either the monoacetylated or parent compound (Lazear et al. 

1979; Reid et al. 1984; Tanaka 198l), diacetylation may be a detoxification reaction for 3,3’­

dichlorobenzidine (see also Sections 2.4.1 and 2.4.2). 

There is no information regarding the metabolism of 3,3’-dichlorobenzidine in children. However, Nacetylation (as discussed above) in humans is likely done by one of two families of Nacetyltransferases. One of these families, NAT2, is developmentally regulated (Leeder and Keams 

1997). Some enzyme activity can be detected in the fetus by the end of the first trimester. Almost all 

infants exhibit the slow acetylator phenotype between birth and 2 months of age. The adult phenotype 

distribution is reached by the age of 4-6 months, whereas adult activity is found by approximately l-3 

years of age. Also, UDPglucuronosyltransferase, responsible for the formation of glucuronide 

conjugates, seems to achieve adult activity by 6-18 months of age (Leeder and Kearns 1997). These 

data suggest that metabolism of 3,3’-dichlorobenzidine by infants will differ from that in adults in 

extent, rate, or both. 

The metabolism of several 3,3’-dichlorobenzidine-based pigments has been studied in animal 

experiments to determine if they are metabolized to 3,3’-dichlorobenzidine. In a study where rats were 

exposed by inhalation to Pigment Yellow 17 (230 mg/m3

 air) for 4 hours, 3,3’-dichlorobenzidine was 

not detected in either urine or blood during the following 14 days (Hofmann and Schmidt 1993). No 

detectable residues of 3,3’-dichlorobenzidine were found in urine samples of hamsters administered a 

single dose of 100 mg/kg purified Yellow 12 (NCTR 1979; Nony et al. 1980). Similarly, 

3,3’-dichlorobenzidine was not detected in 
3,3’-DICHLOROBENZIDINE 39 

2. HEALTH EFFECTS
 

urine samples of rats fed 3,3’-dichlorobenzidine-derived pigments (C.I. Pigment Yellow 12, 16, 

and 83) in the diet at concentrations of 0.1% (1,000 ppm), 0.3% (3,000 ppm), and 0.9% (9,000 

ppm) for 104 weeks (Leuschner 1978). Based on the results of these studies, there is no evidence 

for the metabolic cleavage of tested pigments to 3,3’-dichlorobenzidine in test animals (Hoffman 

and Schmidt 1993; Leuschner 1978; NCTR 1979; Nony et al. 1980). 

2.3.4 Elimination and Excretion 

There is no information regarding the elimination and excretion of 3,3’-dichlorobenzidine or 

metabolites in children following any route of exposure. 

2.3.4.1 Inhalation Exposure 

Less than 0.2 ppb 3,3’-dichlorobenzidine was detected in urine samples of 36 workers exposed to 

3,3’-dichlorobenzidine-derived pigments (Hatfield et al. 1982). However, the authors did not 

clearly identify specific pigments. While the authors did not report exposure route, it was 

presumed to have been by inhalation. Dermal exposure may have also occurred. 

No studies were located regarding excretion in animals after inhalation exposure to 

3,3’-dichlorobenzidine. 

2.3.4.2 Oral Exposure 

Very limited information was located regarding excretion of 3,3’-dichlorobenzidine and/or 

metabolites in humans after oral exposure. In 4 volunteers who ingested a single 250 mg dose of 

3,3’-dichlorobenzidine, the percentage of N-hydroxyacetyl compound excreted free in the urine 

in 24 hours ranged from 0.32 to 1.55%, whereas the percentage of N-hydroxyacetyl compound 

excreted as glucuronide in 24 hours ranged from 0.11 to 0.45% (Belman et al. 1968). Studies on 

the fate of 3,3’-dichlorobenzidinederived pigments fail to provide conclusive evidence that these 

pigments are broken down to release free 3,3’-dichlorobenzidine in humans. 

Results from animal studies show that 3,3’-dichlorobenzidine administered by gavage is excreted 

primarily in feces and to a lesser extent in urine. In rats administered a single oral dose of 

[

14C]-3,3’-dichloro­
3,3’-DICHLOROBENZIDINE 40 

2. HEALTH EFFECTS
 

benzidine (40 mg/kg), the elimination from plasma appeared to be biphasic, with half-lives of 

about 6 and 14 hours for the rapid and slow phases, respectively (Hsu and Sikka 1982). 

Elimination of 3,3’-dichlorobenzidine-derived radioactivity from liver, kidneys, and lungs also 

exhibited rapid and slow phases, with half-lives of 5.8 and 77 hours for the liver, 7.1 and 139 

hours for the kidneys, and 3.8 and 43.3 hours for the lungs. Approximately 58-72% of the 

administered dose was recovered in bile and feces and 23-33% in urine (Hsu and Sikka 1982). 

Most of the material found in bile and feces consisted of conjugated metabolites, while most of 

the material in urine consisted of unconjugated metabolites. No detectable residues of 

3,3’-dichlorobenzidine were found in urine samples of hamsters administered a single dose of 100 

mg/kg purified Yellow 12 (NCTR 1979; Nony et al. 1980). Similarly, 3,3’-dichlorobenzidine was 

not detected in urine samples of rats fed 3,3’-dichlorobenzidine-derived pigments (C.I. Pigment 

Yellow 12, 16, and 83) in the diet at concentrations of 0.1% (1,000 ppm), 0.3% (3,000 ppm), and 

0.9% (9,000 ppm) for 104 weeks (Leuschner 1978). 

2.3.4.3 Dermal Exposure 

No studies were located regarding the excretion of 3,3’-dichlorobenzidine in humans following 

dermal exposure. Fecal excretion in rats at 24 hours following 3,3’-dichlorobenzidine exposure 

was 19% of the administered dose, while urinary excretion accounted for 8% (Shah and Guthrie 

1983). Fifty-one percent of the administered dose was unabsorbed from the site of application at 

24 hours. The remaining 49% was distributed throughout the body, feces and urine. 

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 substance 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. 
3,3’-DICHLOROBENZIDINE 41 

2. HEALTH EFFECTS
 

PBPK/PD models refine our understanding of complex quantitative dose behaviors by helping to 

delineate and characterize the relationships between: (1) the external/exposure concentration and 

target tissue dose of the toxic moiety, and (2) the target tissue dose and observed responses 

(Andersen and Krishnan 1994; Andersen et al. 1987). These models are biologically and 

mechanistically based and can be used to extrapolate the pharmacokinetic behavior of chemical 

substances from high to low dose, from route to route, between species, and between 

subpopulations within a species. The biological basis of PBPK models results in more meaningful 

extrapolations than those generated with the more conventional use of uncertainty factors. 

The PBPK model for a chemical substance is developed in four interconnected steps: (1) model 

representation, (2) model 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 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 2-2 shows a conceptualized representation of a 

PBPK model. If PBPK models for 3,3’-dichloro 

3,3’-DICHLOROBENZIDINE 43 

2. HEALTH EFFECTS
 

benzidine 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. 

No PBPK modeling studies were located for 3,3’-dichlorobenzidine. 

2.4 MECHANISMS OF ACTION 

2.4.1 Pharmacokinetic Mechanisms 

No information was located for the mechanism of inhalation, oral, or dermal absorption of 

3,3’-dichlorobenzidine in humans or animals. Also, no information was located for the 

mechanism by which 3,3’-dichlorobenzidine is transported in the blood. However, a studies in 

rats have shown that 3,3’-dichlorobenzidine forms adducts with hemoglobin (Birner et al. 1990; 

Joppich-Kuhn et al. 1997), indicating that at least a small amount of the chemical is associated 

with red blood cells. 

3,3’-Dichlorobenzidine induces liver microsomal enzymes in a pattern similar to 

3-methylcholanthrene. Liver microsomes from male Sprague-Dawley rats pretreated 

intraperitoneally with 3,3’-dichlorobenzidine yielded information to suggest that the induction 

pattern of P-450 isozymes by 3,3’-dichlorobenzidine resembles that of 3-methylcholanthrene. 

3,3’-Dichlorobenzidine significantly induced ethoxy-coumarin O-deethylase, p-nitrophenetole 

O-deethylase, and arylhydrocarbon hydrolase by 5-, 6-, and 5-fold, respectively (Iba et al. 1983). 

Another study also found that 3,3’-dichlorobenzidine induces P-450 isozymes in a pattern similar 

to 3-methylcholanthrene (i.e., induces P-450c) (CYP2B l), and P-450d (CYPlA2) but mainly 

P-450c (CYP2Bl) (Iba and Thomas 1988). The same authors also conducted studies to identify 

the isozymes involved in NADPH-dependent activation of 3,3’-dichlorobenzidine by rat hepatic 

microsomes to mutagens in the Ames test. 3,3’-Dichlorobenzidine activation was unaffected by 

monoclonal antibodies to P-450b (CYPIAI) or P-450c (CYP2Bl) but was inhibited by 69% by 

polyclonal antibodies to P-450d (CYPlA2). 3,3’-Dichlorobenzidine activation was also inhibited 

46% by antibody specific to NADPH-cytochrome P-450 reductase. Also, addition of 

methimazole, a high affinity substrate for the flavin-containing monooxygenase, reduced the 

residual mutagenicity in the systems containing antibody to P-450b (CYPlA2) and cytochrome 

P-450 reductase to 9% and 19%, respectively, of the appropriate control values. Based on these 

results, Iba and Thomas (1983) concluded that P-450d 
3,3’-DICHLOROBENZIDINE 44 

2. HEALTH EFFECTS
 

(CYPlA2) contributes to the majority of the P-450-dependent activation of 3,3’-dichlorobenzidine in 

hepatic microsomes. 

If 3,3’-dichlorobenzidine is activated to a mutagenic intermediate by CYPlA2, this would have 

relevance to exposure in utero and in neonates. Human fetal liver does not contain appreciable 

amounts of CYPlA2 (Leeder and Kearns 1997). Adult levels of CYPlA2 are reached at about 

4 months of age and may be exceeded in 1-2-year-old children. CYPlA2 levels subsequently decline 

and reach adult levels at the end of puberty. 

2.4.2 Mechanisms of Toxicity 

Although, data from the existing human and animal studies indicate that 3,3’-dichlorobenzidine is 

minimally toxic, its mechanism of toxicity appears to be well defined, deriving mainly from adduction 

of DNA. The available data suggest that the metabolism of 3,3’-dichlorobenzidine begins with the 

formation of nitroso derivatives which yield a sulfinic acid amide with hemoglobin in erythrocytes. 

This has been suggested to be a mechanism for adduct formation. However, N-oxidation at one of the 

two nitrogens could occur in the parent diamine, the monoacetyl, or the diacetyl derivative. 

N-hydroxy-dichlorobenzidine and N-hydroxyhr-acetyl-dichlorobenzidine could arise from either 

direct N-oxidation of the amino group or by deacetylation of the hydroxamic acid. Peroxidative 

activation of 3,3’-dichlorobenzidine will yield 3,3’-dichlorobenzidine diimine which causes DNA 

damage in bladder which might be responsible for tumor formation in this target in dogs and possibly 

humans. In rodents, N-oxidation of the monoacetyl derivative is an important step of metabolic 

activation (Birner et al. 1990). 

Results from a recent study suggest that cytochrome P-450 (specifically CYP4B 1) activity may 

contribute to the initiation of carcinogenesis in rat and mouse bladder by activation of 

3,3’-dichlorobenzidine to mutagenic compounds (Imaoka et al. 1997). The authors demonstrated the 

presence of CYP4B 1 in rat and mouse bladder microsomes by immunoblotting and 

immunohistochemistry. Furthermore, tissue-staining showed that CYP4Bl was present in epithelial 

cells of the bladder. It was also shown in that study that mouse bladder microsomes activated 3,3’­

dichlorobenzidine, although not to the degree observed with renal microsomes and purified CYP4B 1. 

3,3’-Dichlorobenzidine activation was judged by a gene expression test in Salmonella typhimurium 

NM2009 that detects DNA damage. Rat CYP4Bl produced very high mutagenic activity for 

3,3’-dichlorobenzidine. 
3,3’-DICHLOROBENZIDINE 45 

2. HEALTH EFFECTS 

The genotoxicity of 3,3’-dichlorobenzidine is derived from DNA adduction, as suggested by 

positivereverse mutation results in Salmonella typhimurium TA98 strain, since this strain of 

S. typhimurium detects reverse (histidine revertants) mutation in both activated and direct-acting basepair substitution and frameship mutagens (Vithayathil et al. 1983). The extent of covalent binding of a 

compound to DNA and the persistence of the resulting adducts are considered important determinants 

of cancer initiation by genotoxic carcinogens (Ghosal and Iba 1990). As a direct-acting mutagen, 

3,3’-dichlorobenzidine is an effective inducer of its own activation (Iba 1987a). 

It has been suggested that some of the toxicity (carcinogenicity and non-cancer) of polyhalogenated 

aromatics (such as 3,3’-dichlorobenzidine) may be related to the abilities to induce cytochrome 

P-448-mediated (CYPlA2) monooxygenase activities. Therefore, it is reasonable to expect that the 

hepatocarcinogenicity of 3,3’-dichlorobenzidine may be due, at least in part, to the induction of 

hepatic cytochrome P-448 which would have the impact of producing higher amounts of reactive 

metabolites (Iba et al. 1983). The demonstration that 3,3’-dichlorobenzidine both increases lipid 

peroxidation and decreases antioxidant content in vivo in one study may have a bearing on the 

carcinogenicity of this substance because antioxidants protect against the acute and long-term effects 

of lipid peroxidation (Iba 1987b) which may be an important determinant in carcinogenesis. 

There are data to suggest that 3,3’-dichlorobenzidine may act synergistically with other carcinogens. 

No carcinomas were found in any rats administered one of the following in the diet for a period of 40 

weeks: 0.03% 3,3’-dichlorobenzidine in the diet, 0.001% BBN in drinking water, 0.0005% 2-AAF in 

the diet, or 0.04% FANFT (Ito et al. 1983). However, when BBN and 3,3’-dichlorobenzidine were fed 

together at the same dose levels as above, there was a marked increase in papillary or nodular 

hyperplasia in the rat bladder and the appearance of one papilloma. The authors suggested a 

synergistic effect of 3,3’-dichlorobenzidine on the carcinogenicity of BBN. 

2.4.3 Animal-to-Human Extrapolations 

Information on the toxicity of 3,3’-dichlorobenzidine for humans and animals is limited, 

particularly regarding noncancer end points. Therefore, an attempt to discuss potential 

interspecies differences or similarities in 3,3’-dichlorobenzidine noncancer toxicity based on the 

limited information available seems speculative at this time. 3,3’-Dichlorobenzidine is 

carcinogenic in animals (Osanai 1976; Pliss 1959, 1963; 
3,3’-DICHLOROBENZIDINE 46 

2. HEALTH EFFECTS
 

Sellakumar et al. 1969; Stula et al. 1975, 1978). There is no conclusive evidence of carcinogenicity of 

3,3’-dichlorobenzidine in humans (Gadian 1975; Gerarde and Gerarde 1974; MacIntyre 1975; Myslak 

et al. 1991; Ouellet-Hellstrom and Rench 1996); however, there is concern about occupationally 

exposed subjects because of 3,3’-dichlorobenzidine’s structural similarity with the known human and 

animal carcinogen benzidine. However, unless a cohort exposed only to 3,3’-dichlorobenzidine is 

identified and adequate epidemiological studies on such a cohort are conducted, the question will 

remain unsolved. 

2.5 RELEVANCE TO PUBLIC HEALTH 

Overview. 

Most of the information on human health effects of 3,3’-dichlorobenzidine is derived from 

several reports of exposure in the workplace, in which the inhalation and dermal routes represent 

the most likely routes of exposure. Significant exposure to 3,3’-dichlorobenzidine, would impact 

the health of the general population, seems unlikely. The available occupational studies have 

limitations, including lack of precise exposure data and presence of other compounds, as well as 

other confounding factors. No organ or system could be identified as a target for 3,3’­

dichlorobenzidine toxicity in the available studies in humans. Results from cancer studies in 

humans were inconclusive because of possible co-exposure to other chemicals. Studies in animals 

showed that 3,3’-dichlorobenzidine is a multi-site carcinogen in various species following oral 

administration; no data were available following inhalation or dermal exposure. There is some 

evidence, however, of carcinogenicity in rats after subcutaneous injection of 3,3’­

dichlorobenzidine, and in the offspring of mice after subcutaneous dosing to the dams during 

pregnancy. Systemic effects in animals were limited to reports of formation of adducts with proteins 

such as hemoglobin and with DNA and minor liver effects after chronic oral dosing. Also, ocular 

effects were reported in rabbits after direct instillation of the hydrochloric salt of the compound to the 

eye. In most studies in animals, the animals were exposed to levels of 3,3’-dichlorobenzidine several 

orders of magnitude higher than those found in the environment. Almost nothing is known about the 

toxicokinetics of 3,3’-dichlorobenzidine in humans. 3,3’-Dichlorobenzidine has been identified in the 

urine from workers or volunteers exposed to it; therefore, it is absorbed by humans. The primary route 

of absorption could not be ascertained, but it is assumed to have been inhalation and/or dermal. 

Animals can absorb 3,3’-dichlorobenzidine through ingestion or dermal contact with the chemical; no 

information was located regarding inhalation exposure. Based on limited data regarding 

environmental exposure, the most likely exposure route for populations 
3,3’-DICHLOROBENZIDINE 47 

2. HEALTH EFFECTS
 

living near hazardous waste sites is the dermal route. Under these circumstances, assuming that 

3,3’-dichlorobenzidine is present in surrounding environmental media, this route may be of 

concern since animal studies have shown that 3,3’-dichlorobenzidine is absorbed by this route. 

Issues relevant to children are explicitly discussed in Section 2.6, Children’s Susceptibility, and 

Section 5.6, Exposures of Children. 

Minimal Risk Levels for 3,3’-Dichlorobenzidine. 

Inhalation MRLs. 

No acute-duration inhalation MRL was calculated for 3,3’-dichlorobenzidine due to the 

inadequate data. The information provided in the single relevant study in animals that is available 

(Gerarde and Gerarde 1974) is severely limited by lack of detailed reporting of the results. 

Included among the limitations are lack of information concerning exposure concentration and 

failure to use control groups. No intermediateduration inhalation MRL was calculated for 

3,3’-dichlorobenzidine because no intermediate-duration studies in humans or animals were 

located. No chronic-duration inhalation MRL was calculated for 3,3’-dichlorobenzidine because 

the available human studies do not provide quantitative exposure information (Gadian 1975; 

Gerarde and Gerarde 1974; MacIntyre 1975; Myslak et al. 1991). No chronicduration inhalation 

studies in animals were located. 

Oral MRLs. 

No acute-duration oral MRL was calculated for 3,3’-dichlorobenzidine because the available 

studies did not identify appropriate NOAELs or LOAELs (Ashby and Mohammed 1988; Birner 

et al. 1990; Cihak and Vontorkova 1987; Ghosal and Iba 1990). No intermediate-duration oral 

MRL was calculated for 3,3’-dichlorobenzidine because the available studies did not identify 

relevant noncancer effects (Ito et al.1983; Osanai 1976; Pliss 1959, 1963). No chronic-duration 

oral MRL was calculated for 3,3’-dichlorobenzidine because there were no NOAELs identified 

below the lowest available serious LOAEL for convulsions and slight neuronal degeneration in 

dogs (Stula et al. 1978). 

Death. No deaths were reported in humans from inhalation, oral, or dermal exposure to 

3,3’-dichlorobenzidine. In animals, 3,3’-dichlorobenzidine caused no deaths in rats exposed by 

the inhalation route in concentrations as high as 23,700 mg/m3

 for 2 hours per day for 7 days 

(Gerarde and Gerarde 1974). In 
3,3’-DICHLOROl3ENZIDINE 48 

2. HEALTH EFFECTS
 

addition, the estimated acute oral LD50 for rats (7,070 mg/kg for the free base and 3,820 mg/kg for 

thedihydrochloride salt) and the minimum dermal lethal dose for male and female New Zealand albino 

rabbits (>8,000 mg/kg) for 3,3’-dichlorobenzidine suggested that the lethal toxicity of 

3,3’-dichlorobenzidine is minimal (Gerarde and Gerarde 1974). Consequently, it is unlikely that death 

will occur in humans exposed to 3.3’-dichlorobenzidine at the levels at which it occurs at hazardous 

waste sites. 

Systemic Effects. Dermatitis appears to be the only effect of 3,3’-dichlorobenzidine (free base) 

exposure for which evidence exists in humans (Gerarde and Gerarde 1974). Gastrointestinal upset and 

upper respiratory tract infections have also been reported by workers, but the role of 

3,3’-dichlorobenzidine was uncertain. 3,3’-Dichlorobenzidine has not been found to cause these 

effects in experimental animals. 

Respiratory Effects. Upper respiratory infection and sore throat were among several principal reasons 

for frequent visits to a company’s medical clinic by workers handling 3,3’-dichlorobenzidine 

dihydrochloride (dihydro salt of 3,3’-dichlorobenzidine) (Gerarde and Gerarde 1974). However, data 

from animal studies are equivocal regarding the etiology of these symptoms (Gerarde and Gerarde 

1974). While it is possible that these symptoms were due to exposure to 3,3’-dichlorobenzidine 

hydrochloride, the irritant effects of HCl from the compound in combination with particulate toxicity 

could have been responsible for the observed effects in these studies. Therefore, it is not likely that 

respiratory ailments will occur in humans exposed to 3,3’-dichlorobenzidine at hazardous waste sites. 

Cardiovascular Effects. Reports of cardiovascular effects in humans or animals after exposure to 3,3’­

dichlorobenzidine by any route were not found in any of the existing epidemiological and animal 

studies, suggesting that the cardiovascular system is not a target of 3,3’-dichlorobenzidine toxicity. It 

is unlikely that cardiovascular effects will occur in humans exposed to 3,3’-dichlorobenzidine at levels 

found at hazardous waste sites. 

Gastrointestinal Effects. Gastrointestinal upset was one of the symptoms reported by employees who 

worked with 3,3’-dichlorobenzidine dihydrochloride (dihydro salt of 3,3’-dichlorobenzidine) (Gerarde 

and Gerarde 1974). However, there is no conclusive evidence that 3,3’- dichlorobenzidine caused 

these gastrointestinal upsets since there was exposure to other chemicals as well. In addition, 

3,3’-dichlorobenzidine has not been found to cause any of these effects in experimental animals. 

Therefore, it is unlikely 
3,3’-DICHLOROBENZIDINE 49 

2. HEALTH EFFECTS
 

that exposure to 3,3’-dichlorobenzidine at hazardous waste sites will cause gastrointestinal effects in 

humans. 

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

inhalation, oral, or dermal exposure to 3,3’-dichlorobenzidine. Although hematological effects may 

not be sensitive indicators for 3,3’-dichlorobenzidine toxicity, hemoglobin adducts were observed in 

animal studies following single oral exposures to 127 or 253 mg/kg 3,3’-dichlorobenzidine (Bimer et 

al. 1990) and repeated exposures to 0.3 mg/kg/day for up to 4 weeks (Joppich-Kuhn et al. 1997). 

Birner et al. (1990) suggested that metabolically formed nitroso derivatives and the formation of a 

sulfinic acid amide with cysteine residues in hemoglobin may be the mechanism of adduct formation. 

No hematological abnormalities were found in dogs exposed to 10.4 mg/kg/day 

3,3’-dichlorobenzidine for 7 years (Stula et al.1978). Therefore, it is unlikely that blood abnormalities 

will occur in humans exposed to 3,3’-dichlorobenzidine at levels found at hazardous waste sites. 

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

animals after exposure to 3,3’-dichlorobenzidine by any route. However, since this effect was not 

reported in any of the existing epidemiological and animal studies, it is unlikely that musculoskeletal 

effects will occur in humans exposed to 3,3’-dichlorobenzidine at levels found at hazardous waste 

sites. 

Hepatic Effects. No studies were located regarding hepatic effects in humans after exposure to 

3,3’-dichlorobenzidine. Information from animal studies on the liver effects of exposure to 

3,3’-dichlorobenzidine suggests that exposure to sufficiently high levels of thecompound could cause 

liver injury as indicated by modest elevation in serum transaminase activity, fatty liver (Stula et al. 

1978), decrease in hepatic vitamin E, and lipid peroxidation (Iba 1987a; Iba and Lang 1988; Iba and 

Thomas 1988). Some of these effects may contribute to the liver tumors induced. However, it is not 

known whether these liver injuries will occur in humans exposed to 3,3’-dichlorobenzidine at levels at 

which it occurs at hazardous waste sites since these effects were not reported in any worker studies in 

which exposures are significantly higher. 

Renal Effects. No studies were located regarding renal effects in humans after exposure to 

3,3’-dichlorobenzidine by any route. No effects to the kidneys or urinary parameters monitored were 

observed in dogs exposed to 10.4 mg/kg/day for up to 7 years (Stula et al. 1978). Based on these data, 

it is unlikely that 
3,3’-DICHLOROBENZIDINE 50 

2. HEALTH EFFECTS 

kidney effects will occur in humans exposed to 3,3’-dichlorobenzidine at levels found at 

hazardous waste sites. 

Endocrine Effects. No studies were located regarding endocrine effects in humans or animals 

after exposure to 3,3’-dichlorobenzidine by any route. However, since this effect was not reported 

in any of the existing epidemiological and animal studies, it is unlikely that endocrine effects will 

occur in humans exposed to 3,3’-dichlorobenzidine at levels found at hazardous waste sites. 

Dermal Effects. Dermatitis was cited as the only verified health problem encountered by workers 

in contact with the free base of 3,3’-dichlorobenzidine in a dichlorobenzidine manufacturing plant 

(Gerarde and Gerarde 1974). There was no discernable skin irritation when 3,3’­

dichlorobenzidine dihydrochloride (at an unstipulated dose) was applied to the intact and abraded 

skin of rabbits (Gerarde and Gerarde 1974). Similarly, an aqueous suspension of 

3,3’-dichlorobenzidine instilled intradermally into rats at a dose of 700 mg/kg did not produce 

adverse effects (Gerarde and Gerarde 1974). The observations in humans may have been allergic 

dermatitis, and specific protocols are required to make these determinations in laboratory 

animals. 

Ocular Effects. No studies were located regarding ocular effects in humans after exposure to 

3,3’-dichlorobenzidine by any route. No adverse effects on the eye were noted when 

dichlorobenzidine (isomer unspecified, free base) was directly placed in the conjunctival sac of 

the eye of rabbits (Gerarde and Gerarde 1974). However, 0.1 mL 3,3’-dichlorobenzidine 

dihydrochloride (dihydro salt of 3,3’-dichlorobenzidine) in a 20% corn oil suspension produced 

erythema, pus, and cornea1 opacity, giving a 76% score in the Draize test within an hour when 

placed in the conjunctival sac of the eye of the rabbit (Gerarde and Gerarde 1974). Apparently, 

the irritant effects of hydrochloric acid from the salt-compound contributed to the observed 

effects. Based on these data, it is not probable that adverse effects to the eye will occur in 

humans exposed to 3,3’-dichlorobenzidine at levels at which it occurs at hazardous waste sites. 

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

exposure to 3,3’-dichlorobenzidine by any route. No significant difference in body weight was 

observed in dogs exposed to 10.4 mg/kg/day for up to 7 years (Stula et al. 1978). Based on these 

data, it is unlikely that body weight effects will occur in humans exposed to 

3,3’-dichlorobenzidine at levels found at hazardous waste sites. 
3,3’-DICHLOROBENZIDINE 51 

2. HEALTH EFFECTS
 

Metabolic Effects. No studies were located regarding metabolic effects in humans or animals after 

exposure to 3,3’-dichlorobenzidine by any route. However, since this effect was not reported in any of 

the existing epidemiological and animal studies, it is unlikely that metabolic effects will occur in 

humans exposed to 3,3’-dichlorobenzidine at levels found at hazardous waste sites. 

Immunological and Lymphoreticular Effects. No studies were located regarding immunological 

and/or lymphoreticular effects in humans or animals following exposure to 3,3’-dichlorobenzidine by 

any route of exposure. The immune system does not appear to be a sensitive target of 

3,3’-dichlorobenzidine toxicity. Consequently, immune system disruptions are not expected in humans 

exposed to 3,3’-dichlorobenzidine at the levels at which it occurs at hazard waste sites. 

Neurological Effects. Workers exposed to 3,3’-dichlorobenzidine and possibly to other chemicals in a 

chemical manufacturing plant reported headache and dizziness at the company clinic (Gerarde and 

Gerarde 1974). No further information indicated neurological effects in humans following exposure to 

3,3’-dichlorobenzidine. In animal studies, 1 of 6 dogs exhibited convulsions after 21, 28, and 42 

months of oral treatment with 10.4 mg/kg/day 3,3’-dichlorobenzidine for 3.5 years. A necropsy of the 

dog at 42 months revealed slight neuronal degeneration at unspecified sites in the brain and/or spinal 

cord (Stula et al. 1978). In view of the fact that only one dog developed the lesion, direct causality 

cannot be inferred. In addition, based on its chemical structure, 3,3’-dichlorobenzidine does not 

appear to be a neurotoxicant. The information available suggests that at the levels found in the 

environment, 3,3’-dichlorobenzidine is unlikely to constitute a neurological hazard for humans. 

Reproductive Effects. No studies were located regarding reproductive effects in humans or animals 

following exposure to 3,3’-dichlorobenzidine by any route of exposure. Consequently, reproductive 

system disruptions are not expected in humans exposed to 3,3’-dichlorobenzidine at the levels at 

which it occurs at hazard waste sites. 

Developmental Effects. No studies were located regarding developmental effects of 

3,3’-dichlorobenzidine in humans following brief or long-term exposure by any route. Abnormal 

growth was observed in kidneys explanted from fetuses of pregnant mice treated subcutaneously daily 

during the last week of pregnancy at an average daily dose of approximately 421 mg/kg (Shabad et al. 

1972). Similarly, in subcutaneous-injection studies in BALB/C mice, hyperplastic foci and 

hyperchromic glomeruli were 
3,3’-DICHLOROBENZIDINE 52 

2. HEALTH EFFECTS
 

observed in kidneys of offspring of dams administered 2 mg 3,3’-dichlorobenzidine (about 93.5 

mg/kg) 4 or 5 times throughout gestation (Golub 1970). In a study of similar design, by the same 

group of investigators, subcutaneous injection of 3,3’-dichlorobenzidine during pregnancy to mice 

resulted in the induction of tumors in the progeny (Golub et al. 1975). Because the pups were nursed 

by the dams, it is unknown whether these effects may have been caused by transplacental transfer of 

the active principle, through nursing, or both. The significance of these findings to human health is 

unclear, particularly because of the irrelevant route of exposure and the high doses used. 

Genotoxic Effects. Studies in several test systems show 3,3’-dichlorobenzidine to be genotoxic in vivo 

and in vitro (see Tables 2-3 and 2-4). It has been suggested that genotoxicity of 

3,3’-dichlorobenzidine mediates the carcinogenicity of the compound (Imaoka et al. 1997; Ghosal and 

Iba 1990). 

In vivo, micronuclei were induced in polychromatic erythrocytes of the liver of fetal mice exposed 

transplacentally to the compound, and in liver cells of adult male mice treated orally with the 

compound at a maximum tolerated dose reported to be 1,000 mg/kg (Cihak and Vontorkova 1987). A 

sex difference in the genotoxicity of the compound is suggested, since adult male mice, but not 

pregnant females developed erythrocyte micronuclei following 3,3’-dichlorobenzidine exposure. 

However, whether this differential effect extends to carcinogenic effects is unclear. Positive chromatid 

exchange findings in an in vitro test system provide supportive evidence for 3,3’-dichlorobenzidineinduced cytogenetic changes. In a study using type I, II, and III Bloom Syndrome (BS) 

B-lymphoblastoid cell lines, 3,3’-dichlorobenzidine induced sister chromatid exchanges (SCEs) in all 

three types (Shiraishi 1986). However, the induction of SCE was variable among the three types. 

Exposure of BS type II and type III cells to 3,3’-dichlorobenzidine (1x10-8 to 1.3x10-3 M) caused an 

increase in SCEs (120-140/cell) over baseline levels (70/cell) at the highest concentration 

(1.3x10-3 M). BS type II cells required metabolic activation, while BS type III cells were sensitive 

with and without activation. The frequency of SCEs in BS type I cells was lower than in II and III. 

The genotoxic effect of 3,3’-dichlorobenzidine is further supported by positive responses in bacterial 

assays employing Sulmonella tester strains TA1538 and TA98 in the absence of liver activating 

systems (Garner et al. 1975; Iba 1987a; Iba and Thomas 1988; Lazear et al. 1979; Savard and Josephy 

1986). In another study, 3,3’-dichlorobenzidine exhibited both direct and hydrogen peroxidedependent mutagenicity in S. thyphimurium strain TA98, but not Tal00 or TA102, leading the authors 

to suggest that enzymes perhaps 


3,3’-DICHLOROBENZIDINE 55 

2. HEALTH EFFECTS
 

endogenous to the tester strain TA98 may play a role in the activation of 3,3’-dichlorobenzidine 

(Lang and Iba 1987). A mixture containing Arochlor-induced rat liver homogenate and 10 μg 

3,3’-dichlorobenzidine was positive for reverse mutation in S. typhimurium strain TA98 

(histidine revertants) (Vithayathil et al. 1983). A recent study reported DNA damage in S. 

typhimurium NM2009 after incubation with 3,3’-dichlorobenzidine activated by mouse kidney or 

bladder rnicrosomes or rat liver microsomes (Imaoka et al. 1997). 

3,3’-Dichlorobenzidine is an effective inducer of its own activation (Iba 1987a). The enhancing 

effect of 3,3’-dichlorobenzidine pretreatment on the in vitro liver activation of the chemical to 

mutagens has been associated with the induction of cytochrome P-450d (CYPlA2) (Iba and 

Thomas 1988). This action may result in the compound enhancing its own genotoxicity and 

carcinogenicity. 3,3’-Dichlorobenzidine was also shown to be a potent inducer of hepatic 

microsomal enzymic activities mediated by cytochrome-P-448 (CYPlA2) and P-450 in other 

animal studies (Iba and Sikka 1983; Iba and Thomas 1988). In another study to evaluate the 

P-450 induction pattern of 3,3’-dichlorobenzidine, intraperitoneal administration of 20-120 mg/kg 

3,3’-dichlorobenzidine to male Sprague-Dawley rats induced P-450 isozymes in a pattern similar 

to 3-methylcholanthrene (i.e., induced P-450c) (CYP2Bl), and P-450d (CYPlA2) but mainly 

P-450c (CYP2Bl). 3,3’-Dichlorobenzidine activation was unaffected by monoclonal antibodies to 

P-450b (CYPlAl) or P-450c (CYP2Bl) but was inhibited by 69% by polyclonal antibodies to 

P-450d (CYPlA2). 3,3’-Dichlorobenzidine activation was also inhibited by 46% by antibody 

specific to NADPH-cytochrome P-450 reductase. Based on these results, it was concluded that 

P-450d (CYPlA2) is mainly responsible for the activation of 3,3’-dichlorobenzidine to mutagens 

in the Ames test by rat hepatic microsomes (Iba et al. 1983). 

Results of in vivo tests show that 3,3’-dichlorobenzidine induced dose-dependent unscheduled 

DNA synthesis in the liver of male rats treated orally (Ashby and Mohammed 1988). In vitro 

evidence for the genotoxicity of 3,3’-dichlorobenzidine includes the induction of UDS in HeLa 

cells at a concentration range of 10m-7 to 10-4M (Martin et al. 1978), and transformation of high 

passage rat embryo cells infected with the Rauscher leukemia virus (Freeman et al. 1973). In the 

latter system, an effect was observed at 2x10-7 M 3,3’-dichlorobenzidine, but not at 4x10-8 M. 

Also, 3,3’-dichlorobenzidine transformed BHK21 cells (hamster kidney cells) in vitro in the 

presence of metabolic activation (Styles 1978). The UDS assay is used to measure the repair that 

follows DNA damage. However, the relevance of UDS to human health is not known. While 

results were positive in two in vivo assay systems, sufficient data are not available from 
3,3’-DICHLOROBENZIDINE 56 

2. HEALTH EFFECTS
 

more predictive indicator assays to adequately characterize the genotoxic potential for 3,3’­

dichlorobenzidine in humans. 

3,3’-Dichlorobenzidine formed adducts with calf thymus DNA when incubated with rat liver S9 

(Bratcher and Sikka 1982), or horseradish peroxidase (Tsuruta et al. 1985) in vitro. 

3,3’-Dichlorobenzidine was also shown to bind extensively to tissue DNA in rats and mice. Single 

oral administration of 20 or 100 mg/kg radiolabeled 3,3’-dichlorobenzidine to male Sprague-Dawley 

rats or Swiss-Webster mice resulted in extensive binding of the compound to tissue (liver, bladder, 

and intestine) DNA 12, 24, or 96 hours, and 9 or 14 days after treatment (Ghosal and Iba 1990). 

Results from in vitro studies in rats and mice indicated that 3,3’-dichlorobenzidine formed tissue 

DNA-binding derivatives of 3,3’-dichlorobenzidine (Ghosal and Iba 1990). However, the relevance of 

DNA adduct formation to the genotoxicity and carcinogenicity of the compound and to human health 

is not yet established. Therefore, the genotoxicity consequences of 3,3’-dichlorobenzidine in humans 

remain uncertain. 

Cancer. Due, in part, to structure-activity considerations, epidemiological studies of potential cancer 

effects of occupational exposure to 3,3’-dichlorobenzidine have focused upon bladder tumors since 

benzidine is a known bladder carcinogen. One study found an excess incidence of bladder tumors 

among German painters who were exposed to various dyes and pigments derived from benzidine, 

3,3’-dichlorobenzidine, 3,3-dimethylbenzidine (o-tolidine), 3,3-dimethoxybenzidine (o-dianisidine), 

and 2-naphthylamine (Myslak et al. 1991). Because of the potential exposure of the painters to 

multiple chemicals (including some known bladder carcinogens), the role of 3,3’-dichlorobenzidine in 

the increased incidence of bladder tumors, if any, is unknown. A more recent study found a significant 

increase in the incidence of bladder cancers among a group of about 700 employees employed at a 

Connecticut chemical plant (Ouellet-Hellstrom and Rench 1996). In this case there was no exposure to 

benzidine, but the workers were also exposed to several arylamines other than 3,3’-dichlorobenzidine, 

therefore risks from specific chemical exposures could not be evaluated. 

No other epidemiological studies have found bladder tumors or excess tumors at other sites 

(Gadian 1975; Gerarde and Gerarde 1974; MacIntyre 1975). Cancer effects have not been 

satisfactorily investigated in these studies of occupationally exposed workers. These studies were 

conducted with workers who were exposed to 3,3’-dichlorobenzidine for less than 20 years. Since 

the latency period for chemically induced bladder cancer in humans ranges from 5 to 50 years 

(Badalament 1998), the induction period for 
3,3’-DICHLOROBENZIDINE 57 

2. HEALTH EFFECTS
 

3,3’-dichlorobenzidine-induced tumors may not have elapsed for some individuals. Also, the number 

of workers examined in these studies was relatively small, thus limiting the statistical power to detect 

a significant increase in bladder cancer mortality (incidence). 

Some have speculated that 3,3’-dichlorobenzidine may have contributed to the incidence of bladder 

cancer attributed to benzidine in dye industry workers who handled both benzidine and 

3,3’-dichlorobenzidine (Gadian 1975; IARC 1982a). No bladder tumors were observed in a group of 

workers who handled only 3,3’-dichlorobenzidine; in the same plant, bladder tumors were found 

among workers who handled both benzidine and 3,3’-dichlorobenzidine. The investigator reported a 

total exposure time of 68,505 hours for the study population, equivalent to nearly 140 full-time 

working years (Gadian 1975). Cytodiagnostic tests produced no indication of tumors of the bladder in 

an epidemiological study of 259 workers who had been exposed for a total of less than 16 years to 

3,3’-dichlorobenzidine (MacIntyre 1975). In a retrospective epidemiological study, no bladder tumors 

were observed in a cohort of 207 workers, most of whom had been exposed for up to 15 years 

(Gerarde and Gerarde 1974). However, in this study there was no evidence that any valid system of 

medical surveillance of workers ever existed during the years that 3,3’-dichlorobenzidine was used at 

the plant (Gerarde and Gerarde 1974). A number of other inadequacies noted by reviewers of the 

study severely limit the study’s usefulness. 

In animal studies, 3,3’-dichlorobenzidine has been found to cause neoplasia in a variety of target 

organs in several species. The compound produces hepatocellular carcinomas and urinary bladder 

carcinomas in dogs and hamsters (Sellakumar et al. 1969; Stula et al. 1978). Liver cell tumors were 

demonstrated in mice exposed to 3,3’-dichlorobenzidine in the diet (Osanai 1976; Pliss 1959). In rats, 

mammary gland tumors, Zymbal gland tumors, urinary bladder tumors, and leukemias were 

attributable to 3,3’-dichlorobenzidine exposure (Pliss 1959, 1963; Stula et al. 1975). One cancer study 

of dogs which evaluated one sex and used one dose level (precluding dose-response evaluation) shows 

a sufficient number of animals survived to develop tumors (Stula et al. 1978). The results of a study in 

rats suggested that 3,3’-dichlorobenzidine may have a synergistic effect on the bladder carcinogenicity 

of other chemicals (Ito et al. 1983). 

Because of the increased use of closed systems and protective clothing, dermal absorption of 

3,3’-dichlorobenzidine probably represents a relatively minor route of exposure (EPA 1980b). 

However, there is experimental evidence that under certain environmental conditions favoring moist 

skin conditions, such as high relative humidity and high air temperature, dermal absorption of 

3,3’-dichlorobenzidine by 
3,3’-DICHLOROBENZIDINE 58 

2. HEALTH EFFECTS
 

humans may be enhanced (Meigs et al. 1954). Studies have not been located which investigate 

the carcinogenic potential of 3,3’-dichlorobenzidine following dermal exposure in laboratory 

animals. 

Further evidence of the carcinogenic potential of 3,3’-dichlorobenzidine is provided by studies 

where 3,3’-dichlorobenzidine was administered subcutaneously. Following subcutaneous 

administration in rats for 10 to 13 months, the compound was found to cause tumors of the skin, 

sebaceous and mammary glands, and urinary bladder (Pliss 1963). These sites were in addition to 

tumors of the hematopoietic tissues and Zymbal gland which were observed following oral 

exposure (Pliss 1959). Pliss (1963) further indicated that oral exposure to 3,3’-dichlorobenzidine 

resulted in a higher incidence of tumors in rats than after subcutaneous injection of the 

compound. Pliss (1963) also noted that the introduction of chlorine into the benzidine molecule 

resulted in an increased carcinogenic response in the skin and the urinary bladder. Local 

subcutaneous sarcomas and liver tumors were observed in 13/28 strain D mice following 

subcutaneous administration of 3,3’-dichlorobenzidine for 11 months (Pliss 1959). 

In subcutaneous injection studies, induction of tumors in the progeny of BALB/c mice 

administered 2 mg 3,3’-dichlorobenzidine (about 93.5 mg/kg) 4 or 5 times during the last week of 

pregnancy suggest that the chemical may be a transplacental carcinogen (Golub et al. 1975). 

There was an increased incidence of lymphatic leukemias (7 of 24, 29%), lung adenomas 

(5 of 24, 20%), and adenocarcinomas of the mammary gland (4 of 11 female offspring, 36%) in 

the treated group. Lung tumors (3 of 30 offspring, 10%) and mammary gland tumors 

(3 of 19 female offspring, 16%) were observed in untreated controls (Golub et al. 1975). It should 

be noted that since the offspring were nursed by the treated dams, transfer of 

3,3’-dichlorobenzidine to the offspring through maternal milk may have also occurred. 

3,3’-Dichlorobenzidine is an effective inducer of its own metabolic activation (Iba 1987a). The 

enhancement of 3,3’-dichlorobenzidine mutagenesis has been associated with the induction of 

cytochrome P-450d (Iba and Thomas 1988), and may result in the elevation of its carcinogenicity. 

In other animal studies, 3,3’-dichlorobenzidine was also shown to be a potent inducer of hepatic 

microsomal enzymic activities mediated by cytochrome-P-448 and P-450 (Iba and Sikka 1983; 

Iba and Thomas 1988). Consequently, it has been suggested that the hepatocarcinogenicity of 

3,3’-dichlorobenzidine may be due, at least in part, to the induction of hepatic cytochrome P-488 

and DNA-adduction. 
3,3’-DICHLOROBENZIDINE 59 

2. HEALTH EFFECTS
 

While concordance between tumor sites in experimental animals and humans cannot be assumed, the 

occurrence of tumors in multiple organs in several species of experimental animals should be regarded 

as evidence for the potential carcinogenicity of 3,3’-dichlorobenzidine to humans. 

The Environmental Protection Agency (EPA) has determined that 3,3’-dichlorobenzidine is a probable 

human carcinogen. The U.S. Department of Health and Human Services (DHHS) has determined that 

3,3’-dichlorobenzidine and its dihydrochloride salt may reasonably be expected to be carcinogens. 

IARC (1987) has determined that 3,3’-dichlorobenzidine is possibly carcinogenic to humans. 

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 (Morselli et al. 1980; NRC 1993); the gastrointestinal 

absorption of lead is greatest in infants and young children (Ziegler et al. 1978). Distribution of 

xenobiotics may be different; for example, infants have a larger proportion of 
3,3’-DICHLOROBENZIDINE 60 

2. HEALTH EFFECTS
 

their bodies as extracellular water and their brains and livers are proportionately larger (Altman and 

Dittmer 1974; Fomon 1966; Fomon et al. 1982; Owen and Brozek 1966; Widdowson and Dickerson 

1964). The infant also has an immature blood-brain barrier (Adinolfi 1985; Johanson 1980) and 

probably an immature blood-testis barrier (Setchell and Waites 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 (Komori 1990; Leeder and Kearns 1997; NRC 

1993; Vieira et al. 1996). Whether differences in xenobiotic metabolism make the child more or less 

susceptible also depends on whether the relevant enzymes are involved in activation of the parent 

compound to its toxic form or in detoxification. There may also be differences in excretion, 

particularly in the newborn who has a low glomerular filtration rate and has not developed efficient 

tubular secretion and resorption capacities (Altman and Dittmer 1974; NRC 1993; West et al. 1948). 

Children and adults may differ in their capacity to repair damage from chemical insults. Children also 

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 studies were located that specifically addressed the health effects of exposure to 

3,3’-dichlorobenzidine in children. Limited data in adults are mostly derived from occupational 

studies with limitations including lack of precise exposure data and presence of other compounds. As 

a result, no organ or system has been identified as a target for 3,3’-dichlorobenzidine in humans, 

although dermatitis caused by skin contact with the free base was reported in one study (Gerarde and 

Gerarde 1974). It is reasonable to assume that the same effect would be seen in children similarly 

exposed. Because of the structural similarity of 3,3’-dichlorobenzidine with the known human bladder 

carcinogen benzidine, special attention has been paid to the incidence of bladder cancer among 

subjects occupationally exposed to 3,3’-dichlorobenzidine. Thus far, largely because of study 

limitations, there is no conclusive evidence that exposure to 3,3’-dichlorobenzidine increases the risk 

of bladder cancer in humans (Gadian 1975; Gerarde and Gerarde 1974; Myslak et al. 1991; 

Ouellet-Hellstrom and Rench 1996). 
3,3’-DICHLOROBENZIDINE 61 

2. HEALTH EFFECTS
 

No studies were available that provided information on possible adverse developmental effects in 

humans exposed to 3,3’-dichlorobenzidine. The few available studies in animals were inadequate 

since they used parenteral administration of high doses of 3,3’-dichlorobenzidine 

(Golub 1970; Golub et al. 1975; Shabad et al. 1972). 

There is no information regarding pharmacokinetics of 3,3’-dichlorobenzidine in children nor it is 

known whether 3,3’-dichlorobenzidine can be stored and excreted in breast milk. Although there 

have been no direct measurements to determine whether 3,3’-dichlorobenzidine can cross the 

placenta, there is some indirect evidence that it or its metabolites do. The evidence is based on the 

results of a study in which oral administration of 3,3’-dichlorobenzidine to pregnant mice resulted 

in the induction of micronuclei in the liver of fetuses (Cihak and Vontorvoka 1987). The results 

of another study in which subcutaneous administration of 3,3’-dichlorobenzidine to pregnant 

mice induced abnormal growth of the kidneys explanted from the fetuses also suggest that 

3,3’-dichlorobenzidine or a metabolite can cross the placenta (Shabad et al. 1972). There is no 

information on whether 3,3’-dichlorobenzidine can be stored in maternal tissues and be mobilized 

during pregnancy or lactation, or whether it can reach parental germ cells. 

There is no information on the metabolism of 3,3’-dichlorobenzidine in children. Limited data in 

humans suggest that N-acetylation is an important metabolic pathway (Belman et al. 1968), and a 

detoxification mechanism. N-Acetylation in humans is likely done by one of two families of 

N-acetyltransferases. One of these families, NAT2, is developmentally regulated 

(Leeder and Kearns 1997). Some enzyme activity can be detected in the fetus by the end of the 

first trimester. Almost all infants exhibit the slow acetylator phenotype between birth and 2 

months of age. The adult phenotype distribution is reached by the age of 4-6 months, whereas 

adult activity is found by approximately l-3 years of age. Also, UDP-glucuronosyltransferase, 

responsible for the formation of glucuronide conjugates, seems to achieve adult activity by 618 

months of age (Leeder and Keams 1997). These data suggest that metabolism of 3,3’­

dichlorobenzidine by infants will differ from that in adults in extent, rate, or both. 

There are no biomarkers of exposure or effect for 3,3’-dichlorobenzidine that have been validated 

in children or adults exposed as children. There are no biomarkers in adults that identify previous 

childhood exposure. No studies were located regarding interactions of 3,3’-dichlorobenzidine 

with other chemicals in children or adults. No studies were located that examined possible 

differential susceptibility between young and older organisms. 
3,3’-DICHLOROBENZIDINE 62 

2. HEALTH EFFECTS
 

No information was located regarding pediatric-specific methods for reducing peak absorption 

following exposure to 3,3’-dichlorobenzidine, reducing body burden, or interfering with the 

mechanism of action for toxic effects. In addition, no data were located regarding whether methods for 

reducing toxic effects of 3,3’-dichlorobenzidine used in adults might be contraindicated in children. 

There is no information regarding possible transgenerational effects of 3,3’-dichlorobenzidine in 

humans or animals. 

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 

(NASLNRC 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 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 (NRC 

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 3,3’­

dichlorobenzidine 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 
3,3’-DICHLOROBENZIDINE 63 

2. HEALTH EFFECTS 

potential health impairment (e.g., DNA adducts). Biomarkers of effects caused by 

3,3’-dichlorobenzidine 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. 

2.7.1 Biomarkers Used to Identify or Quantify Exposure to 3,3’-Dichlorobenzidine 

A test system that involves extracting dichlorobenzidine or its metabolite 

(monoacetyldichlorobenzidine) from urine and reacting it with Chloramine-T has been developed 

to screen for dichlorobenzidine exposure in workers (Hatfield et al. 1982). An amperometric 

method has been developed for the detection of 3,3’-dichlorobenzidine in the urine as a 

quantitative assay for the biological monitoring of people occupationally exposed to this 

substance or a metabolic precursor such as certain pigments. This method is based on the 

possibility of two electron oxidation at carbon electrodes by aromatic diamines (TrippelSchulte et al. 1986). 

Hemoglobin adducts have been detected in female Wistar rats orally administered single 127 or 

253 mg/kg doses of 3,3’-dichlorobenzidine (Birner et al. 1990). The investigators suggested that 

metabolically formed nitroso derivatives can result in the formation of a sulfinic acid amide with 

cysteine residues in hemoglobin (Birner et al. 1990). Hydrolysis yielded mainly 

3,3’-dichlorobenzidine; N-acetylated 3,3’-dichlorobenzidine was also detected. Using a more 

sensitive analytical method, Joppich-Kuhn et al. (1997) also detected 3,3’-dichlorobenzidinehemoglobin adducts in rats treated repeatedly with much lower doses (0.3-5.8 mg/kg/day) of 

3,3’-dichlorobenzidine in the drinking water. The limit of detection of the method 

was below 0.1 ng/g hemoglobin and was linear up to 150 ng/g hemoglobin. Although these 

methods have not yet been validated in an occupationally exposed population, they appear 

potentially suitable for use as a biomarker of human exposure to 3,3’-dichlorobenzidine. 
3,3’-DICHLOROBENZIDINE 64 

2. HEALTH EFFECTS 

2.7.2 Biomarkers Used to Characterize Effects Caused by 3,3’-Dichlorobenzidine 

For more information on biomarkers for renal and hepatic effects of chemicals, see ATSDR/CDC 

Subcommittee Report on Biological Indicators of Organ Damage (1990). For information on 

biomarkers for neurological effects, see OTA (1990). 

Currently no disease states in humans are clearly associated with exposure to 

3,3’-dichlorobenzidine. There is evidence that 3,3’-dichlorobenzidine is carcinogenic in animals 

(Golub et al. 1975; Osanai 1976; Pliss 1959, 1963; Sellakumar et al. 1969; Stula et al. 1975, 

1978) and that it is genotoxic in test systems (Ashby and Mohammed 1988; Cihak and 

Vontorkova 1987; Ghosal and Iba 1990; Shiraishi 1986). Hemoglobin adducts have been isolated 

from the blood of 3,3’-dichlorobenzidine-treated animals (Birner et al. 1990; Joppich-Kuhn et al. 

1997), although further studies are needed to determine the associations between blood levels of 

these adducts and specific adverse effects. 

2.8 INTERACTIONS WITH OTHER CHEMICALS 

In contrast to its effects on other mutagens and carcinogens, di-tert,-butylated hydroxytoluene 

(BHT), an antioxidant and a free radical scavenger-considered to be a cancer chemopreventative 

agent based on its ability to inhibit various phases of the carcinogenic process including the 

bioactivation and binding of carcinogenic chemical compounds to DNA-was shown to increase 

the mutagenicity of 3,3’-dichlorobenzidine to Salmonella TA98 by 21-32% and the covalent 

binding of 3,3’-dichlorobenzidine to added DNA by 32-76% (Ghosal and Iba 1992). 

A synergistic role for 3,3’-dichlorobenzidine and other aromatic amines in the development of 

bladder cancer has been suggested. This was proposed in a study in which no carcinomas were 

found in any rats administered one of the following: 0.03% 3,3’-dichlorobenzidine in the diet, 

0.001% BBN (N-butyl-N-(hydroxybutyl)nitrosamine) in drinking water, 0.0005% 

2-AAP (2-acetylaminofluorene) in the diet, or 0.04% FANFT (N-[4-(5-nitro-2-furyl)-2­

thiazolyllformamide) in the diet for a period of 40 weeks (Ito et al. 1983). However, when BBN 

and 3,3’-dichlorobenzidine were fed together at the same dose levels as above, there was a 

marked increase in papillary or nodular hyperplasia in the rat bladder and the appearance of one 

papilloma. Based on these findings, the authors suggested that 3,3’-dichlorobenzidine had a 

synergistic effect on the carcinogenicity of BBN. In rats sequentially administered BBN (0.01%), 
3,3’-DICHLOROBENZIDINE 65 

2. HEALTH EFFECTS 

FANFT (0.15%) 2-AAF (0.025%), and 3,3’-dichlorobenzidine (0.03%) for 4 weeks, the 

incidence ofbladder cancer after administration of the four chemicals was no different than after 

administration of the first three, suggesting no additive or antagonistic effect for 

3,3’-dichlorobenzidine (Ito et al. 1983). 

2.9 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE 

A susceptible population is defined as one which will exhibit a different or enhanced response to 

a chemical compared to most persons exposed to the same level of exposure. Reasons may 

include genetic makeup, age, health and nutritional status, and exposure to other toxic substances 

(e.g., cigarette smoke). For this chemical, these parameters may result in reduced detoxification 

or excretion of 3,3’-dichlorobenzidine, or compromised function of target organs affected by 

3,3’-dichlorobenzidine. Populations who are at greater risk due to their unusually high exposure 

to 3,3’-dichlorobenzidine are discussed in Section 5.6, Populations With Potentially High 

Exposure. 

No information was located that identified any human population that is exceptionally susceptible 

to the toxicity of 3,3’-dichlorobenzidine. See Section 2.6, Children’s Susceptibility, for a 

discussion of that topic. 

2.10 METHODS FOR REDUCING TOXIC EFFECTS 

This section will describe clinical practice and research concerning methods for reducing toxic 

effects of exposure to 3,3’-dichlorobenzidine. 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 3,3’-dichlorobenzidine. When specific exposures have occurred, poison control 

centers and medical toxicologists should be consulted for medical advice. 

2.10.1 Reducing Peak Absorption Following Exposure 

The only information in the literature regarding reducing the absorption of 3,3’-dichlorobenzidine 

was found in a Fact Sheet published by the State of New Jersey (State of New Jersey 1997). The 

recommendations source indicate that following eye contact, eyes should immediately be flushed 

with large amounts of water for at least 15 minutes, occasionally lifting upper and lower lids. It is 

also recommended that after skin contact contaminated clothing should be quickly removed and 

contaminated skin should be 
3,3’-DICHLOROBENZIDINE 66 

2. HEALTH EFFECTS 

immediately washed with large amounts of soap and water. A person exposed to 

3,3’-dichlorobenzidine in the air should be removed from the source of exposure promptly. 

Other information specific for 3,3’-dichlorobenzidine, aimed at minimizing exposure, was found 

in the HSDB database (HSDB 1997). This information indicates that full body protective clothing 

and gloves should be used by those employed in handling operations. Full face supplied air 

respirators of continuous flow or pressure demand should also be used. In addition, employees 

working with 3,3’-dichlorobenzidine (or its salts) within an isolated system, such as “glove box,” 

should wash their hands and arms upon completion of the assigned task and before engaging in 

other activities not associated with the isolated system. 

2.10.2 Reducing Body Burden 

There are no established methods for reducing the body burden of 3,3’-dichlorobenzidine. 

2.10.3 Interfering with the Mechanism of Action for Toxic Effects 

There are no known methods for interfering with the toxic effects of 3,3’-dichlorobenzidine. 

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 3,3’-dichlorobenzidine 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 

3,3’-dichlorobenzidine. 

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 
3,3’-DICHLOROBENZIDINE 67 

2. HEALTH EFFECTS
 

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 .l Existing Information on Health Effects of 3,3’-Dichlorobenzidine 

The existing data on health effects of inhalation, oral, and dermal exposure of humans and 

animals to 3,3’-dichlorobenzidine are summarized in Figure 2-3. The purpose of this figure is to 

illustrate the existing information concerning the health effects of 3,3’-dichlorobenzidine. 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 Zdentifiing Substance-Specific Data Needs Related to 

Toxicological Profiles (ATSDR 1989), is substance-specific information necessary to conduct 

comprehensive public health assessments. Generally, ATSDR defines a data gap more broadly as 

any substance-specific information missing from the scientific literature. 

Essentially no studies of human exposure to 3,3’-dichlorobenzidine were located by specific 

routes, except for occupational data on direct dermal effects following dermal exposure and a 

recent carcinogenicity study in which skin contact with 3,3’-dichlorobenzidine and other 

arylamines was found to be the most important exposure route (see Figure 2-3). Although there 

are studies of workers in the United States exposed to 3,3’-dichlorobenzidine, these reports are 

limited by the fact that exposure often involved other compounds, and both the route and extent 

of exposure are largely unknown. Dermal effects have also been investigated in experimental 

animals as well as ocular irritant properties of 3,3’-dichlorobenzidine exposure. There is 

no evidence to suggest that the non-ocular systemic toxicological effects of 

3,3’-dichlorobenzidine may be route- or species-specific. 

Additional information on health effects following dermal exposure is sparse. The majority of 

animal studies of 3,3’-dichlorobenzidine have focused on carcinogenic effects following oral 

exposure, whereas data on noncarcinogenic effects are limited. 

3,3’-DICHLOROBENZIDINE 69 

2. HEALTH EFFECTS 

2.11.2 Identification of Data Needs 

Acute-Duration Exposure. One study in humans showed that the compound may cause respiratory 

effects when inhaled and that application of 3,3’-dichlorobenzidine base causes skin irritation 

(Gerarde and Gerarde 1974). Thus, this limited information in humans is insufficient to conclusively 

identify target organs, other than the skin, following exposure by any route. Acute-duration exposure 

can cause eye damage (erythema, pus, cornea1 opacity) in rabbits following conjunctival application. 

However, the relevance of these findings for the general population is unknown since conjunctival 

application is not a typical route of exposure, and exposure by the inhalation route is unlikely. 

3,3’-Dichlorobenzidine can be lethal following oral and dermal exposure at very high doses. In most 

animal studies, comprehensive gross and histopathological evaluations have not been conducted and 

clinical signs have not been monitored. Such studies may provide insight into systemic toxicity and 

potential health threat associated with acute-duration exposure. With the exception of effects caused 

by direct contact of 3,3’-dichlorobenzidine with the skin or the eyes, the limited pharmacokinetic data 

do not suggest route-specific target organs. The available data were inadequate for derivation of either 

inhalation or oral acute MRLs. 

Intermediate-Duration Exposure. No intermediate-duration studies in humans were located. 

Intermediate-duration oral studies have been performed in rats without adverse systemic effects, but 

these studies used only one dose level (Griswold et al. 1968; Ito et al. 1983; Osanai 1976; Pliss 1959, 

1963). Organs and/or tissues from the reproductive, neurological, and immunological systems have 

not been examined in the available intermediate-duration studies; such information would be useful. 

No intermediate-duration inhalation or dermal studies were found. Animal studies evaluating 

toxicological parameters at several dose levels would provide dose-response data which could prove 

more predictive when assessing potential adverse effects in humans following intermediate-duration 

exposure. No oral intermediate MRL was derived because the available studies did not identify 

relevant noncancer effects. 

Chronic-Duration Exposure and Cancer. No studies were located that examined noncancer end points 

in humans following chronic exposure to 3,3’-dichlorobenzidine. Available chronic-duration oral 

studies provide information regarding systemic and carcinogenic effects in rats and dogs (Stula et al. 

1975, 1978). These studies employed one dose level and toxicological parameters measured were 

limited. The inadequacies of these studies precluded derivation of a chronic oral MRL. 

No chronic-duration animal inhalation or dermal exposure studies were located. Well conducted 

chronic-duration inhalation, dermal, 
3,3’-DICHLOROBENZIDINE 70 

2. HEALTH EFFECTS 

and oral studies involving low-dose exposure in animals might provide dose-response data on 

potential systemic effects of exposure in humans. The available data are insufficient to establish a 

relationship between the concentration of 3,3’-dichlorobenzidine and/or its metabolites in the body 

and the levels that are associated with adverse effects. Studies that provide data on the body burden of 

3,3’-dichlorobenzidine associated with toxicity may prove useful. 

Various studies have assessed the potential carcinogenicity of 3,3’-dichlorobenzidine in workers 

exposed to it (Gadian 1975; Gerarde and Gerarde 1974; MacIntyre 1975; Myslak et al. 1991; OuelletHellstrom and Rench 1996). However, many confounders have rendered the results inconclusive. A 

major difficulty in such studies is the simultaneous exposure to several potential or known 

carcinogens. The carcinogenicity of 3,3’-dichlorobenzidine has been well established in animals after 

oral administration of the compound (Osanai 1976; Pliss 1959, 1963; Sellakumar et al. 1969; Stula et 

al. 1975, 1978), but no information is available regarding inhalation and dermal exposure. There is 

suggestive evidence that 3,3’-dichlorobenzidine may cause cancer in animals when applied dermally 

since tumors were found in rats injected with the compound subcutaneously (Pliss 1963). Of particular 

interest would be additional studies, using relevant routes of exposure, to confirm the findings that 

3,3’-dichlorobenzidine causes cancer in offspring of rats injected with the chemical subcutaneously 

during pregnancy (Golub et al. 1975) 

Genotoxicity. Available studies in animals and in bacterial systems show that 3,3’-dichlorobenzidine 

does alter genetic material (Ashby and Mohammed 1988; Bratcher and Sikka 1982; Cihak and 

Vontorkova 1987; Garner et al. 1975; Iba 1987a; Iba and Thomas 1988; Imaoka et al. 1997; Lang and 

Iba 1987; Lazear et al. 1979; Savard and Josephy 1986; Shiraishi 1986; Styles 1978). Studies 

involving more predictive indicator test systems may allow a better assessment of mutagenic potential. 

Reproductive Toxicity. No studies were found regarding reproductive toxicity of 

3,3’-dichlorobenzidine. Should data suggesting that reproductive organs are affected in a 90-day 

study become available, multigenerational reproductive studies in animals may be warranted. 

Developmental Toxicity. No studies were found regarding developmental toxicity of 

3,3’-dichlorobenzidine in humans. Animal studies have shown that 3,3’-dichlorobenzidine and/or 

metabolites may be transferred across the placenta and or through maternal milk to the offspring and 

may affect the growth of the kidneys after parenteral exposure during pregnancy (Golub 1972; Shabad 

et al. 1972) or induce tumors 
3,3’-DICHLOROBENZIDINE 71 

2. HEALTH EFFECTS
 

in the offspring (Golub et al. 1975). The effects of the compound on development following oral, 

inhalation, or dermal exposure have not been studied. Well conducted animal studies employing 

various dose levels and relevant exposure routes during critical developmental periods may provide 

information on potential fetotoxicity, embryotoxicity, and teratogenic effects in humans. Also, 

cross-fostering studies may help determine the relative impacts of in utero transfer of the chemical and 

transfer through nursing. Further animal data may provide dose-response information if studies are 

conducted to determine what dose of 3,3’-dichlorobenzidine, or its metabolites, reaches the fetus. 

Immunotoxicity. No studies were located assessing the potential effect on the immune system during 

3,3’-dichlorobenzidine exposure. Studies that examine antibody levels and responses to bacterial 

infections after exposure to 3,3’-dichlorobenzidine would provide valuable information on the 

immune system. Also, evaluation of morbidity among individuals exposed to 

3,3’-dichlorobenzidine in the workplace may provide important indirect evidence regarding their 

immune status. 

Neurotoxicity. Based on its chemical structure, 3,3’-dichlorobenzidine does not appear to be 

neurotoxicant, but the nervous system has not been carefully evaluated after exposure to this chemical. 

Workers exposed to 3,3’-dichlorobenzidine (and to other chemicals as well) complained of headache 

and dizziness (Gerarde and Gerarde 1974). A chronic-duration oral study in dogs reported convulsions 

in one of six dogs treated orally with 3,3’-dichlorobenzidine (Stula et al. 1978). Upon necropsy, the 

authors noticed slight neuronal degeneration in tissues (unspecified) of the nervous system from this 

dog. However, the effect was seen in only one of the six dogs and only one dose level was tested. The 

limited information available does not suggest that 3,3’-dichlorobenzidine is a neurotoxicant, and 

studies aimed exclusively to evaluate this end point seem unnecessary at this time. However, any 

future long-term toxicity study on 3,3’-dichlorobenzidine in animals should include histological 

evaluation of representative elements of the nervous system. Furthermore, evaluation of neurological 

end points in offspring from animals exposed during gestation would provide information that may be 

relevant to children of pregnant women exposed to 3,3’-dichlorobenzidine in the workplace. 

Epidemiological and Human Dosimetry Studies. The potential for occupational exposure exists in the 

use of 3,3’-dichlorobenzidine in the synthesis of 3,3’-dichlorobenzidine-based pigments for printing 

ink applications and to a lesser extent in paints. Workers exposed to 3,3’-dichlorobenzidine (and 

simultaneously to other chemicals) have complained of gastrointestinal upset, upper respiratory 

infection, 
3,3’-DICHLOROBENZIDINE 72 

2. HEALTH EFFECTS
 

sore throat, caustic burns, headache, dizziness, and dermatitis (Gerarde and Gerarde 1974). The only 

one of these effects that appeared to be associated with 3,3’-dichlorobenzidine exposure with 

reasonable certainty is dermatitis, which was attributed to a manufacturing process change that 

resulted in exposure to dichlorobenzidine-freebase (Gerarde and Gerarde 1974). Studies of 

occupationally exposed individuals are complicated by the fact that there is usually simultaneous 

exposure to other chemicals. Based on available data, the potential for nonindustrial exposure to the 

general population by air, soil, or water is expected to be negligible. Epidemiological studies of people 

who live in areas where 3,3’-dichlorobenzidine has beendetected in groundwater, near industries 

releasing 3,3’-dichlorobenzidine, or near hazardous waste sites could provide information on whether 

3,3’-dichlorobenzidine exposure produces effects in humans. In the unlikely event that exposure of the 

general population (in the past or present) primarily to 3,3’-dichlorobenzidine is identified, individuals 

should be monitored for gastrointestinal, respiratory, dermal, and neurological effects (as reported 

earlier by Gerarde and Gerarde 1974). 

No studies were located that monitored human tissues for content of 3,3’-dichlorobenzidine or its 

metabolites. 3,3’-Dichlorobenzidine is excreted in urine. If 3,3’-dichlorobenzidine and metabolites can 

be detected and correlated with exposure, it may be possible to correlate urinary levels of 

3,3’-dichlorobenzidine or its metabolites, with systemic effects. 

Biomarkers of Exposure and Effect. 

Exposure. A test system that involves extracting dichlorobenzidine or its metabolite 

(monoacetyldichlorobenzidine) from urine and reacting it with Chloramine-T has been developed to 

screen for dichlorobenzidine exposure in workers (Hatfield et al. 1982). In addition, an amperometric 

method has been developed for the detection of 3,3’-dichlorobenzidine in the urine as a quantitative 

assay for the biological monitoring of occupationally exposed persons to this substance. This method 

is based on the two electron oxidation at carbon electrodes by aromatic diamines (Trippel-Schulte et 

al. 1986). Hemoglobin adducts have been detected in female Wistar rats orally administered single 

doses of 127 or 253 mg/kg 3,3’-dichlorobenzidine (Birner et al. 1990) and to repeated doses of 0.3 

mg/kg/day (Joppich-Kuhn et al. 1997). Birner et al.(1990) suggested that metabolically formed nitroso 

derivatives can result in the formation of a sulfinic acid amide with cysteine residues in hemoglobin. 

Hydrolysis yielded mainly 3,3’-dichlorobenzidine; N-acetylated-3,3’-dichlorobenzidine was also 

detected. This method has not yet been validated in an occupationally exposed population. More 

research is needed to determine if this method is suitable for use 
3,3’-DICHLOROBENZIDINE 73 

2. HEALTH EFFECTS
 

as a biomarker of human exposure to 3,3’-dichlorobenzidine. Further studies to develop simpler, more 

sensitive biomarkers of exposure that are specific for 3,3’-dichlorobenzidine would be useful in 

monitoring exposure of people living near hazardous waste sites containing 

3,3’-dichlorobenzidine. 

Effect. There are no specific disease states in humans or animals that have been associated with 

exposure to 3,3’-dichlorobenzidine. Hemoglobin adducts have been isolated from the blood of 

3,3’-dichlorobenzidine-treated animals (Birner et al. 1990; Joppich-Kuhn et al. 1997). It is not known 

what relationship exists between adduct levels in the blood and 3,3’-dichlorobenzidine toxicity. 

Further research in animal models is needed to determine if these adducts could be correlated with 

effects of 3,3’-dichlorobenzidine exposure. Further studies to identify more sensitive toxic effects 

(noncancer) that are specific for 3,3’-dichlorobenzidine would be useful in monitoring effects in 

people living near hazardous waste sites containing 3,3’-dichlorobenzidine. 

Absorption, Distribution, Metabolism, and Excretion. Available data are insufficient to allow 

accurate evaluation of absorption, metabolism, or persistence of 3,3’-dichlorobenzidine in human 

tissues. Additional studies to identify and quantify metabolites of 3,3’-dichlorobenzidine in humans 

and animals would be useful in establishing the relevance of animal studies in predicting human health 

effects. Metabolic handling of 3,3’-dichlorobenzidine in humans needs to be better characterized 

before urinary levels of the compound or its metabolites can be used to quantitate human exposure. 

Comparative Toxicokinetics. Pharmacokinetics studies have not been performed under conditions 

analogous to those of the carcinogenicity studies. Therefore, it is not possible to determine systemic 

levels of the compound associated with the reported effects. Pharmacokinetics data developed under 

exposure conditions associated with biological effects would markedly increase the possibility of 

improved species extrapolation for evaluating the true potency of 3,3’-dichlorobenzidine. 

Methods for Reducing Toxic Effects. There are no disease states in humans that are associated with 

exposure to 3,3’-dichlorobenzidine. Therefore, studies that further characterize means of assessing 

human exposures (biomonitoring) along with identification of programs designed to minimize this 

exposure would be effective for mitigation of potential effects resulting from accidental exposure in 

occupational settings or exposure to humans living near hazardous waste sites where 

3,3’-dichlorobenzidine might be stored. 
3,3’-DICHLOROBENZIDINE 74 

2. HEALTH EFFECTS
 

Children’s Susceptibility. The information on health effects of 3,3’-dichlorobenzidine in humans is 

derived exclusively from studies of occupational exposure (Gadian 197.5; Gerarde and Gerarde 1974; 

MacIntyre 1975; Myslak et al. 1991; Quellet-Hellstron and Rench 1996). Because of study limitations 

such as simultaneous exposure to other chemicals, no target organ or system has been identified for 

3,3’-dichlorobenzidine. In one occupational study it was reported that contact with the free base 

caused dermatitis (Gerarde and Gerarde 1974); it is reasonable to assume that children will respond in 

a similar manner under similar exposure conditions, although such exposure scenarios for children 

seem unrealistic. There is no information available to determine whether children and adults are 

equally susceptible to the toxic effects of 3,3’-dichlorobenzidine. No studies in animals have 

addressed this issue either, but given the unlikelihood of exposure to 3,3’-dichlorobenzidine by the 

general population, such studies do seem warranted at this time. 

There is no information on whether the developmental process is altered in humans exposed to 3,3’-

dichlorobenzidine. Studies in animals have been inadequate (Golub 1970; Golub et al. 1975; Shabad 

et al. 1972) and further well conducted research would be helpful to clarify whether the developmental 

process can be affected in animals exposed to 3,3’-dichlorobenzidine by a relevant route of exposure. 

This also includes information on whether 3,3’-dichlorobenzidine (or metabolites) can cross the 

placenta and/or be transferred to offspring via breast milk. There are no data to evaluate whether 

harmacokinetics of 3,3’-dichlorobenzidine in children are different from adults. There are no PBPK 

models for 3,3’-dichlorobenzidine, but a need for such a model is not apparent at this time. There is no 

information to evaluate whether metabolism of 3,3’-dichlorobenzidine in children is different than in 

adults, but there are some theoretical reasons to suspect that it might be different. 

Continued research into the development of sensitive and specific biomarkers of exposure and effect 

for 3,3’-dichlorobenzidine, and the validation of these biomarkers in occupationally exposed 

individuals would be valuable. Since at this point there are no validated biomarkers of exposure and 

effect in adults, it makes sense to focus efforts on occupationally exposed adults rather than children 

who are unlikely to be exposed. There are no data on interactions of 3,3’-dichlorobenzidine with 

other chemicals in children or adults. There are no pediatric-specific methods to reduce peak 

absorption for 3,3’-dichlorobenzidine following exposure, to reduce body burdens, or to interfere with 

3,3’-dichlorobenzidine’s mechanism of action, but it is reasonable to assume that exposure avoidance 

measures should be applied to children where needed. 
3,3’-DICHLOROBENZIDINE 75 

2. HEALTH EFFECTS 

Child health data needs relating to exposure are discussed in Section 5.8.1, Data Needs: 

Exposures of Children. 

2.11.3 Ongoing Studies
 

No ongoing studies were located for 3,3’-dichlorobenzidine (FEDRIP 1998).
 

3,3’-DICHLOROBENZIDINE 77 

3. CHEMICAL AND PHYSICAL INFORMATION 

3.1 CHEMICAL IDENTITY
 

Information regarding the chemical identity of 3,3’-dichlorobenzidine is located in Table 3-l.
 

3.2 PHYSICAL AND CHEMICAL PROPERTIES
 

Information regarding the physical and chemical properties of 3,3’-dichlorobenzidine is located in 

Table 3-2. 



3,3’-DICHLOROBENZIDINE 81 

4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

4.1 PRODUCTION 

3,3’-Dichlorobenzidine is commercially produced by reduction of o-nitrochlorobenzene through 

various reduction procedures to form a hydrazo compound, which is rearranged in the presence of 

mineral acids to form 3,3’-dichlorobenzidine (DCMA 1989; Sax 1987). Commercial supplies are 

usually provided in the form of the dihydrochloride salt because of its greater stability. 

According to the 1997 Directory of Chemical Producers (SRI 1997), only one company, Lomac, 

Inc. of Muskegon, Michigan, manufactures (that is, produces) 3,3’-dichlorobenzidine. By 

contrast, in 1986, there were approximately 10 suppliers of the chemical listed in the United 

States (NTP 1994). Current production volumes of 3,3’-dichlorobenzidine for individual 

companies are considered confidential business information and cannot be reported. The United 

States International Trade Commission (USITC 1984a) reported a 1983 production volume of 

3,3’-dichlorobenzidine-based dyes of over 18 million pounds in the United States. However, 

3,3’-dichlorobenzidine is no longer used to manufacture dyes in the United States (CPMA 1998). 

Consumption of 3,3’-dichlorobenzidine in the United States amounted to 9.9 million pounds in 

1987 (Hopmeier 1988). 

Table 4-l lists the facilities in each state that manufacture 3,3’-dichlorobenzidine or process the 

compound for further distribution, the range of maximum amounts of 3,3’-dichlorobenzidine 

on-site, and the activities and uses of the product. “Processing” means the further distribution of 

the compound either as the same physical compound, in a different form or physical state, or as 

part of another article or mixture (40 CFR 372.3). In 1996, there was one facility in the United 

States that manufactured or used 3,3’-dichlorobenzidine. The data listed in Table 4-1 are derived 

from the 1996 Toxics Release Inventory (TR196 1998). Only certain types of facilities were 

required to report. Therefore, this is not an exhaustive list. 

4.2 IMPORT/EXPORT 

Imports of 3,3’-dichlorobenzidine base and salts were 1.1 million pounds in 1983, while pigments 

derived from 3,3’-dichlorobenzidine were about 129,000 pounds in 1983 (USITC 1984b). 

3,3’-DICHLOROBENZIDINE 83 

4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

4.3 USE 

3,3’-Dichlorobenzidine is used primarily in the production of yellow, and some red and orange 

pigments for the printing ink, textile, paper, paint, rubber, plastic, and related industries (EPA 1979a). 

As of 1983, 7 specified pigments were commercially available. The yellow pigments derived from 

3,3’-dichlorobenzidine can be used as substitutes for lead chromate pigments (HSDB 1996). Little, if 

any, dye is prepared from this compound. The chemical also has application as a compounding 

ingredient for rubber and plastics (HSDB 1996), and can be used to test for the presence of gold 

(Searle 1976). 3,3’-Dichlorobenzidine is used in the manufacture of the raw material 

tetraminobiphenyl which is used to produce polybenzimidazole (PBI). PBI fiber is used in many 

protective clothing applications, such as firefighter’s apparel, welder’s garments, high-temperature 

gloves, and crash rescue garments (Celanese 1985). 

3,3’-Dichlorobenzidine is also used with 4,4’-methylenebis (2-chloroaniline) as a curing agent for 

liquidcastable polyurethane elastomers (HSDB 1996). 

4.4 DISPOSAL 

3,3’-Dichlorobenzidine is treated in the workplace as a controlled substance under OSHA. Therefore, 

strict requirements have been made to minimize exposure to the chemical in the workplace air and 

contact with the skin and eyes. Nonetheless, some releases may occur in wastewater effluents. 

One company which purchases 3,3’-dichlorobenzidine as the dihydrochloride salt in sealed fiber in 

drums rinses the empty drums with water, adds the rinse water to the product stream, then sprays the 

drums with a sodium hypochlorite bleach solution (converting the 3,3’-dichlorobenzidine to a 

quinone-type compound), and places them in polyethylene bags for disposal (London and Boiano 

1986). 

3,3’-Dichlorobenzidine is listed as a toxic substance 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 3,3’­

dichlorobenzidine is controlled by a number of federal regulations (see Chapter 7). The current 

recommended technologies specified for treating 3,3’-dichlorobenzidine-containing wastes (waste 

waters and nonwastewaters) prior to land disposal include wet air oxidation, chemical or 

electrolytic oxidation, and carbon adsorption and incineration (EPA 
3,3’-DICHLOROBENZIDINE 84 

4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 

1986). Facilities which generate 3,3’-dichlorobenzidine-containing wastes, and owners and 

operators of hazardous waste treatment, storage, and disposal facilities must also comply with 

regulations promulgated under the authority of the Resource Conservation and Recovery Act 

(RCRA). 
3,3’-DICHLOROBENZIDINE 85 

5. POTENTIAL FOR HUMAN EXPOSURE 

5.1 OVERVIEW 

3,3’-Dichlorobenzidine is currently used in the production of insoluble dyes and pigments. 

Almost all 3,3’-dichlorobenzidine is now manufactured outside the United States and is imported 

for on-site processing or for use as a reactant to synthesize pigments. “Processing” means 

preparing a chemical after its manufacture for commercial distribution either as the same physical 

compound, in a different form or physical state, or as part of another article (for instance, a 

mixture) containing the chemical (40 CFR 372.3). 

Use of the compound to synthesize soluble dyes ceased as of 1986, when better dyes from other 

sources were introduced. The distinction between dyes and pigments is not always clear. 

Pigments are almost without exception insoluble and exist as finely divided solid powders that are 

insoluble but wettable under the conditions of use. Dyes are almost always soluble organic 

substances used in coloring textiles or other fibrous substances. 

Release routes of 3,3’-dichlorobenzidine to the environment appear to be waste waters, sludges, 

and solid wastes where emissions are not properly controlled during the use of 

3,3’-dichlorobenzidine or during its chemical transformation to pigments. The compound has 

been found in water and soil at hazardous waste sites, a result of the improper land disposal of 

solid wastes. 

Concern for human health derives primarily from inhalation of airborne dust or skin contact 

during careless handling or accidental spillage in occupational settings or drinking of 

contaminated well water by persons living in the proximity of hazardous waste sites. However, 

occupational case reports suggest that risk to workers exposed to 3,3’-dichlorobenzidine through 

the use of 3,3’-dichlorobenzidine-based pigments may be minimal. No adverse health effects 

were reported among 20 workers engaged in the manufacture and handling of 

3,3’-dichlorobenzidine alone (concentration not specified) in a Japanese facility (DCMA 1989). 

No detectable levels of 3,3’-dichlorobenzidine or its monoacetyl metabolite (at a detection limit 

of 0.2 ppb) were seen in urine samples of workers who were exposed to pigments derived from 

3,3’-dichlorobenzidine on the day the samples were collected (Hatfield et al. 1982). The urine 

analysis results for workers with 
3,3’-DICHLOROBENZIDINE 86 

5. POTENTIAL FOR HUMAN EXPOSURE
 

high potential for pigment exposure suggest that these pigments are not metabolized in humans 

although, without pigment exposure data, this conclusion is somewhat tentative. 

The hydrochloric acid salt of 3,3’-dichlorobenzidine readily photolyses in water exposed to natural 

sunlight, but may not readily biodegrade in soil and acclimated sludges. It has a strong tendency to 

partition to soils and sediments, a property which reduces the potential for human exposure (Boyd et 

al. 1984; Chung and Boyd 1987; Sikka et al. 1978). Once partitioned to soil, the compound apparently 

binds further with humic substances to form humic-like materials that presumably would be 

non-hazardous (Sikka et al. 1978). However, in a recent paper, Nyman et al. (1997) stated that 

dehalogenation of 3,3’-dichlorobenzidine to form benzidine (also a toxic substance) occurs in 

sediment/water mixtures under anaerobic conditions. The compound does not volatilize or hydrolyze 

in solution, but it may slowly oxidize (Banerjee et al. 1978; Callahan et al. 1979). 

3,3’-Dichlorobenzidine may be bioconcentrated by aquatic organisms (Appleton and Sikka 1980), but 

it is not certain if it is biomagnified by transfer through the food chain. 3,3’-Dichlorobenzidine 

accumulates in freshwater fish during aquatic exposure to either 5 ppb or 0.1 ppm concentrations of 

the chemical. After returning the fish to fresh, uncontaminated water, clearance of the compound from 

edible flesh was initially rapid (half-life of approximately 48 hours), but residues remained even after 

14 days (Appleton and Sikka 1980). Steady-state concentrations in fish from ambient (unspiked) water 

exposures would be expected to be very low. 

The reductive cleavage in vivo of azo dyes in general was first observed by Rinde and Troll (1975). 

Since then, several research groups have published articles that relate to the potential for human 

exposure to 3,3’-dichlorobenzidine that might arise via various chemical and biochemical mechanisms 

that degrade 3,3’-dichlorobenzidine-based synthetic dyes. A study by Hoffman and Schmidt (1993) 

found no evidence for metabolic cleavage of Pigment Yellow 17 to produced 3,3’-dichlorobenzidine 

in rats that inhaled the pigment. However, Zwirner-Baier and Neumann (1994), based on analysis of 

hemoglobin adducts from rats that drank the pigments, concluded that intestinal cleavage processes 

release very small amounts of 3,3’-dichlorobenzidine from Pigment Yellow 17 and Direct Red 46 

(0.6% and 3%, respectively, of the total dose administered over 4 weeks). In another study 

(Sagelsdorff et al. 1996), the lack of appearance of 3,3’-dichlorobenzidine from Pigment Yellow 13 

and 17 is shown, but a marked formation of 3,3’-dichlorobenzidine occurs from a soluble azo dye, 

C. I. Direct Red 46, which was an impurity in the pigments they studied. 
3,3’-DICHLOROBENZIDINE 87 

5. POTENTIAL FOR HUMAN EXPOSURE
 

In metabolism studies of azo dyes and pigments in the hamster, in viva cleavage of the benzidinebased dye, Direct Black 38, to benzidine was shown by analysis of the urine. However, studies of the 

3,3’-dichlorobenzidine-based pigment, Pigment Yellow 12, showed no evidence for in vivo cleavage 

to release 3,3’-dichlorobenzidine (Nony et al. 1980). 

3,3’-Dichlorobenzidine has been identified in at least 32 of the 1,467 current or former EPA National 

Priorities List (NPL) hazardous wastes sites (HazDat 1998). However, the number of sites evaluated 

for 3,3’-dichlorobenzidine is not known. The frequency of these sites within the United States can be 

seen in Figure 5-l. The manufacture and use of 3,3’-dichlorobenzidine has been strictly regulated by 

OSHA since 1974. All work with the compound is done in closed systems and any residues are 

destroyed by chemical reaction. Such precautions, if conscientiously practiced, make it unlikely that 

significant quantities of 3,3’-dichlorobenzidine have been disposed of in landfills or at NPL sites after 

1974. 

NPL Superfund Records of Decision (RODS) were located for 24 of the 27 currently listed NPL sites 

where the HazDat database lists 3,3’-dichlorobenzidine as a contaminant. A ROD is a legally binding 

document that states the results of investigation and feasibility testing at hazardous waste sites and 

tells what techniques will be used to remediate the site. At four of the sites, 3,3’-dichlorobenzidine 

was verified as a contaminant. The RODS for the other 20 sites did not mention 3,3’­

dichlorobenzidine as a contaminant of concern (i.e., one that warrants development of cleanup criteria 

and a choice of remedy). Affected soil was removed from three of the four contaminated sites. Only 

one site, Bofors Nobel in Michigan, required development of a cleanup criteria (CPMA 1998). 

5.2 RELEASES TO THE ENVIRONMENT 

According to the Toxics Release Inventory (TRI), in 1996, a total of 2 pounds (1 kg) of 3,3’­

dichlorobenzidine was released to the environment from one processing facility (TR196 1998). 

Table 5-l lists amounts released from this facility. In addition, an estimated 250 pounds (118 kg) 

were released by manufacturing and processing facilities to publicly owned treatment works 

(POTWs), and an estimated 51,550 pounds (23,432 kg) were transferred offsite (TR196 1998). 

The TRI data should be used with caution because only certain types of facilities are required to 

report. Therefore, this is not an exhaustive list. 

3,3’-DICHLOROBENZIDINE 89 

5. POTENTIAL FOR HUMAN EXPOSURE
 

3,3’-Dichlorobenzidine has been identified in a variety of environmental media (air, surface 

water, groundwater, soil, and sediment) collected at 32 of the 1,467 current or former NPL 

hazardous waste sites (HazDat 1998). The frequency of these sites within the United States can be 

seen in Figure 5-l. 

5.2.1 Air 

The free base form of 3,3’-dichlorobenzidine is no longer utilized by industry in the United 

States. It is primarily supplied as the dihydrochloride salt (CPMA 1998). When it was used as the 

free base, it was handled as a powder or a moist paste (NIOSH 1980). 3,3’-Dichlorobenzidine is 

not a volatile chemical. A vapor pressure of 4.5x10x-9 mm Hg at 20 oC has been reported (DCMA 

1989). Prior to OSHA 1974 regulations, benzidine and 3,3’-dichlorobenzidine were manufactured 

in open systems that permitted atmospheric releases of suspended particles at the work site 

(Shriner et al. 1978), but no historical data were located specifically for 3,3’-dichlorobenzidine 

emissions (atmospheric or in water). The absence of data may be attributed to analytical methods 

used at that time that could not distinguish benzidine from its derivatives or many other aromatic 

amines (Shriner et al. 1978). Under OSHA regulations adopted in 1974, only closed 

manufacturing systems are permitted, and atmospheric emissions are presumably reduced 

because of this regulation. 

Estimated releases of 2 pounds (0.9 kg) of 3,3’-dichlorobenzidine to the atmosphere from one 

facility in 1996, accounted for 100% of the estimated total environment releases (TR196 1998). 

These releases are summarized in Table 5-1. The TRI data should be used with caution because 

only certain types of facilities are required to report information to the Toxics Release Inventory 

only if they employ more than 10 full-time employees, if their facility is classified under Standard 

Industrial Classification (SIC) codes 20 through 39, and if their facility produces, imports, or 

processes 25,000 or more pounds of any TRI chemical or otherwise used more than 10,000 

pounds of a TRI chemical in a calendar year (EPA 1997). A member company of the Color 

Pigment Manufacturers Association, Inc., which monitors 3,3’-dichlorobenzidine under state 

regulations, reports that only de minimus values are found (CPMA 1998). 

3,3’-Dichlorobenzidine was not identified in any air samples collected at any of the 32 NPL 

hazardous waste sites where it was detected in some other environmental media (HazDat 1998). 

3,3’-DICHLOROBENZIDINE 91 

. 

5. POTENTIAL FOR HUMAN EXPOSURE 

5.2.2 Water 

The free base form of 3,3’-dichlorobenzidine is sparingly soluble in water. The solubility of 

3,3’-dichlorobenzidinem-2HCl in water is 4 mg/L at a pH of 6.9 (Banerjee et al. 1978). A 

solubility of 3.1 mg/L is also quoted (CPMA 1998). 3,3’-Dichlorobenzidine may be released into 

the environment in waste waters generated by the production of dyes and pigments. 

No releases of 3,3’-dichlorobenzidine to the surface water were reported in 1996 (TR196 1998). 

Two hundred and fifty pounds (550 kilograms) were released to publicly owned treatment works 

(POTWs) (TR196 1998). These releases are summarized in Table 5-1. The TRI data should be 

used with caution because only certain types of facilities are required to report information to the 

Toxics Release Inventory only if they employ more than 10 full-time employees, if their facility 

is classified under Standard Industrial Classification (SIC) codes 20 through 39, and if their 

facility produces, imports, or processes 25,000 or more pounds of any TRI chemical or otherwise 

used more than 10,000 pounds of a TRI chemical in a calendar year (EPA 1997). As a result of 

secondary treatment processes in POTWs, only a small percentage of any 3,3’-dichlorobenzidine 

that might enter POTWs is subsequently released into surface water. 

3,3’-Dichlorobenzidine has been identified in surface water and groundwater samples collected at 

19 of the 32 NPL hazardous waste sites where it was detected in some other environmental media 

(HazDat 1998). 

5.2.3 Soil 

According to the Toxics Release Inventory, in 1996, there were no reported releases of 3,3’­

dichlorobenzidine to soil from any large processing facilities (TR196 1998). The TRI data should 

be used with caution because only certain types of-facilities are required to report information to 

the Toxics Release Inventory only if they employ more than 10 full-time employees; if their 

facility is classified under Standard Industrial Classification (SIC) codes 20 through 39; and if 

their facility produces, imports, or processes 25,000 or more pounds of any TRI chemical or 

otherwise used more than 10,000 pounds of a TRI chemical in a calendar year (EPA 1997). 
3,3’-DICHLOROBENZIDINE 92 

5. POTENTIAL FOR HUMAN EXPOSURE
 

3,3’-Dichlorobenzidine has been identified in soil and sediment samples collected at 18 of the 32 NPL 

hazardous waste sites where it was detected in some environmental media (HazDat 1998). 

5.3 ENVIRONMENTAL FATE 

Because 3,3’-dichlorobenzidine adsorbs to airborne dust particles or is otherwise bound to particulate 

matter, it is subject to dispersion, gravitational settling, and wash-out by rain. In water, 

3,3’-dichlorobenzidine is sparingly soluble, does not volatilize or hydrolyze, and may slowly oxidize 

in solution (Banerjee et al. 1978; Callahan et al. 1979; Mabey et al. 1982). 3,3’-Dichlorobenzidine 

may be strongly adsorbed to soils, clays, and sediments, depending on the pH of the soil-water system. 

It may be strongly bound by soil organic matter (Boyd et al. 1984; Chung and Boyd 1987; Sikka et al. 

1978). Although earlier research indicates that the compound does not appear to be readily 

biodegradable in soil or waste water sludges, recent work by Nyman (Nyman et al. 1997) indicates 

that more than 80% of 3,3’-dichlorobenzidine may be microbially degraded to benzidine under 

anaerobic conditions. 3,3’-Dichlorobenzidine is bioconcentrated by aquatic organisms under 

experimental conditions (Appleton and Sikka 1980), but it is not certain if it is bioaccumulated or 

transferred through the natural food chain. 

5.3.1 Transport and Partitioning 

In the atmosphere, 3,3’-dichlorobenzidine stays attached to dust particles or bound to particulate 

matter. As such, suspended 3,3’-dichlorobenzidine is subject to atmospheric convection, dispersion, 

gravitational settling, and wash-out by rain. 

The Henry’s law constant for a compound is useful in estimating the partitioning of the compound 

between its vapor phase and aqueous media. At 25 oC, a value of 5.11x10-11 atm-m3

/mole has been 

estimated (SRC 1994). This very low value suggests that 3,3’-dichlorobenzidine essentially remains 

dissolved in water, and does not migrate from water into air. 

3,3’-Dichlorobenzidine in solution has a strong tendency to be adsorbed onto soils and sediments. The 

extent of adsorption of hydrophobic (sparingly water soluble) compounds has been shown to be highly 

correlated with the organic carbon content of the adsorbents (Hassett et al. 1983). When adsorption is 

expressed as a function of organic carbon content, an organic carbon-water partition coefficient (Koc) 

is 
3,3’-DICHLOROBENZIDINE 93 

5. POTENTIAL FOR HUMAN EXPOSURE
 

generated, which is a unique property of the compound and may be used to rank the relative mobility 

of organic contaminants in saturated soil-water systems. A Koc value for 

3,3’-dichlorobenzidine of 1,553 (based on an octanol-water partition coefficient (Kow) of 3,236) was 

calculated by Mabey et al. (1982). This relatively high value implies that 

3,3’-dichlorobenzidine would exhibit “low” mobility in soil (see Roy and Griffin 1985). However, 

3,3’-dichlorobenzidine is not strictly a hydrophobic compound but can exist as a weak base in water, 

and exists in both neutral and cationic forms. Written as an acid-base reaction, the amine groups may 

be protonated as follows: 

3,3’-DCB +H2O ↔ 3,3’-DCBH+

 + OH3,3’-DCBH’ + Hz0 ↔ 3,3’-DCBH2

2+ + OHpKa values reported for the conjugate acids (DCBH’ and DCBH,“) vary somewhat. Sikka (Sikka et al. 

1978) and Boyd (Boyd et al. 1984) reported that they are <4. Nyman (Nyman et al. 1997) reported 

pKa,1 and pKa,2 values of 1.6 and 3.2, respectively. Thus, in the pH range of most environmental 

situations (pH 6-8) the dominant state of 3,3’-dichlorobenzidine in water would be the non-ionic form. 

As pH increases, the proportion of cationic forms of 3,3’-dichlorobenzidine decreases, and the extent 

of adsorption to sediments via Coulombic interactions would also decrease and 3,3’-dichlorobenzidine 

adsorption would be dominated by hydrophobic processes. This expectation was demonstrated by 

Sikka and coworkers (Sikka et al. 1978), who found that the adsorption constant (Kf ) decreased with 

increasing pH; the decrease was more rapid in the range of pH 7-9. The adsorption data conformed to 

the Freundlich equation, Ca = KfCs

1/n where Ca is the concentration of 3,3’-dichlorobenzidine adsorbed 

per mass of adsorbent, and Cs is the equilibrium concentration of 3,3’-dichlorobenzidine in solution. 

Kf and l/n are empirically derived constants. No correlation was found between Kf and the organic 

carbon content of the sediments (Boyd et al. 1984; Sikka et al. 1978). Similarly, the extent of 

benzidine adsorption does not correlate to the organic carbon content of soils and sediments (Graveel 

et al. 1986; Zierath et al. 1980). It was concluded that nonprotonated 3,3’-dichlorobenzidine is subject 

to hydrophobic bonding to some extent (Boyd et al. 1984). It is clear from these studies that 

adsorption constants for 3,3’-dichlorobenzidine cannot be accurately predicted for a given soil based 

only on a Koc value. 

The adsorption of 3,3’-dichlorobenzidine by soils and sediments may not be readily reversible (Boyd 

et al. 1984; Chung and Boyd 1987; Sikka et al. 1978). The extent of 3,3’-dichlorobenzidine desorption 

decreased with an increase in the age of the sample. Also, the adsorbed 3,3’-dichlorobenzidine was 
3,3’-DICHLOROBENZIDINE 94 

5. POTENTIAL FOR HUMAN EXPOSURE
 

resistant to extraction. After 24 hours of 3,3’-dichlorobenzidine-sediment contact, only 36% of the 

parent compound could be extracted by methanol. It is speculated that 3,3’-dichlorobenzidine forms 

covalent bonds with soil humic components (Sikka et al. 1978; Boyd et al. 1984). Experiments have 

indicated that covalent binding of ring-substituted anilines to humates is not a readily reversible 

reaction (Parris 1980). 3,3’-Dichlorobenzidine was highly immobile in soil column experiments 

(Chung and Boyd 1987). Water was passed through sandy soil (Entic Haplorthod) and 

3,3’-dichlorobenzidine-contaminated sewage sludge samples. Only small amounts of radioactive 

3,3’-dichlorobenzidine added to columns of sandy soil or sewage sludge were eluted with water over 

extended time periods. Extractable radioactivity from these soils and sludge samples decreased with 

time of chemical contact. There was greater adsorption of 3,3’-dichlorobenzidine to soil than to 

sludge, apparently as a result of the greater humus content of the soil samples, which suggested that 

the compound may favor migration from sludge to soil substrates (Chung and Boyd 1987). 

Since 3,3’-dichlorobenzidine is lipophilic, it may be concentrated from aqueous media by aquatic 

organisms. Bluegill sunfish were exposed to radiolabeled 3,3’-dichlorobenzidine in dynamic-flow 

experiments for 130-168 hours (Appleton and Sikka 1980) . Moderately low bioconcentration factors 

(BCF) of 495-507 were calculated for the whole fish. BCFs in fish (golden ide) of 610 and in green 

algae of 940 have been reported (Freitag et al. 1985). A BCF in edible portions of bluegill sunfish of 

114-170 has also been reported (EPA 1980b). Bioaccumulation by plants or terrestrial animals has not 

been studied. Assuming a log Kow (range, 3.02-3.78) (DCMA 1989; Mabey et al. 1982) 

3,3’-dichlorobenzidine is not likely to bioaccumulate appreciably. However, Law states that some 

bioaccumulation in aquatic organisms might be expected (Law 1995). The flesh of freshwater fish 

exposed to 5 ppb or 0.1 ppm concentrations of the chemical in water showed some accumulation. 

After returning the fish to clean water, clearance of the compound was rapid (a half-life of 

approximately 48 hours), but residues remained even after 14 days (Appleton and Sikka 1980). 

5.3.2 Transformation and Degradation 

5.3.2.1 Air 

3,3’-Dichlorobenzidine in the sunlit, ambient air atmosphere may react with photochemically 

produced hydroxyl radicals and ozone, but there are no quantitative data on reaction rates. The 

persistence of “all 
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5. POTENTIAL FOR HUMAN EXPOSURE
 

benzidines” in the atmosphere has been estimated by assuming a hydroxyl radical concentration 

of 8x10-10 mole/L (an average value in a 24-hour day-night cycle) (EPA 1975). Treating the 

oxidation process as a first-order reaction, the rate constant was 7.2x1012/mole-hour and the 

corresponding half-life was 12 hours. This estimation approach was based on data on the rates of 

reaction of hydroxyl radicals with olefins, aromatics, and alkanes in the atmosphere. The 

estimated half-life of 3,3’-dichlorobenzidine in air has ranged from 1 to 60 days (EPA 1980b; 

Shriner et al. 1978). The most recently published value for the degradation half-life in air via 

reaction with OH radicals is 9.7 hours (SRC 1995a). The reason for this disparity among the halflife estimates is not known. No other information on the fate of 3,3’-dichlorobenzidine in the 

atmosphere was located. 

5.3.2.2 Water 

The limited information that is available suggests that 3,3’-dichlorobenzidine may photolyze in 

water to yield benzidine, which is more photostable yet still toxic. It does not appear that the 

chemical is susceptible to any other transformations in water except protonation by acid-base 

reactions. 

There are no data to suggest that the hydrolysis of 3,3’-dichlorobenzidine is significant (Callahan 

et al. 1979). A hydrolysis rate constant of 0/mole-hour for 3,3’-dichlorobenzidine has been 

proposed (Mabey et al. 1982). 

It has been speculated that aqueous solutions of aromatic amines can be oxidized by organic 

radicals, but there are no actual data on reaction rates. Based on a study of reaction rate data for 

compounds with structures similar to 3,3’-dichlorobenzidine, an estimate of the half-life of 

aromatic amines in water is approximately 100 days, assuming a peroxy radical concentration of 

10-10 mole/L in sunlit, oxygenated water (EPA 1975). Based on the oxidation rates of similar 

compounds, the direct oxidation of 3,3’-dichlorobenzidine by singlet oxygen in solution may be 

treated as a first-order reaction, to arrive at an estimated reaction constant of <4x107

/mole-hour 

(Mabey et al. 1982). The oxidation rate constant with peroxy radicals was estimated to be 

approximately 4x107

/mole-hour. However, no information was located that demonstrates that 

3,3’-dichlorobenzidine is significantly oxidized in water. 

3,3’-Dichlorobenzidine was found to be extremely photolabile in water (Sikka et al. 1978; 

Banerjee et al. 1978). 3,3’-Dichlorobenzidine photolyzed yielding monochlorobenzidine, 

benzidine, and a number of 
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5. POTENTIAL FOR HUMAN EXPOSURE
 

colored, water-insoluble products. In natural sunlight, the half-life of 3,3’-dichlorobenzidine in 

water was determined to be approximately 90 seconds. While 3,3’-dichlorobenzidine is very 

rapidly photolyzed under environmental conditions, the process may yield benzidine, a relatively 

photostable carcinogen (Banerjee et al. 1978). 

3,3’-Dichlorobenzidine in lake water samples was not metabolized by microorganisms over a 

4-week period (Sikka et al. 1978) although 1 lake sample of the 2 tested contained approximately 

5 million microorganisms per mL. The composition of the biological community was not 

described. Minor decreases in 3,3’-dichlorobenzidine concentrations were attributed to adsorption 

onto suspended sediment. 

5.3.2.3 Sediment and Soil 

Earlier reports gave little indication that 3,3’-dichlorobenzidine is significantly degraded in soil or 

that it is transformed to other substances. More recent research (Nyman et al. 1997) reports that 

sediment/water mixtures spiked with 3,3’-dichlorobenzidine display evidence of the chemical’s 

degradation. In the experiments reported by these authors, silty-clay to sandy sediments collected 

from a lake near Holland, Michigan, were spiked with 3,3’-dichlorobenzidine and incubated at 20 

°C for 12 months under anaerobic conditions. Time-course analysis of this mixture showed that 

dehalogenation of 3,3’-dichlorobenzidine to produce benzidine appears to take place through a 

transient intermediate, 3-monochlorobenzidine. Up to 80% of the 3,3’-dichlorobenzidine was 

transformed to benzidine over a l-year incubation period. No metabolites were observed in 

autoclaved samples, suggesting that dehalogenation is mediated by microbial activity. The final 

product, benzidine, shows more affinity for the solution (aqueous) phase and thus has a 

greater potential for transport in the environment. 

Unsubstituted benzidine may be oxidized at clay surfaces when mixed with some types of clay 

minerals (Tennakoon et al. 1974; Theng 197 1). Benzidine is oxidized to a monovalent radical 

cation by iron (III) in the silicate lattice and by aluminum at crystal edges. However, there is no 

experimental evidence that demonstrates that 3,3’-dichlorobenzidine is subject to the same type 

of surface oxidation at solid-liquid interfaces. 
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5. POTENTIAL FOR HUMAN EXPOSURE
 

Activated sludge did not degrade 3,3’-dichlorobenzidine after weekly subculturing. The sludge 

was not described or chemically characterized. Observed decreases in 3,3’-dichlorobenzidine 

concentration were attributed to adsorption by the sludge. 

The results of seven laboratories conducting aerobic biodegradation experiments with 

3,3’-dichlorobenzidine have been summarized (Brown and Laboureur 1983). There was a clear 

dependence of the extent of degradation on the concentration of yeast extract added to the batch 

containers. The role of the extract was uncertain, but without it, no degradation was detected. The 

authors hypothesize that the yeast may be a food source to allow buildup of large concentrations 

of active bacteria that are able to break down the amines. The authors felt that these results 

showed the “inherent biodegradability” of 3,3’-dichlorobenzidine, but that the compound should 

not be classified as “readily biodegradable” (Brown and Laboureur 1983). Possible degradation 

mechanisms and degradation by-products were not discussed. 

3,3’-Dichlorobenzidine degraded very little when incubated with soil. In a study by Boyd et al. 

(1984), a Brookston clay loam soil (a typic Argiaquoll fine loamy, mixed mesic) containing 

[

14C]-3,3’-dichlorobenzidine at concentrations of 40 and 4 mg/kg of dry soil was incubated 

aerobically and anaerobically in batch experiments (Boyd et al. 1984). Under aerobic conditions, 

3,3’-dichlorobenzidine degradation occurred at a very slow rate; accumulative 14CO2 production 

was approximately 2% after 32 weeks. Under anaerobic conditions, no gas evolution was 

detected after 1 year of incubation. The authors did not comment on the population or type of 

microorganisms in the soil sample (Boyd et al. 1984). Additional studies indicated that 3,3’­

dichlorobenzidine was very persistent in soil and sludge-amended soil (Chung and Boyd 1987). 

Biodegradation of [14C]-3,3’-dichlorobenzidine was evaluated during a 182-day incubation period 

in a sandy soil (Entic Haplorthod) amended with sewage sludge. The total amount of 

[

14C]-3,3’-dichlorobenzidine recovered as 14CO2 was <2%. It should be noted that biodegradation 

when measured by 14CO2 evolution may provide a conservative estimate of the extent of 

decomposition. This technique does not account for carbon that is incorporated into the biomass 

or into soil organic matter, or for the compound being only partially metabolized (Graveel et al. 

1986). The disparity between the results of this work and the results of Nyman (Nyman et al. 

1997) is probably related to the nature of their respective biotic communities. 
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5. POTENTIAL FOR HUMAN EXPOSURE 

5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT 

Reliable evaluation of the potential for human exposure to 3,3’-dichlorobenzidine depends in part 

on the reliability of supporting analytical data from environmental samples and biological 

specimens. In reviewing data on 3,3’-dichlorobenzidine levels monitored or estimated in the 

environment, it should also be noted that the amount of chemical identified analytically is not 

necessarily equivalent to the amount that is bioavailable. The analytical methods available for 

detection and measurement of 3,3’-dichlorobenzidine are detailed in Chapter 6. 

3,3’-Dichlorobenzidine was not detected in the ambient air at production facilities at detection 

limits of 0.1-5.0 ng/m3

 (Narang et al. 1982; Riggin et al. 1983). The median concentration of 

3,3’-dichlorobenzidine in waste effluents (<10 ppb), groundwater (<10 ppb), surface water (<10 

ppb), and soils (<1 ppb) is very low, although significant contamination may be associated with 

hazardous waste sites (Staples et al. 1985). Moreover, the production and use of 

3,3’-dichlorobenzidine-based dyes has decreased to zero over the last 30 years, while 

environmental and health regulations have been implemented to reduce the release of 

3,3’-dichlorobenzidine to the environment. 

5.4.1 Air 

3,3’-Dichlorobenzidine does not naturally occur in the environment (IARC 1982a). 

3,3’-Dichlorobenzidine was not detected in ambient air of two dyestuff production plants at 

detection limits of 5 (Narang et al. 1982) and 0.1 ng/m3

 (Riggin et al. 1983). More recent data on 

occupational exposure levels indicate the presence of levels ≤0.6-2.5 μg/m3

 in 

3,3’-dichlorobenzidine production and pigment manufacturing plants in Germany (DCMA 1989). 

The concentration of 3,3’-dichlorobenzidine in the Canadian environment was estimated by 

Liteplo and Meek (1994) by applying the Level III Fugacity Computer Model of Mackay and 

Paterson (Mackay and Paterson 1991). Assuming that 1% of the total amount produced in and 

imported to Canada is released into various media in proportions similar to those given in the 

U.S. TRI, the average concentration of 3,3’-dichlorobenzidine in air, as estimated by the model, is 

7.6x10-16 μg/m3

. 
3,3’-DICHLOROBENZIDINE 99 

5. POTENTIAL FOR HUMAN EXPOSURE 

5.4.2 Water 

EPA’s computerized water quality database (STORET) was used to determine the median 

concentration of 3,3’-dichlorobenzidine in surface water, groundwater, and municipal and 

industrial inflow and outflow (Staples et al. 1985). The median concentration of 

3,3’-dichlorobenzidine detected in 12 of 1,239 samples of waste effluent collected from about 

1980 to 1984, was reported to be <10 ppb. The median concentration of 3,3’-dichlorobenzidine in 

both surface and groundwater was also reported to be <10 ppb. The EPA reported that water 

samples collected from drinking-water wells near a waste disposal lagoon that contained 

3,3’-dichlorobenzidine-manufacturing wastes had concentrations of the chemical ranging from 

0.13 to 0.27 ppm (EPA 1980b). EPA indicated that 3,3’-dichlorobenzidine concentrations in 

waste waters from metal finishing operations were 0.07 ppb or less (EPA 1983c). Discharge 

concentrations from other industrial sources were at most 10 ppb. Using a Fugacity Computer 

Model, Liteplo and Meek estimated the concentration of 3,3’-dichlorobenzidine in Canadian 

water to be 3.4x10-7 ng/L (Liteplo and Meek 1994). Because the model does not address the 

possibility of bound residue in sediment, the concentration in water is certainly overestimated. 

Capillary gas chromatography/mass spectrometry (GC/MS) was used to identify, but not 

quantify, 3,3’-dichlorobenzidine in the dissolved phase (that is, smaller particles and dispersed 

colloids not retained by the filter) of water concentrates from the Besos River in Spain (Grifoll et 

al. 1992). Valls et al. identified 3,3’-dichlorobenzidine in urban wastewater in the same region 

(Valls et al. 1990). 

5.4.3 Sediment and Soil 

The estimated median concentration of 3,3’-dichlorobenzidine in sediments in the United States 

has been reported to be <l ppm on a dry sediment basis (Staples et al. 1985). Of the 34 sediment 

or soil measurements recorded in the STORET database, none of the samples contained 

detectable concentrations of 3,3’-dichlorobenzidine. 

5.4.4 Other Environmental Media 

There is a potential for 3,3’-dichlorobenzidine to occur in waste water sludges and industrial solid 

wastes. A 3,3’-dichlorobenzidine concentration of 16 ppm in municipal sludge from Michigan 

has been reported 
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5. POTENTIAL FOR HUMAN EXPOSURE
 

(Chung and Boyd 1987). 3,3’-Dichlorobenzidine was detected at concentrations of 3.13 mg/kg 

dry sewage sludge in 2 of a total of 253 sewage treatment plants examined (Fricke et al. 1985). 

These plants were all in the United States (Arizona, Indiana, Michigan, Missouri, New Mexico, 

New York, and Texas). Concentrations up to 535 μg/L were detected in a communal sewage 

treatment plant (Lopez-Avila et al. 1981). The chemical was detected at 8.55 mg/kg in sewage 

sludge of an aeration basin in Muskegon, Michigan (Demirjian et al. 1984). 

Because the chemical has no agricultural or food chemical application, it is very unlikely that 

3,3’-dichlorobenzidine occurs in food in general. [14C]-3,3’-Dichlorobenzidine was found to 

rapidly accumulate in bluegill sunfish as a result of their exposure to water in which either 5 or 

100 μg/L of the chemical was intentionally added. Residues were distributed in both the edible 

and nonedible portions (Appleton and Sikka 1980). However, 3,3’-dichlorobenzidine was not 

detected in fish samples obtained from rivers near nine textile dyestuff manufacturers known to 

use 3,3’-dichlorobenzidine-based pigments (Diachenko 1979). 

5.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE 

Years ago, benzidine and its congeners such as 3,3’-dichlorobenzidine were likely to be found 

only in the vicinity of pigment plants (EPA 1980b; Shriner et al. 1978) where wastes may escape 

or be discharged. 3,3’-Dichlorobenzidine may also be found in locations where it is used in 

formulating other products such as rubber and plastic (HSDB 1996) or in producing 

polybenzimidazole (PBI) (Celanese 1985). However, 3,3’-dichlorobenzidine is no longer used to 

manufacture soluble dyes in the United States (CPMA 1998). Based on available data, the 

potential for nonindustrial exposure via air, soil, or water is expected to be negligible. The 

greatest chance of exposure by the general public is from the improper land disposal of 

compounds. The significance of this exposure route can only be evaluated on a site-by-site basis. 

No uses of 3,3’-dichlorobenzidine in commonplace consumer products are known. In the past, the 

general public may have been exposed to minute amounts of 3,3’-dichlorobenzidine during the 

use of pressurized spray containers of paints, lacquers, and enamels containing traces of 

benzidine yellow, a pigment derived from 3,3’-dichlorobenzidine (Shriner et al. 1978). 

3,3’-Dichlorobenzidine-based pigments are normally used in printing ink applications; their use 

in paints is rare and, thus, its presence in present-day pressurized paint spray would not be 

expected (CPMA 1998). 
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5. POTENTIAL FOR HUMAN EXPOSURE 

Today the most likely possibilities for occupational exposure exist in the processing of 

3,3’-dichlorobenzidine in the synthesis of pigments, the compounding of PBI, and for workers in the 

garment, leather, printing, paper, and homecraft industries where benzidine-based pigments are used. 

However, there appears to be no information available on current levels of occupational exposure in 

the United States. Since 1974, OSHA regulations have set strict standards for worker protection, 

required the use of closed manufacturing vessels, and prescribed methods to chemically destroy 

residues. Although there is limited evidence for in vivo cleavage of 3,3’-dichlorobenzidine-derived 

pigments to free 3,3’-dichlorobenzidine in animals, urinary tract data from pigment workers suggest 

that 3,3’-dichlorobenzidine-derived pigments are not significantly metabolized in humans. Less than 

0.2 ppb of 3,3’-dichlorobenzidine was detected in urine samples of 36 workers exposed to pigments 

derived from the compound (Hatfield et al. 1982). 

In Canada, the estimated daily intake of 3,3’-dichlorobenzidine by various segments of the population 

has been calculated. The calculations are based on the predicted levels of 3,3’-dichlorobenzidine in 

air, water, and soil, as well as on the estimated daily intake of each (air, water, soil) by Canadians 

(Government of Canada 1993). The predicted concentrations or human intake levels are not measured 

values but rather predicted values based on output from mathematical models using worst-case 

assumptions that do not take into consideration removal mechanisms such as photolysis, oxidation, or 

irreversible binding to substrates. The total intake by adults (20 or more years of age) is predicted to 

be 7.4x10-9 ng/kg body weight/day. For infants up to 6 months of age (the group with the greatest 

predicted exposure on the basis of body weight), the total intake is estimated at 3.6x10-8 ng/kg body 

weight/day. 

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 Section 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 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 
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5. POTENTIAL FOR HUMAN EXPOSURE
 

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 judgment of adults in avoiding hazards 

(NRC 1993). 

No specific references on exposures of infants or children to 3,3’-dichlorobenzidine were located. 

Young children may be exposed to 3,3’-dichlorobenzidine by ingesting paint chip debris, colorful 

objects or paints, and soil if the material contains the chemical. Mathematical models (using 

somewhat unrealistic worstcase assumptions) predict that the estimated total intake of 

3,3’-dichlorobenzidine by infants up to 6 months of age would be 3.6x10-8 ng/kg bodyweight/day, 

about 5 times greater than the estimate of 7.4x 10-9 ng/kg body weight/day for adults age 20 or older 

(Government of Canada 1993). 

Children sometimes put dirt in their mouths. Because the adsorption of 3,3’-dichlorobenzidine to soils 

and sediments may not be readily reversible (Boyd et al. 1984; Chung and Boyd 1987; Sikka et al. 

1978), the bioavailability of the compound is limited. A child who ingested contaminated dirt would 

be expected to incur less exposure as compared to that from other, more direct routes. 

Another potential exposure route for children is through exposure to clothing and tracked-in dirt 

brought in by parents who work in factories that produce 3,3’-dichlorobenzidine. A public health 

assessment study conducted in Michigan in 1981 (ATSDR 1996) found the compound in the homes of 

9 employees. Samples collected from vacuum cleaner bags had up to 10.5 ppm and dryer lint 

contained up to 0.74 ppm. If these homes have not been adequately cleaned, exposure could continue. 

5.7 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES 

In addition to individuals who are occupationally exposed to 3,3’-dichlorobenzidine (see Section 5.5) 

there are several groups within the general population that have the potential for exposures to 

3,3’-dichlorobenzidine at levels above those of the general population. These groups include 

individuals living in proximity to sites where 3,3’-dichlorobenzidine was produced or sites where 

3,3’-dichlorobenzidine was disposed, and individuals living near one of the 32 NPL hazardous waste 

sites where 3,3’-dichlorobenzidine has been detected in some environmental media (HazDat 1998). 

3,3’-Dichlorobenzidine was not detected in fish samples obtained from rivers near nine textile 

dyestuff manufacturers known to use 3,3’-dichloro­
3,3’-DICHLOROBENZIDINE 103 

5. POTENTIAL FOR HUMAN EXPOSURE
 

benzidine-based pigments (Diachenko 1979), nor were there any fish consumption advisories for 

3,3’-dichlorobenzidine in 1996. Therefore, recreational and subsistence fishers are not at risk. 

NIOSH, in 1980, concluded that during the use of benzidine-based dyes, the greatest potential for 

exposurewould be expected to be by dermal absorption or inhalation by personnel who routinely 

handle dry powders (NIOSH 1980). However, EPA (1980b) has generalized that dermal 

absorption in the workplace is probably a minor route of 3,3’-dichlorobenzidine exposure, 

although dermatitis has occurred in workers in plants where 3,3’-dichlorobenzidine and 

3,3’-dichlorobenzidine-based pigments were manufactured. It may be that health risks with 

regard to 3,3’-dichlorobenzidine exposure depend on the specific operations of the individual 

plant and the extent of personal protective practices of the individual operator. 

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 3,3’-dichlorobenzidine 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 3,3’-dichlorobenzidine. 

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. 

5.8.1 Identification of Data Needs 

Physical and Chemical Properties. It has been demonstrated that 3,3’-dichlorobenzidine is 

strongly adsorbed by soils and sediments, and that it may not readily desorb. Adsorption cannot 

be accurately predicted a priori; such data are soil-system specific and must be determined 

experimentally for each 
3,3’-DICHLOROBENZIDINE 104 

5. POTENTIAL FOR HUMAN EXPOSURE
 

system under study. Because there is some discrepancy regarding the volatility of the free base form 

of 3,3’-dichlorobenzidine (Gerarde and Gerarde 1974; CPMA 1998) research in this area is indicated. 

Production, Import/Export, Use, Release, and Disposal. According to the Toxics Release 

Inventory (TRI) report (TR196 1998), 3,3’-dichlorobenzidine is manufactured at one facility in 

Michigan. Three of the five facilities listed by TR196 that process the compound depend on imports 

for their supply. The chemical is no longer used to produce dyes in the United States (better dyes 

based on other chemicals are available); its main use is in the production of pigments (DCMA 1989). 

It also finds some use in the formulation of rubber and plastic (HSDB 1996) and in the production of 

PBI (Celanese 1985). The compound is not used in the home or in the open environment; however, 

there is evidence that the compound can be brought into the home on the shoes and clothing of adults 

who work with 3,3’-dichlorobenzidine (ATSDR 1996) but the quantity that might be present is 

unknown. In the workplace, OSHA regulations require that the compound be handled in closed 

systems and that shipping containers be cleaned thoroughly (again, within a closed system) before 

disposal (DCMA 1989). The free base or salt form of the compound is not used in the home or in the 

general environment. It is handled only by industry to make pigments; thus there seems to be little 

chance the chemical could contaminate the food supply. No evidence of the compound in fish taken 

downstream from nine facilities known to handle 3,3’-dichlorobenzidine was found (Diachenko 

1979). Citations regarding disposal techniques for 3,3’-dichlorobenzidine are found in the Hazardous 

Substances Data Base (HSDB). Small quantities can be destroyed by chemical reaction, for example, 

with sodium hypochlorite solution, which converts 3,3’-dichlorobenzidine to a quinone-type 

compound. Incineration at high temperatures can be used to destroy work garments and miscellaneous 

solid wastes exposed to the compound. Presumably only small amounts would need to be disposed 

since the compound is mainly consumed by conversion to pigments. 

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 TRI, which contains this information for 1996, became available in May of 1998. This database 

will be updated yearly and should provide a list of industrial production facilities and emissions. 

Environmental Fate. It is not known if 3,3’-dichlorobenzidine, like benzidine, is oxidized by clay 

minerals or if cations in water can have the same oxidizing effect. 3,3’-Dichlorobenzidine does not 

appear to biodegrade easily, but the few studies in this area did not state the type(s) or concentrations 

of 
3,3’-DICHLOROBENZIDINE 105 

5. POTENTIAL FOR HUMAN EXPOSURE
 

microorganisms used in each study. More systematic studies with other organisms may prove useful. 

A recent study (Nyman et al. 1997) provides evidence that in the span of a year up to 80% of 

3,3’-dichlorobenzidine can degrade to benzidine in anaerobic mixtures of sediment/water. Further 

research to identify the pathways and products of decomposition of 3,3’-dichlorobenzidine in various 

soils is needed. The toxicological profile for benzidine contains information on the environmental fate 

of that compound (ATSDR 1995). 

Bioavailability from Environmental Media. No information on the presence of 

3,3’-dichlorobenzidine in foods was located in the available literature. The Canadian Government’s 

Priority Substances List Assessment Report for 3,3’-dichlorobenzidine (Government of Canada 1993) 

also reports that no data on the levels of 3,3’-dichlorobenzidine in drinking water or foodstuffs were 

identified within either Canada or the United States. Because 3,3’-dichlorobenzidine has been found to 

bind strongly to soil constituents (Berry and Boyd 1985; Chung and Boyd 1987), Law (1995) 

concluded that it would also bind strongly to sedimentary material in the marine aquatic environment 

and thus may have limited bioavailability. 

Food Chain Bioaccumulation. 3,3’-Dichlorobenzidine is bioconcentrated by aquatic organisms under 

experimental conditions. Whole-fish BCFs of around 500, with equilibration occurring in 96-168 

hours, have been published (Appleton and Sikka 1980). In view of the n-octanoywater partition 

coefficient for 3,3’-dichlorobenzidine, limited bioaccumulation could be expected (Law 1995) 

because the retention time of the chemical in exposed fish is short (Appleton and Sikka 1980). The 

ability of aquatic organisms to concentrate the compound could present a human health hazard if 

contaminated fish were eaten. However, 3,3’-dichlorobenzidine was not found in fish taken from 

waters in the vicinity of dye or textile manufacturing plants on the Buffalo and Delaware rivers in the 

United States (Diachenko 1979). It was concluded that monitoring for 3,3’-dichlorobenzidine in 

marine waters of the United Kingdom is unwarranted at present (Law 1995). 

Exposure Levels in Environmental Media. There were no quantitative data on current 

atmospheric levels of 3,3’-dichlorobenzidine emissions or on the chemical’s potential to act as a 

surface contaminant of soil environments. It is difficult to determine 3,3’-dichlorobenzidine levels in 

the aquatic environment because the concentrations tend to be at or below analytical detection limits. 

In general, it may only be possible to ascertain fully the environmental fate of 3,3’-dichlorobenzidine 

as analytical advances permit the routine determination of very low concentrations. Moreover, 

determination of the nature and environmental fate of breakdown products of 3,3’-dichlorobenzidine 

would be useful. 
3,3’-DICHLOROBENZIDINE 106 

5. POTENTIAL FOR HUMAN EXPOSURE
 

Reliable monitoring data for the levels of 3,3’-dichlorobenzidine in contaminated media at hazardous 

waste sites are needed so that the information obtained on levels of 3,3’-dichlorobenzidine in the 

environment can be used in combination with the known body burdens of 3,3’-dichlorobenzidine to 

assess the potential risk of adverse health effects in populations living in the vicinity of hazardous 

waste sites. 

Exposure Levels in Humans. It has been speculated that the 1974 OSHA regulations have reduced 

workplace air levels of 3,3’-dichlorobenzidine (CPMA 1998). However, it would be important to 

conduct exposure studies to monitor air levels in the workplace to confirm this premise. The need for 

more information on the extent of air, water, and soil contamination by industrial plant emissions or 

waste sites containing 3,3’-dichlorobenzidine continues. There is little information on exposure of 

children to 3,3’-dichlorobenzidine (or products derived from the compound). The compound has a 

very limited distribution and is not present in consumer goods (other than in insoluble pigmented 

forms). This information is necessary for assessing the need to conduct health studies on these 

populations. 

Exposures of Children. There is no available information on exposure of children to 

3,3’-dichlorobenzidine (or products derived from the compound). The compound has a very limited 

distribution and is not present in consumer goods (other than in insoluble pigmented forms). Thus, 

there is no pressing need to gather data related to children’s exposure. However, given sufficient 

resources, the topic of inadvertent take-home exposure by occupationally exposed parents could be 

explored. A public health assessment (ATSDR 1996) found measurable levels of 

3,3’-dichlorobenzidine (10.5 ppm in vacuum cleaner bags and 0.74 ppm in clothes dryer lint) in the 

homes of workers who were employed in manufacturing or processing the compound. 

Exposure Registries. No exposure registries for 3,3’-dichlorobenzidine were located. This substance is 

not 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 the epidemiological research needed to assess adverse health outcomes that may be related 

to exposure to this substance. 

5.8.2 Ongoing Studies
 

No information was located regarding ongoing studies.
 
3.3’-DICHLOROBENZIDINE 107 

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 3,3’-dichlorobenzidine, its metabolites, and other 

biomarkers of exposure and effect to 3,3’-dichlorobenzidine. 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 

Methods for the determination of 3,3’-dichlorobenzidine and its metabolites in biological 

materials are summarized in Table 6- 1. 

The compound 3,3’-dichlorobenzidine has been measured most often in urine and serum using 

gas chromatography (GC) (Bowman and Nony 1981; Bowman and Rushing 1981; Hoffman and 

Schmidt 1993; Joppich-Kuhn et al. 1997; Nony and Bowman 1980; Nony et al. 1980) and high 

performance liquid chromatography (HPLC) (Birner et al. 1990; Bowman and Nony 1981; 

CPMA 1998; Nony and Bowman 1980; Nony et al. 1980; Zwirner-Baier and Neumann 1994). A 

method for 3,3’-dichlorobenzidine in fish using GC (Diachenko 1979) has been reported. GC 

methods usually relied upon selective detection of the fluorinated derivatives while HPLC 

methods relied on absorbence or electrochemical detection. In addition, one method of analysis in 

urine used a spectrophotometric approach (Roberts and Rossano 1982). Several of the reported 

methods can also be used to determine the mono- and di-acetylated metabolites. The studies of 

Birner et al. (1990), Joppich-Kuhn et al. 1997, and Zwirner-Baier and Neumann (1994) reported 

the determination of 3,3’-dichlorobenzidine and monoacetyl-3,3’-dichlorobenzidine following 

hydrolysis of the analyte-hemoglobin adducts (the adduct is a marker of exposure). Although 

most of these methods have been developed using animal samples, they should also be applicable 

to the determination of 3,3’-dichlorobenzidine and its metabolites in samples of human origin. 

Limits of detection in the low to mid ppb range 


3,3’-DICHLOROBENZIDINE 110 

6. ANALYTICAL METHODS
 

have been reported, although the hemoglobin adduct method of Joppich-Kuhn et al. (1997) reported a 

limit of detection of less than 0.1 ng/g (ppb). These sensitive methods are potentially useful for the 

assessment of human exposure to 3,3’-dichlorobenzidine. 

6.2 ENVIRONMENTAL SAMPLES 

Methods for the determination of 3,3’-dichlorobenzidine in environmental samples are summarized in 

Table 6-2. 

The determination of 3,3’-dichlorobenzidine in environmental samples is most commonly achieved by 

GC/mass spectrometry (GC/MS) (Diachenko 1979; EPA 1982b, 1986a, 1984a; Greenberg et al. 1992) 

and HPLC (Armentrout and Cutie 1980; EPA 1982a; Morales et al. 1981; NIOSH 1994; Riggin and 

Howard 1979). Sample preparation typically employs liquid-liquid or liquid-solid extractions for 

water, waste water, soils, sediments, and solid waste. Supercritical fluid extraction has also been 

shown to provide good recovery of 3,3’-dichlorobenzidine from a spiked, dried soil (Oostdyke et al. 

1995). Lopez-Avila et al. (1996) demonstrated that microwave-assisted extraction using a hexaneacetone solvent system gave recoveries from spiked (5 mg/kg), standard soil of 96%. The same 

solvent system in Soxhlet extraction resulted in only 47% recovery. 

Solid phase extraction followed by capillary zone electrophoresis with UV absorbence detection has 

been shown to be applicable to the isolation and determination of 3,3’-dichlorobenzidine in water at 

ppm levels (Cavallaro et al. 1995). 

For the HPLC determination of 3,3’-dichlorobenzidine in water, a relatively complicated 

procedure may be used (EPA 1982a) in which the analyte is extracted into chloroform, backextracted with acid, neutralized, and extracted with chloroform. The chloroform is exchanged to 

methanol and concentrated using a rotary evaporator and nitrogen blowdown, then brought to a 5 

mL volume with an acetate buffer. HPLC with electrochemical detection is used, providing for a 

method detection limit of 0.13 μg/L; single operator accuracy and precision for 30 analytes of 5 

different types of water samples over a spike range of l-5 μg/L gave an average recovery of 65% 

and a standard deviation of 9.6% (EPA 1982a). The more complicated the matrix, the more 

extensive the sample preparation methods generally need to be. In certain circumstances (i.e., 

relatively clean water samples), water matrices can be introduced directly into the 


3,3’-DICHLOROBENZIDINE 113 

6. ANALYTICAL METHODS
 

analysis step without prior treatment (Armentrout and Cutie 1980). GC separation methods can be 

applied also to the extracts obtained for HPLC analyses. Detection of the free amine, in addition 

to fluorinated derivatives, has been demonstrated by GC methods. 

Dichlorobenzidine and its salts are collected from air matrices using adsorption/filtration 

approaches (Morales et al. 1981; NIOSH 1994) and recovered from the adsorbent using methanol 

containing a small amount of triethylamine (TEA). The addition of TEA converts any salt to the 

corresponding amine, thus rendering it soluble in the organic solvent. Limits of detection in the 

low μg/m3

 (low to sub-ppb) range have been reported. The compound 4,4’-methylenebis(2­

chloroaniline) was reported to interfere with 3,3’-dichlorobenzidine (Morales et al. 1981; NIOSH 

1994). 

6.3 ADEQUACY OF THE DATABASE 

Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation 

with theAdministrator of EPA and agencies and programs of the Public Health Service) to assess 

whether adequate information on the health effects of 3,3’-dichlorobenzidine 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 3,3’-dichlorobenzidine. 

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. Methods for the determination 

of 3,3’-dichlorobenzidine in urine and serum have been reported (Birner et al. 1990; Bowman and 

Nony 1981; Bowman and Rushing 1981; Hoffman and Schmidt 1993; CPMA 1998; Nony and 

Bowman 1980; Nony et al. 1980; Zwirner-Baier and Neumann 1994). Some of the methods have 

been shown to be 
3,3’-DICHLOROBENZIDINE 114 

6. ANALYTICAL METHODS
 

suitable for the determination of the acetylated metabolites (Bowman and Nony 198 1; Nony and 

Bowman 1980; Nony et al. 1980). The methods of Birner et al. (1990), Joppich-Kuhn et al. 

(1997), and Zwirner-Baier and Neumann (1994) permit the analysis of hemoglobin adducts of 

3,3’-dichlorobenzidine and its monoacetyl metabolite. Limits of detection for 

3,3’-dichlorobenzidine in urine and serum were reported to be as low as 1 to 5 ppb (Bowman and 

Rushing 1981; Hoffman and Schmidt 1993; Roberts and Rossano 1982), with detectable 

concentrations of the acetylated metabolites somewhat higher. Most of these studies were 

performed with samples from rats; the methods should be tested to determine if they are 

applicable to samples of human origin. In addition, the levels of these biomarkers associated with 

exposures to 3,3’-dichlorobenzidine of toxicological concern should be defined in order to 

increase their utility. 

Methods for Determining Parent Compounds and Degradation Products in Environmental 

Media. Methods have been described for the determination of 3,3’-dichlorobenzidine in air, with 

reported limits of detection of 0.5 μg/m3 (NIOSH 1994) and 3 μg/m3

 (Morales et al. 1981). 

Methods for the analysis of 3,3’-dichlorobenzidine in water and waste water have also been 

described, with reported detection limits of 16.5 μg/L (ppb) (EPA 1982b; Greenberg et al. 1992), 

50 μg/L (ppb) (EPA 1984a), 3 ppb (Armentrout and Cutie 1980), 0.13 μg/L (ppb) (EPA 1982a), 

and 50 to 100 ng/L (ppt) (Riggin and Howard 1979). The only method found for 

3,3’-dichlorobenzidine in food (fish) reported a limit of detection of less than 20 ppb (Diachenko 

1979). It does not appear that additional methods for the determination of 3,3’-dichlorobenzidine 

in air or water are needed. Additional methods for 3,3’-dichlorobenzidine in foods are needed. If 

MRLs were established, the needs could be defined more precisely. 

6.3.2 Ongoing Studies 

No ongoing studies in which new methods for the determination of 3,3’-dichlorobenzidine are 

being developed were found in a search of the Federal Research in Progress database 

(FEDRIP 1998). 
3,3’-DICHLOROBENZIDINE 115 

7. REGULATIONS AND ADVISORIES
 

The national and state regulations and guidelines pertaining to 3,3’-dichlorobenzidine in air, 

water, and other media are summarized in Table 7- 1. 

There is no oral reference dose (RfD) for 3,3’-dichlorobenzidine. The health effects data for 

3,3’-dichlorobenzidine were reviewed by the EPA RfD/RfC Work Group and determined to be 

inadequate for derivation of an inhalation RfC (IRIS 1998). 

The EPA has determined that 3,3’-dichlorobenzidine is a probable human carcinogen, B2 

classification (IRIS 1998). The International Agency for Research on Cancer (IARC) has 

classified 3,3’-dichlorobenzidine as a Group 2B carcinogen-possibly carcinogenic to humans 

(IARC 1987). The American Conference of Governmental Industrial Hygienists (ACGIH) 

classifies 3,3’-dichlorobenzidine as A3, which indicates that the chemical is carcinogenic in 

experimental animals when administered at a relatively high dose (ACGIH 1997). The National 

Toxicology Program (NTP) of the U.S. Department of Health and Human Services has 

determined that 3,3’-dichlorobenzidine and its salt may reasonably be expected to be 

cancer-causing agents (NTP 1998). 

3,3’-Dichlorobenzidine is on the list of chemicals subject to the requirements of the Emergency 

Planning and Community Right-to-Know Act of 1986 (EPCRA) (EPA 1988a). Section 3 13 of 

Title III of EPCRA requires owners and operators of certain facilities that manufacture, import, 

process, or otherwise use the chemicals on this list to report annually their release of those 

chemicals to any environmental media (U.S. Congress 1986). 

3,3’-Dichlorobenzidine is one of a number of carcinogenic compounds regulated by OSHA. To 

control exposures to 3,3’-dichlorobenzidine in workplace air and to protect the health of workers, 

OSHA’s regulatory standards provide strict guidelines for manufacturing, processing, 

repackaging, handling, using, and storing the compound (OSHA 1996). These standards also 

include the requirements for personal protective equipment, training, labeling, posting, and 

engineering controls. In addition to engineering controls such as continuous local exhaust 

ventilation and workplace practices such as full body protective clothing, the employer must 

maintain medical surveillance records (OSHA 1996). OSHA requires that initial medical 

screening and regular medical examinations be made available to any employee who is 
3,3’-DICHLOROBENZIDINE 116 

7. REGULATIONS AND ADVISORIES 

exposed to 3,3’-dichlorobenzidine at potentially hazardous levels. The employer must also 

provide a training program that informs its employees of the carcinogenic hazards of 

3,3’-dichlorobenzidine, the nature of the operation involving the chemical that could result in 

exposure, decontamination procedures,and specific emergency procedures to be used if exposure 

does occur (OSHA 1996). OSHA also regulates 3,3’-dichlorobenzidine under the Hazard 

Communication Standard (HCS) and as a chemical hazard in laboratories (NTP 1998). The HCS 

has established uniform requirements to make sure that the hazards of all chemicals imported 

into, produced, or used in workplaces are evaluated and that information on the hazards they pose 

is transmitted to affected employers and exposed employees (OSHA 1998). 

EPA regulates 3,3’-dichlorobenzidine under the Clean Air Act (CAA) and has designated 

3,3’-dichlorobenzidine as a hazardous air pollutant (HAP) (EPA 1994; U.S. Congress 1990). The 

major source category for which the national emissions standards for new stationary sources are 

applicable to 3,3’-dichlorobenzidine emissions is the synthetic organic chemicals manufacturing 

industry (SOCMI)-equipment leaks (EPA 1994). 

3,3’-Dichlorobenzidine is regulated by the Clean Water Effluent Guidelines in Subchapter N of 

Title 40 of the Code of Federal Regulations. Electroplating is the point source category for which 

3,3’-dichlorobenzidine is controlled as a total toxic organic (EPA 1981). The point source 

categories for which 3,3’-dichlorobenzidine has a specific regulatory limitation are steam electric 

power generation (EPA 1982) and metal finishing (EPA 1983a). The EPA has proposed a 

reportable quantity of 10 pounds for 3,3’-dichlorobenzidine for its water quality criteria for the 

protection of human health (IRIS 1998). 

The Resource Conservation and Recovery Act (RCRA) identifies 3,3’-dichlorobenzidine as the 

hazardous constituent in various hazardous wastes. It is the regulated constituent in hazardous 

wastes assigned the waste code U073 (EPA 1988b). 

Under the Comprehensive Environmental Response, Compensation, and Liability Act 

(CERCLA), owners of vessels or facilities are required to immediately report releases of 

3,3’-dichlorobenzidine equal to or greater than the reportable quantity of 1 pound (0.454 kg) 

(EPA 1985). It is subject to the requirements under the Superfund Amendments and 

Reauthorization Act (SARA) of 1986 (IRIS 1998). 
3,3’-DICHLOROBENZIDINE 117 

7. REGULATIONS AND ADVISORIES 

Although the Food and Drug Administration (FDA) classifies 3,3’-dichlorobenzidine as a 

carcinogen, the agency has not enacted regulatory guidelines (NTP 1998) or issued advisories 

specifically targeting 3,3’-dichlorobenzidine as being a danger in the food supply. 

EPA has selected 3,3’-dichlorobenzidine and its mixtures for priority consideration for testing 

under the Toxic Substances Control Act (TSCA) (IRIS 1998). 





_____________________________ 

3,3’-DICHLOROBENZIDINE 123 

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Organization. 

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Comar CL, Bronner F, eds. Mineral metabolism: An advanced treatise volume II - the elements 

part A. New York, NY: Academic Press. 

*Ziegler EE, Edwards BB, Jensen RL et al. 1978. Absorption and retention of lead by infants. 

Pediatr Res 12:29-34. Zierath DL, Hassett JJ, Banwart WL, et al. 1980. Sorption of benzidine by 

sediments and soils. Soil Sci 129(5):277-281. 

*Zwirner-Baier I, Neumann HG. 1994. Biomonitoring of aromatic amines IV: Use of hemoglobin 

adducts to demonstrate the bioavailability of cleavage products from diarylide azo pigments in 

vivo. Arch Toxicol 68( 1):8-14. 
3,3’-DICHLOROBENZIDINE 139 

9. GLOSSARY
 

Absorption-The taking up of liquids by solids, or of gases by solids or liquids. 

Acute Exposure-Exposure to a chemical for a duration of 14 days or less, as specified in the 

Toxicological Profiles. 

Adsorption-The adhesion in an extremely thin layer of molecules (as of gases, solutes, or liquids) 

to the surfaces of solid bodies or liquids with which they are in contact. 

Adsorption Coefficient (Koc)-The ratio of the amount of a chemical adsorbed per unit weight of 

organic carbon in the soil or sediment to the concentration of the chemical in solution at 

equilibrium. 

Adsorption Ratio (Kd)-The amount of a chemical adsorbed by 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. 

Benchmark Dose (BMD) -is usually defined as the lower confidence limit on the dose that 

produces a specified magnitude of changes in a specified adverse response. For example, a 

BMD10 would be the dose at the 95% lower confidence limit on a 10% response, and the 

benchmark response (BMR) would be 10%. The BMD is determined by modeling the dose 

response curve in the region of the dose response relationship where biologically observable data 

are feasible. 

Benchmark Dose Model-is a statistical dose-response model applied to either experimental 

toxicological or epidemiological data to calculate a BMD. 

Bioconcentration Factor (BCF) -The quotient of the concentration of a chemical in aquatic 

organisms at a specific time or during a discrete time period of exposure divided by the 

concentration in the surrounding water at the same time or during the same period. 

Biomarkers-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. 

Cancer Effect Level (CEL)-The lowest dose of chemical in a study, or group of studies, that 

produces significant increases in the incidence of cancer (or tumors) between the exposed 

population and its appropriate control. 

Carcinogen-A chemical capable of inducing cancer. 

Case-Control Study-A type of epidemiological study which examines the relationship between a 

particular outcome (disease or condition) and a variety of potential causative agents (such as toxic 

chemicals). In a case-controlled study, a group of people with a specified and well-defined 

outcome is identified and compared to a similar group of people without outcome. 

Case Report-describes a single individual with a particular disease or exposure. These may 

suggest some potential topics for scientific research but are not actual research studies. 
3,3’-DICHLOROBENZIDINE 140 

9. GLOSSARY
 

Case Series-describes the experience of a small number of individuals with the same disease or 

exposure. These may suggest potential topics for scientific research but are not actual research 

studies. 

Ceiling Value-A concentration of a substance that should not be exceeded, even instantaneously. 

Chronic Exposure-Exposure to a chemical for 365 days or more, as specified in the Toxicological 

Profiles. 

Cohort Study-A type of epidemiological study of a specific group or groups of people who have 

had a common insult (e.g., exposure to an agent suspected of causing disease or a common 

disease) and are followed forward from exposure to outcome. At least one exposed group is 

compared to one unexposed group. 

Cross-sectional Study-A type of epidemiological study of a group or groups which examines the 

relationship between exposure and outcome to a chemical or to chemicals at one point in time. 

Data Needs-substance-specific informational needs that if met would reduce the uncertainties of 

human health assessment. 

Developmental Toxicity-The occurrence of adverse effects on the developing organism that may 

result from exposure to a chemical prior to conception (either parent), during prenatal 

development, or postnatally to the time of sexual maturation. Adverse developmental effects may 

be detected at any point in the life span of the organism. 

Dose-Response Relationship-The quantitative relationship between the amount of exposure to a 

toxicant and the incidence of the adverse effects. 

Embryotoxicity and Fetotoxicity- Any toxic effect on the conceptus as a result of prenatal 

exposure to a chemical; the distinguishing feature between the two terms is the stage of 

development during which the insult occurs. The terms, as used here, include malformations and 

variations, altered growth, and in utero death. 

Environmental Protection Agency (EPA) Health Advisory-An estimate of acceptable drinking 

water levels for a chemical substance based on health effects information. A health advisory is 

not a legally enforceable federal standard, but serves as technical guidance to assist federal, state, 

and local officials. Epidemiology-refers to the investigation of factors that determine the 

frequency and distribution of disease or other health-related conditions within a defined human 

population during a specified period. 

Genotoxicity-a specific adverse effect on the genome of living cells that, upon the duplication of 

affected cells, can be expressed as a mutagenic, clastogenic or carcinogenic event because of 

specific alteration of the molecular structure of the genome. 

Half-life-a measure of rate for the time required to eliminate one half of a quantity of a chemical 

from the body or environmental media. 
3,3’-DICHLOROBENZIDINE 141 

9. GLOSSARY
 

Immediately Dangerous to Life or Health (IDLH)-The maximum environmental concentration of 

a contaminant from which one could escape within 30 minutes without any escape-impairing 

symptoms or irreversible health effects. 

Incidence--The ratio of individuals in a population who develop a specified condition to the total 

number of individuals in that population who could have developed that condition in a specified 

time period. 

Intermediate Exposure-Exposure to a chemical for a duration of 15-364 days, as specified in the 

Toxicological Profiles. 

Immunological Effects-are functional changes in the immune response. 

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 Concentratioq(LO) (LCLO) -The lowest concentration of a chemical in air which has been 

reported to have caused death in humans or animals. 

Lethal Concentratioq(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(50) (LD50) -The lowest dose of a chemical introduced by a route other than inhalation 

that has been reported to have caused death in humans or animals. 

Lethal Dose(50) ( LD50)-The dose of a chemical which has been calculated to cause death in 50% 

of a defined experimental animal population. 

Lethal Tlme(50) (LT50)-A calculated period of time within which a specific concentration of a 

chemical isexpected to cause death in 50% of a defined experimental animal population. 

Lowest-Observed-Adverse-Effect Level (LOAEL)-The lowest exposure level of chemical in a 

study, or group of studies, that produces statistically or biologically significant increases in 

frequency or severity of adverse effects between the exposed population and its appropriate 

control. 

Lymphoreticular Effects-represent morphological effects involving lymphatic tissues such as the 

lymph nodes, spleen, and thymus. Malformations-Permanent structural changes that may 

adversely affect survival, development, or function. 

Minimal Risk Level (MRL) -An estimate of daily human exposure to a hazardous substance that 

is likely to be without an appreciable risk of adverse noncancer health effects over a specified 

route and duration of exposure. 
3,3’-DICHLOROBENZIDINE 142 

9. GLOSSARY
 

Modifying Factor (MF)-A value (greater than zero) that is applied to the derivation of a minimal 

risk level (MRL) to reflect additional concerns about the database that are not covered by the 

uncertainty factors. The default value for a MF is 1. 

Morbidity-State of being diseased; morbidity rate is the incidence or prevalence of disease in a 

specific population. 

Mortality-Death; mortality rate is a measure of the number of deaths in a population during a 

specified interval of time. 

Mutagen-A substance that causes mutations. A mutation is a change in the DNA sequence of a 

cell’s DNA. Mutations can lead to birth defects, miscarriages, or cancer. 

Necropsy-The gross examination of the organs and tissues of a dead body to determine the cause 

of death or pathological conditions. 

Neurotoxicity-The occurrence of adverse effects on the nervous system following exposure to a 

chemical. 

No-Observed-Adverse-Effect Level (NOAEL)-The dose of a chemical at which there were no 

statistically or biologically significant increases in frequency or severity of adverse effects seen 

between the exposed population and its appropriate control. Effects may be produced at this dose, 

but they are not considered to be adverse. 

Octanol-Water Partition Coefficient (Kow)-The equilibrium ratio of the concentrations of a 

chemical in n-octanol and water, in dilute solution. 

Odds Ratio-a means of measuring the association between an exposure (such as toxic substances 

and a disease or condition) which represents the best estimate of relative risk (risk as a ratio of the 

incidence among subjects exposed to a particular risk factor divided by the incidence among 

subjects who were not exposed to the risk factor). An odds ratio of greater than 1 is considered to 

indicate greater risk of disease in the exposed group compared to the unexposed. 

Organophosphate or Organophosphorus Compound-a phosphorus containing organic compound 

and especially a pesticide that acts by inhibiting cholinesterase. 

Permissible Exposure Limit (PEL)-An Occupational Safety and Health Administration (OSHA) 

allowable exposure level in workplace air averaged over an &hour shift of a 40 hour workweek. 

Pesticide-general classification of chemicals specifically developed and produced for use in the 

control of agricultural and public health pests. 

Pharmacokinetics-is the science of quantitatively predicting the fate (disposition) of an exogenous 

substance in an organism. Utilizing computational techniques, it provides the means of studying 

the absorption, distribution, metabolism and excretion of chemicals by the body. 
3,3’-DICHLOROBENZIDINE 143 

9. GLOSSARY
 

Pharmacokinetic Model - is a set of equations that can be used to describe the time course of a 

parent chemical or metabolite in an animal system. There are two types of pharmacokinetic 

models: data-based and physiologically-based. A data-based model divides the animal system 

into a series of compartments which, in general, do not represent real, identifiable anatomic 

regions of the body whereby the physiologically-based model compartments represent real 

anatomic regions of the body. 

Physiologically Based Pharmacodynamic (PBPD) Model - is a type of physiologically-based 

doseresponse model which quantitatively describes the relationship between target tissue dose 

and toxic end points. These models advance the importance of physiologically based models in 

that they clearly describe the biological effect (response) produced by the system following 

exposure to an exogenous substance. 

Physiologically Based Pharmacokinetic (PBPK) Model - is comprised of a series of 

compartments representing organs or tissue groups with realistic weights and blood flows. These 

models require a variety of physiological information: tissue volumes, blood flow rates to tissues, 

cardiac output, alveolar ventilation rates and, possibly membrane permeabilities. The models also 

utilize biochemical information 4such as air/blood partition coefficients, and metabolic 

parameters. PBPK models are also called biologically based tissue dosimetry models. 

Prevalence-The number of cases of a disease or condition in a population at one point in time. 

Prospective Study--a type of cohort study in which the pertinent observations are made on events 

occurring after the start of the study. A group is followed over time. 

q1*-The upper-bound estimate of the low-dose slope of the dose-response curve as determined by 

the multistage procedure. The ql* can be used to calculate an estimate of carcinogenic potency, 

the incremental excess cancer risk per unit of exposure (usually μg/L for water, μg/kg/day for 

food, and μg/m3 for air). 

Recommended Exposure Limit (REL)-A National Institute for Occupational Safety and Health 

(NIOSH) time-weighted average (TWA) concentrations for up to a 10-hour workday during a 40­

hour workweek. 

Reference Concentration (RfC)-An estimate (with uncertainty spanning perhaps an order of 

magnitude) of a continuous inhalation exposure to the human population (including sensitive 

subgroups) that is likely to be without an appreciable risk of deleterious noncancer health effects 

during a lifetime. The inhalation reference concentration is for continuous inhalation exposures 

and is appropriately expressed in units of mg/m3

 or ppm. 

Reference Dose (RfD)-An estimate (with uncertainty spanning perhaps an order of magnitude) of 

the daily exposure of the human population to a potential hazard that is likely to be without risk 

of deleterious effects during a lifetime. The RfD is operationally derived from the No-ObservedAdverse-Effect Level (NOAEL- from animal and human studies) by a consistent application of 

uncertainty factors that reflect various types of data used to estimate RfDs and an additional 

modifying factor, which is based on a professional judgment of the entire database on the 

chemical. The RfDs are not applicable to nonthreshold effects such as cancer. 
3,3’-DICHLOROBENZIDINE 144 

9. GLOSSARY
 

Reportable Quantity (RQ)-The quantity of a hazardous substance that is considered reportable under 

the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA). 

Reportable quantities are (1) 1 pound or greater or (2) for selected substances, an amount established 

by regulation either under CERCLA or under Section 311 of the Clean Water Act. Quantities are 

measured over a 24-hour period. 

Reproductive Toxicity-The occurrence of adverse effects on the reproductive system that may result 

from exposure to a chemical. The toxicity may be directed to the reproductive organs and/or the 

related endocrine system. The manifestation of such toxicity may be noted as alterations in sexual 

behavior, fertility, pregnancy outcomes, or modifications in other functions that are dependent on the 

integrity of this system. 

Retrospective Study-A type of cohort study based on a group of persons known to have been exposed 

at some time in the past. Data are collected from routinely recorded events, up to the time the study is 

undertaken. Retrospective studies are limited to casual factors that can be ascertained from existing 

records and/or examining survivors of the cohort. 

Risk-the possibility or chance that some adverse effect will result from a given exposure to a 

chemical. 

Risk Factor-An aspect of personal behavior or lifestyle, an environmental exposure, or an inborn or 

inherited characteristic, that is associated with an increased occurrence of disease or other healthrelated event or condition. 

Risk Ratio--The ratio of the risk among persons with specific risk factors compared to the risk among 

persons without risk factors. A risk ratio greater than 1 indicates greater risk of disease in the exposed 

group compared to the unexposed. 

Short-Term Exposure Limit (STEL)-The American Conference of Governmental Industrial Hygienists 

(ACGIH) maximum concentration to which workers can be exposed for up to 15 min continually. No 

more than four excursions are allowed per day, and there must be at least 60 min between exposure 

periods. The daily Threshold Limit Value - Time Weighted Average (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)-An American Conference of Governmental Industrial Hygienists 

(ACGIH) concentration of a substance to which most workers can be exposed without adverse effect. 

The TLV may be expressed as a Time Weighted Average (TWA), as a Short-Term Exposure Limit 

(STEL), or as a ceiling limit (CL). 

Time-Weighted Average (TWA)-An allowable exposure concentration averaged over a normal &hour 

workday or 40-hour workweek. . 

Toxic Dose(50) (TD50) - A calculated dose of a chemical, introduced by a route other than inhalation, 

which is expected to cause a specific toxic effect in 50% of a defined experimental animal population. 
3,3’-DICHLOROBENZIDINE 145 

9. GLOSSARY
 

Toxicokinetic-The study of the absorption, distribution and elimination of toxic compounds in the 

living organism. 

Uncertainty Factor (UF)-A factor used in operationally deriving the Minimal Risk Level (MRL) 

or Reference Dose (RfD) or Reference Concentration (RfC) from experimental data. UFs are 

intended to account for (1) the variation in sensitivity among the members of the human 

population, (2) the uncertainty in extrapolating animal data to the case of human, (3) the 

uncertainty in extrapolating from data obtained in a study that is of less than lifetime exposure, 

and (4) the uncertainty in using Lowest-Observed-Adverse-Effect Level (LOAEL) data rather 

than No-Observed-Adverse-Effect Level (NOAEL) data. A default for each individual W is 10; if 

complete certainty in data exists, a value of one can be used; however a reduced UF of three may 

be used on a case-by-case basis, three being the approximate logarithmic average of 10and 1. 

Xenobiotic-any chemical that is foreign to the biological system. 

3.3’-DICHLOROBENZIDINE A-l 

APPENDIX A
 

ATSDR MINIMAL RISK LEVELS AND WORKSHEETS 

The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) [42 

U.S.C. 9601 et seq.], as amended by the Superfund Amendments and Reauthorization Act 

(SARA) [Pub. L. 99-4991, 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 substancespecific 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 (l-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. 
3,3’-DICHLOROBENZIDINE 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, MRLs 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 E29, Atlanta, Georgia 30333. 
3,3’-DICHLOROBENZIDINE A-3 

APPENDIX A 

MINIMAL RISK LEVEL (MRL) WORKSHEET 

No MRLs were derived for 3,3’-dichlorobenzidine. 

3,3’-DICHLOROBENZIDINE 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 (2-1, 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), LowestObserved-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 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. 
3,3’-DICHLOROBENZIDINE B-2 

APPENDIX B 

(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, 1 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 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. MRLs are not derived from Serious LOAELs. 

(10)	 Reference The complete reference citation is given in chapter 8 of the profile. 
3,3’-DICHLOROBENZIDINE B-3 

APPENDIX B
 

(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 footnotes. Footnote “b” indicates the NOAEL of 3 ppm in key number 18 

was used to derive an MRL 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 opencircle symbol indicates to a 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) Kev to LSE Figure The Key explains the abbreviations and symbols used in the figure. 


3,3’-DICHLOROBENZIDINE B-6 

APPENDIX B
 

Chapter 2 (Section 2.5) 

Relevance to Public Health 

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. 

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). 
3,3’-DICHLOROBENZIDINE B-7 

APPENDIX B
 

To derive an MRL, ATSDR generally selects the most sensitive endpoint which, in its best 

judgment, 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 (UF) of 10 must be employed. Additional uncertainty 

factors of 10 must be used both for human variability to protect sensitive subpopulations (people 

who are most susceptible to the health effects caused by the substance) and for interspecies 

variability (extrapolation from animals to humans). In deriving an MRL, these individual 

uncertainty factors are multiplied together. The product is then divided into the inhalation 

concentration or oral dosage selected from the study. Uncertainty factors used in developing a 

substance-specific MRL are provided in the footnotes of the LSE Tables.