TOXICOLOGICAL PROFILE FOR 4,4'-METHYLENEBIS(2-CHLOROANILINE) MBOCA Prepared by: Clement International Corporation Under Contract No. 205-88-0608 Prepared for: U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Agency for Toxic Substances and Disease Registry May 1994 MBOCA ii CAT. FOR PUBLIC HEALTH 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. MBOCA iii UPDATE STATEMENT 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 RA 2Y 7 |99¢ Fu BL FOREWORD The Superfund Amendments and Reauthorization Act (SARA) of 1986 (Public Law 99-499) extended and amended the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or Superfund). This public law directed the Agency for Toxic Substances and Disease Registry (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 Environmental Protection Agency (EPA). The revised list of the 275 most hazardous substances was published in the Federal Register on October 28, 1992 (57 FR 48801). For prior versions of the list of substances, sce Federal Register notices dated April 17, 1987 (52 FR 12866); October 20, 1988 (53 FR 41280); October 26, 1989 (54 FR 43619); October 17, 1990 (55 FR 42067); and October 17, 1991 (56 FR 52166). Section 104(i)(3) of CERCLA, as amended, directs the Administrator of ATSDR to prepare a toxicological profile for each substance on the list. Each profile must include the following: (A) The examination, summary, and interpretation of available toxicological information and epidemiological evaluations on a hazardous substance in order to ascertain the levels of significant human exposure for the substance and the associated acute, subacute, and chronic health effects. (B) A determination of whether adequate information on the health effects of each substance is available or in the process of development to determine levels of exposure that present a significant risk to human health of acute, subacute, and chronic health effects. (C) Where appropriate, identification of toxicological testing needed to identify the types or levels of exposure that may present significant risk of adverse health effects in humans. This toxicological profile is prepared in accordance with guidelines developed by ATSDR and EPA. The original guidelines were published in the Federal Register on April 17, 1987. Each profile will be revised and republished as necessary. The ATSDR toxicological profile is intended to succinctly characterize the toxicological and adverse health effects information for the hazardous substance being described. Each profile identifies and reviews the key literature (that has been peer-reviewed) that describes a hazardous substance’s toxicological properties. Other pertinent literature is also presented, but described in less detail than the key studies. The profile is not intended to be an exhaustive document; however, more comprehensive sources of specialty information are referenced. Each toxicological profile begins with a public health statement, that describes in nontechnical language, a substance’s relevant toxicological properties. Following the public health statement is information concerning levels of significant human exposure and, where known, significant health effects. The adequacy of information to determine a substance’s health effects is described in a health effects summary. Data needs that are of significance to protect public health will be identified by ATSDR and EPA. The focus of the profiles is on health and toxicological information; therefore, we have included this information in the beginning of the document. Foreword The principal audiences for the toxicological profiles are health professionals at the federal, state, and local levels, interested private sector organizations and groups, and members of the public. This profile reflects our assessment of all relevant toxicological testing and information that has been peer reviewed. It has been reviewed by scientists from ATSDR, the Centers for Disease Control and Prevention (CDC), and other federal agencies. It bas also been reviewed by a panel of nongovernment peer reviewers and was made available for public review. Final responsibility for the contents and views expressed in this toxicological profile resides with ATSDR. David Satcher, M.D., Ph.D. Administrator Agency for Toxic Substances and Disease Registry MBOCA Vii CONTRIBUTORS CHEMICAL MANAGER(S)/AUTHOR(S): Penny Duerksen-Hughes, Ph.D. ATSDR, Division of Toxicology, Atlanta, GA Vera Brankovan, Ph.D. Clement International Corporation THE PROFILE HAS UNDERGONE THE FOLLOWING ATSDR INTERNAL REVIEWS: 1. Green Border Review. Green Border Review assures the consistency with ATSDR policy. 2. 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. 3. Minimal Risk Level Review. The Minimal Risk Level Workgroup considers issues relevant to substance-related minimal risk levels (MRLs), reviews the health effects database of each profile, and makes recommendations for derivation of MRLs. 4. Quality Assurance Review. The Quality Assurance Branch assures that consistency across profiles is maintained, identifies any significant problems in format or content, and establishes that Guidance has been followed. MBOCA ix PEER REVIEW A peer review panel was assembled for MBOCA. The panel consisted of the following members: 1. Dr. W. Emmett Braselton, Jr., Professor of Pharmacology/Toxicology, Michigan State University, East Lansing, Michigan. 2. Dr. Arthur Gregory, private consultant, Techto Enterprises, Sterling, Virginia. 3. Dr. Fumio Matsumura, Assistant Director of Toxic Substances Program, Institute of Toxicology and Environmental Health, University of California, Davis, California. These experts collectively have knowledge of MBOCA's physical and chemical properties, toxicokinetics, key health end points, mechanisms of action, human and animal exposure, and quantification of risk to humans. All reviewers were selected in conformity with the conditions for peer review specified in Section 104(i)(13) of the Comprehensive Environmental Response, Compensation, and Liability Act, as amended. Scientists from the Agency for Toxic Substances and Disease Registry (ATSDR) have reviewed the peer reviewers’ comments and determined which comments will be included in the profile. A listing of the peer reviewers’ comments not incorporated in the profile, with a brief explanation of the rationale for their exclusion, exists as part of the administrative record for this compound. A list of databases reviewed and a list of unpublished documents cited are also included in the administrative record. The citation of the peer review panel should not be understood to imply its approval of the profile’s final content. The responsibility for the content of this profile lies with the ATSDR. MBOCA xi CONTENTS UPDATE STATEMENT eee iil FOREWORD . . oe v CONTRIBUTORS ee ee ees vii PEER REVIEW ix LIST OF FIGURES . . . eee XV LIST OF TABLES . «oe eee xvii I. PUBLIC HEALTH STATEMENT ©... ns 1 1.1 WHAT IS MBOCAY? eens 1 1.2 WHAT HAPPENS TO MBOCA WHEN IT ENTERS THE ENVIRONMENT? ees 1 1.4 HOW CAN MBOCA ENTER AND LEAVE MY BODY? ................... 2 1.5 HOW CAN MBOCA AFFECT MY HEALTH? ........................... 2 1.6 IS THERE A MEDICAL TEST TO DETERMINE WHETHER I HAVE BEEN EXPOSED TO MBOCA? ..................... PIPE 3 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN HEALTH? ............... 3 1.8 WHERE CAN I GET MORE INFORMATION? .......................... 3 2. HEALTH EFFECTS... eee 5 2.1 INTRODUCTION . . «eee 5 2.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE ......... 5 2.2.1 Inhalation Exposure ............. i 6 22.1.1 Death ..... 7 2.2.1.2 Systemic Effects . ........ 7 2.2.1.3 Immunological Effects ................................ 8 2.2.1.4 Neurological Effects .................................... 8 2.2.1.5 Reproductive Effects ............. 8 2.2.1.6 Developmental Effects .................................. 8 2.2.1.7 Genotoxic Effects ......... 8 22.1.8 CANCE «veut 8 22.2 Oral EXpOsure . . . o.oo 9 222.1 Death ... 9 2.222 Systemic Effects . ........ 9 2.2.2.3 Immunological Effects ................................... 11 2.2.2.4 Neurological Effects ................ 11 2.2.25 Reproductive Effects ........... 12 22.2.6 Developmental Effects ..................... 12 2.2.2.7 Genotoxic Effects ......... 12 222.8 CANCEI «ttt 13 2.23 Dermal Exposure ....... LL... 21 MBOCA 3. 4. xii 223.1 Death .. 21 22.3.2 Systemic Effects... . Lo. 21 2.23.3 Immunological Effects ............. 22 2.23.4 Neurological Effects ........... 22 2.2.3.5 Reproductive Effects ....... ii 22 223.6 Developmental Effects .............. LL. 22 2.23.7 Genotoxic Effects ...... 22 2238 Cancer ..... oo... 22 23 TOXICOKINETICS . eee 23 23.1 Absorption... 25 2.3.1.1 Inhalation Exposure ............. .... LL... 25 23.1.2 Oral Exposure ....... 26 23.1.3 Dermal Exposure ......... 26 23.2 Distribution . . ....L 27 2.3.2.1 Inhalation Exposure ............ a... 27 23.22 Oral EXposure ...... oo. 27 23.23 Dermal Exposure ........ 28 2.3.24 Other Routes of Exposure ........................ ........ 28 23.3 Metabolism ..... LL 28 2.3.3.1 [Inhalation Exposure ............. Li... 29 23.3.2 Oral EXposure . ...... oot 29 23.3.3 Dermal Exposure . ...... 30 2.3.4 EXCretion ..... oii 31 2.3.4.1 [Inhalation Exposure .................................... 31 23.42 Oral Exposure ........ i 32 23.43 Dermal Exposure ......... 33 2.3.44 Other Routes of Exposure ................................ 34 2.3.5 Mechanisms of Action .......... 35 2.4 RELEVANCE TO PUBLICHEALTH .................................. 36 2.5 BIOMARKERS OF EXPOSURE AND EFFECT .......................... 48 2.5.1 Biomarkers Used to Identify or Quantify Exposure to MBOCA ........... 49 2.5.2 Biomarkers Used to Characterize Effects Caused by MBOCA ............ 49 2.6 INTERACTIONS WITH OTHER CHEMICALS .......................... 50 2.7 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE ................ 50 2.8 METHODS FOR REDUCING TOXIC EFFECTS ......................... S51 2.8.1 Reducing Peak Absorption Following Exposure . ...................... 51 2.8.2 Reducing Body Burden .......... 52 2.8.3 Interfering with the Mechanism of Action for Toxic Effects . ............. 53 29 ADEQUACY OF THE DATABASE ........ a. 53 29.1 Existing Information on Health Effects of MBOCA ................... 53 PP 53 2.93 On-going Studies . . . . «oo 59 CHEMICAL AND PHYSICAL INFORMATION .............................. 61 3.1 CHEMICAL IDENTITY . ... 61 3.2 PHYSICAL AND CHEMICAL PROPERTIES ............................ 61 PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL .................... 65 4.1 PRODUCTION ©... 65 MBOCA xiii 42 IMPORT/EXPORT . .. ss 65 43 USE 65 4.4 DISPOSAL . .. 67 5. POTENTIAL FOR HUMAN EXPOSURE ............ i. 69 5.1 OVERVIEW . Lo. 69 5.2 RELEASES TO THE ENVIRONMENT ......... i. 69 S21 AIT Lo 69 522 WALT ooo 69 523 Soil oe 69 53 ENVIRONMENTAL FATE . 72 5.3.1 Transport and Partitioning ...................................... 72 5.3.2 Transformation and Degradation .................................. 72 S320 Ar 72 5322 Water ....... 72 5.3.23 Sediment and Soil .......... 73 5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT ........ 73 SAT AIT 73 SA2 Waar . o.oo 74 5.4.3 Sediment and Soil ......... 74 5.4.4 Other Environmental Media ................ an... 74 5.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE ............ 74 5.6 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES ............... 76 5.7 ADEQUACY OF THE DATABASE . ..... i. 76 5.7.1 Identification of Data Needs ............... nnn. . 76 5.7.2 On-going Studies . .......... 78 6. ANALYTICAL METHODS . . . . sd 79 6.1 BIOLOGICAL MATERIALS . ..... 79 6.2 ENVIRONMENTAL SAMPLES . . . 86 6.3 ADEQUACY OF THE DATABASE . . . . 91 6.3.1 Identification of Data Needs ............... een... 91 6.3.2 On-going Studies . ....... 92 7. REGULATIONS AND ADVISORIES . .... 93 8. REFERENCES . 97 9. GLOSSARY . «i 117 APPENDICES A. USER'S GUIDE . ... A-1 MBOCA i" LIST OF FIGURES 2-1 Levels of Significant Exposure to MBOCA - Oral .............................. 20 2-2 Proposed Metabolic Pathway of MBOCA .................................... 24 2-3 Existing Information on Health Effects of MBOCA ............................ 54 5-1 Frequency of NPL Sites with MBOCA Contamination .......................... 70 MBOCA xvii LIST OF TABLES Levels of Significant Exposure to MBOCA - Oral .............................. 16 Genotoxicity of MBOCA In Vivo ....... eee 41 Genotoxicity of MBOCA In Vitro ......... ieee 42 Chemical Identity of MBOCA ©... 62 Physical and Chemical Properties of MBOCA ................................. 63 Facilities That Manufacture or Process MBOCA ............................... 66 Releases to the Environment from Facilities That Manufacture or Process MBOCA ..................... 71 Analytical Methods for Determining MBOCA in Biological Materials ............... 80 Analytical Methods for Determining MBOCA in Environmental Samples ............ 87 Regulations and Guidelines Applicable to MBOCA ............................ 94 MBOCA 1 1. PUBLIC HEALTH STATEMENT This Statement was prepared to give you information about 4,4-methylenebis(2- chloroaniline) (MBOCA) and to emphasize the human health effects that may result from exposure to it. The Environmental Protection Agency (EPA) has identified 1,350 hazardous waste sites as the most serious in the nation. These sites comprise the "National Priorities List" (NPL): Those sites which are targeted for long-term federal cleanup activities. MBOCA has been found in at least 4 of the sites on the NPL. However, the number of NPL sites evaluated for MBOCA is not known. As EPA evaluates more sites, the number of sites at which MBOCA is found may increase. This information is important because exposure to MBOCA may cause harmful health effects and because these sites are potential or actual sources of exposure to MBOCA. 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 can be exposed to a substance only when you come in contact with it. You may be exposed by breathing, eating, or drinking substances containing the substance or by skin contact with it. If you are exposed to a substance such as MBOCA, many factors will determine whether harmful health effects will occur and what the type and severity of those health effects will be. These factors include the dose (how much), the duration (how long), the route or pathway by which you are exposed (breathing, eating, drinking, or skin contact), the other chemicals to which you are exposed, and your individual characteristics, such as age, gender, nutritional status, family traits, life-style, and state of health. 1.1 WHAT IS MBOCA? MBOCA is a synthetic chemical used in industry primarily to produce castable polyurethane parts. It also has a coating application in chemical reactions to "set" glues, plastics, and adhesives. Since plastics have many uses, MBOCA is used very widely. Other names for MBOCA include 4,4’-methylenebis(2-chloroaniline), bis amine, DACPM, MCA, methylenebis ortho chloroaniline, and MOCA. The name MBOCA comes from methylene bis ortho chloro aniline. Pure MBOCA is a colorless solid, but MBOCA is usually made and used as yellow, tan, or brown pellets. If MBOCA is heated above 205°C, it may decompose. MBOCA has no odor or taste. Chapter 3 contains more information on the chemical and physical properties of MBOCA. Chapter 4 contains more information on its production and use. 1.2 WHAT HAPPENS TO MBOCA WHEN IT ENTERS THE ENVIRONMENT? MBOCA may enter the environment through disposal of solid waste from manufacturing plants that use MBOCA in castable polyurethane processing. MBOCA is not likely to evaporate from the soil or water into the air. However, it may enter the air as dust when it is made at production plants or during mixing and grinding operations at polyurethane plants. It may enter surface waters from the waste streams of these plants. Some of the MBOCA 2 1. PUBLIC HEALTH STATEMENT MBOCA may be broken down by sunlight or by microscopic organisms. Chapter 4 contains more information on the production, use, and disposal of MBOCA, while Chapter 5 has information on the release of MBOCA to the environment. 1.3 HOW MIGHT | BE EXPOSED TO MBOCA? Most exposure to MBOCA occurs in the workplace. If you work with MBOCA, you may breathe small particles of it in the air or get it on your skin if you brush against a surface covered by MBOCA dust. There are several ways to be exposed to MBOCA outside of the workplace. For example, you may be exposed to MBOCA if you live in an area where the soil is contaminated with MBOCA. You may also be exposed if you eat foods grown in soils that contain MBOCA. However, you are unlikely to drink water contaminated with MBOCA because it does not dissolve in water. Chapter 5 contains more information on the potential exposure of humans to MBOCA. 1.4 HOW CAN MBOCA ENTER AND LEAVE MY BODY? MBOCA can enter your bloodstream if you breathe it in the air, eat it, or get it on your skin. Results of studies in humans and animals show that MBOCA can enter your body very quickly through the skin or lungs. Once MBOCA is in your body, most of it leaves your body quickly. MBOCA and its breakdown products leave the body through urine and feces. Results of studies in humans and animals show that most MBOCA leaves the body within a few days of exposure. The small amount of MBOCA that may remain in your body after you are exposed is likely to break down or leave your body at a slower rate. Chapter 2 contains more information on how MBOCA can enter and leave the body. 1.5 HOW CAN MBOCA AFFECT MY HEALTH? Studies of human exposure suggest that the small amounts of MBOCA usually found in the air or on surfaces in or near factories do not cause toxic effects. However, it is possible that acute exposure to a large amount of MBOCA, such as an industrial accident, may produce effects that we do not know very much about. Information on how MBOCA can affect your health is very limited, and we do not know if there are any other long-term human health effects of exposure to MBOCA. MBOCA is suspected of causing bladder cancer and is considered a probable human carcinogen. Information is being gathered to determine whether bladder cancer in humans may result from short-, medium-, or long-term exposures to MBOCA. We do not know if MBOCA causes birth defects in humans. Chapter 2 contains more information on the human health effects of MBOCA. Studies in animals show that MBOCA can be harmful to the blood cells and livers of dogs and rats. MBOCA also causes lung, liver, breast, and bladder cancers. The Department of Health and Human Services, the International Agency for Research on Cancer, and EPA consider MBOCA to be a probable human carcinogen. MBOCA 3 1. PUBLIC HEALTH STATEMENT 1.6 IS THERE A MEDICAL TEST TO DETERMINE WHETHER | HAVE BEEN EXPOSED TO MBOCA? Your urine can be tested for MBOCA within a few hours of exposure. This test, however, will not detect MBOCA days after exposure has ceased. This test may not be readily available in your doctor’s office. Chapters 2 and 6 contain more information on testing for MBOCA in humans. 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN HEALTH? The government has developed regulations and guidelines for handling MBOCA. They are designed to protect the public from the potential harmful health effects of this chemical. EPA has classified MBOCA as a hazardous waste that must meet specific disposal requirements. The Occupational Safety and Health Administration (OSHA) regulates MBOCA in the workplace. The maximum allowable amount of MBOCA in workroom air, assuming an 8- hour workday and a 40-hour workweek, is 0.22 milligrams per cubic meter. However, the court has suspended such regulations for the present. Chapter 7 contains more information on international, national, and state regulations and guidelines for handling MBOCA in air, water, and other environments. 1.8 WHERE CAN | 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, E-29 Atlanta, Georgia 30333 (404) 639-6000 This agency can also provide you with information on the location of occupational and environmental health clinics. These clinics specialize in the identification, evaluation, and treatment of illness resulting from exposure to hazardous substances. MBOCA 5 2. HEALTH EFFECTS 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 4,4’- methylene-bis(2-chloroaniline) (MBOCA). It contains descriptions and evaluations of toxicological and epidemiological investigations and provides conclusions, where possible, on the relevance of MBOCA toxicity to public health. A glossary and list of acronyms, abbreviations, and symbols can be found at the end of this profile. MBOCA is an aromatic diamine that is used as a curing agent for polyurethane and epoxy resins. It has some of the general toxicity characteristics of aromatic amines. However, methemoglobinemia and the resulting cyanosis, hallmark symptoms of exposure to aromatic amines, are reported with much less frequency after exposure to MBOCA (Linch et al. 1971). The majority of studies on MBOCA were undertaken to investigate its potential to cause cancer in different animal species because MBOCA was suspected of being a human carcinogen. A large number of studies focused on tumor incidence and did not investigate other toxic effects of MBOCA. Another limitation of the animal studies is the information on acute toxicity. An acute effect of MBOCA in the dog was marked methemoglobinemia after a single oral dose (Barnes 1964). Information on intermediate and chronic exposure, however, indicates that MBOCA is carcinogenic in animals. 2.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE To help public health professionals and others address the needs of persons living or working near hazardous waste sites, the information in this section is organized first by route of exposure — inhalation, oral, and dermal; and then by health effect — death, systemic, immunological, neurological, reproductive, developmental, genotoxic, and carcinogenic effects. These data are discussed in terms of three exposure periods — acute (14 days or less), intermediate (15-364 days), and chronic (365 days or more). Levels of significant exposure (LSE) for each route and duration are presented in Table 2-1 and illustrated in Figure (2-1). The points in the figure showing no-observable-adverse-effect levels (NOAELSs) or lowest-observable-adverse-effect levels (LOAELS ) reflect the actual doses (levels of exposure) used in the studies. LOAELSs 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 a significant dysfunction. However, the Agency has established guidelines and policies that are used to classify these end points, which can be found in Tables 2-1, 2-2, 2-3, and 2-4. ATSDR believes that there is sufficient merit in this approach to warrant an attempt at distinguishing between "less serious” and "serious" effects. The distinction between "less serious” and "serious" effects MBOCA 6 2. HEALTH 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 NOAELSs 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 LSE tables and figures may differ depending on the user’s perspective. Public health officials and others concerned with taking appropriate actions to take at hazardous waste sites may want information on levels of exposure associated with more subtle effects in humans or animals (LOAELs) or exposure levels below which no adverse effects (NOAELS) have been observed. Estimates of levels posing minimal risk to humans (Minimal Risk Levels of MRLs) may be of interest to health professionals and citizens alike. Levels of exposure associated with carcinogenic effects (Cancer Effect Levels, CELs) of MBOCA are reported 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° to 1077), as developed by EPA. Estimates of exposure levels posing minimal risk to humans (Minimal Risk Levels of MRLs) have been made for MBOCA. An MRL is defined as an estimate of daily human exposure to a substance that is likely to be without an appreciable risk of adverse effects (noncarcinogenic) over a specified duration of exposure. MRLs are derived when reliable and sufficient data exist to identify the target organ(s) of effect or the most sensitive health effect(s) for a specific duration within a given route of exposure. MRLs are based on noncancerous health effects only and do not consider carcinogenic effects. MRLs can be derived for acute, intermediate, and chronic duration exposures for inhalation and oral routes. Appropriate methodology does not exist to develop MRLs for dermal exposure. Although methods have been established to derive these levels (Barnes and Dourson 1988; EPA 1989a), uncertainties are associated with these techniques. Furthermore, ATSDR acknowledges additional uncertainties inherent in the application of the procedures to derive less than lifetime MRLs. As an example, acute inhalation MRLs may not be protective for health effects that are delayed in development or are acquired following repeated acute insults, such as hypersensitivity reactions, asthma, or chronic bronchitis. As these kinds of health effects data become available and methods to assess levels of significant human exposure improve, these MRLs will be revised. A User's Guide has been provided at the end of this profile (see Appendix A) to aid in the interpretation of the tables and figures for Levels of Significant Exposure ant the MRLs. 2.2.1 Inhalation Exposure The studies presented in the section on inhalation exposure address occupational or accidental exposures. In occupational settings, dermal, inhalation and oral exposures often occur simultaneously. Therefore, many of the effects described in this section may be partially due to dermal or oral exposures to MBOCA. Exact levels of occupational and accidental exposures are not known. For this reason, the results in this section are not presented in table or figure form. MBOCA 7 2. HEALTH EFFECTS 2.2.1.1 Death No studies were located regarding death in humans or animals after inhalation exposure to MBOCA. A literature search did not reveal any human or animal studies that reported death due to MBOCA inhalation. 2.2.1.2 Systemic Effects No studies were located regarding respiratory, cardiovascular, hematological, musculoskeletal, or hepatic effects in humans or animals after inhalation exposure to MBOCA. Other systemic effects observed following inhalation exposure are discussed below. Gastrointestinal Effects. Information on the gastrointestinal effects of MBOCA following inhalation exposure is extremely limited. In a case study of accidental occupational exposure, a worker complained of feeling ill in the stomach after he was sprayed in the face with molten MBOCA (Hosein and Van Roosmalen 1978). Some of the MBOCA entered his mouth. He was wearing overalls, gloves, and safety glasses, but no respirator or face shield. He washed his face and eyes immediately. No follow-up information was given about his condition. There was no information on the dose to which he was exposed. No studies were located regarding gastrointestinal effects in animals after inhalation exposure to MBOCA. Renal Effects. Five hours after a worker was accidentally sprayed in the face with molten MBOCA, his urine contained 220 mg/L of protein, indicating damage to the renal tubules (Hosein and Van Roosmalen 1978). However, 11 hours after the accident, there was only a trace of protein in the urine. Two urine specimens collected within 24 hours after the accident had low specific gravities, suggesting that damage to the renal tubules was transitory. The level of exposure was not reported. In a retrospective bladder cancer incidence study conducted with 532 workers exposed to MBOCA from 1968 to 1979 and 20 workers who were first employed in 1980 and 1981, 385 participated in a urine screening test (Ward et al. 1990). Exfoliative cytology revealed that the morning urine samples obtained from 21 workers contained atypical cells. No cytology readings were positive for cancer. Urine samples obtained from 16 workers were positive for heme (Ward et al. 1990). Almost 90% of the exposed workers were males, and the median duration of exposure was greater than 3 months. This study gathered information on a relatively large group of workers exposed solely to MBOCA (and not to benzidine and B-naphthylamine, which can confound exposures in other occupational settings) and because it alerted medical professionals to the limitations of follow-up procedures generally used after MBOCA exposure. In the course of screening, one participant who was negative for cytology and heme impairment was diagnosed with bladder cancer. Cystoscopy was introduced as a part of the follow-up procedure, and two additional low-grade papillary tumors of the bladder were detected. Data on MBOCA in urine from workers in companies belonging to the Polyurethane Manufacturers Association (PMA) confirmed the importance of biological monitoring (Lowry and Clapp 1992). The percentage of urine samples with <25 pg MBOCA/L increased from 77% in 1985 to 86% in 1990. The percentage of urine samples with >5 pg MBOCA/L decreased from 12% in 1985 to 8% in 1990. No studies were located regarding renal effects in animals after inhalation exposure to MBOCA. MBOCA 8 2. HEALTH EFFECTS Dermal/Ocular Effects. Very limited information is available regarding dermal/ocular effects after inhalation exposure to MBOCA. When a worker was accidentally sprayed with molten MBOCA, he experienced burning of the face and eyes shortly thereafter (Hosein and Van Roosmalen 1978). The worker was wearing gloves and safety glasses but no respirator or face shield, so the exposure may have occurred by ingestion or dermal absorption as well. The limitations of this study are: (1) no further follow-up information was provided on the worker’s condition; (2) the dose of MBOCA was not known; (3) exposure involved mixed routes; and (4) only one exposed individual was described. No studies were located regarding dermal/ocular effects in animals after inhalation exposure to MBOCA. No studies were located regarding the following health effects in humans or animals after inhalation exposure to MBOCA: 2.2.1.3 Immunological Effects 2.2.1.4 Neurological Effects 2.2.1.5 Reproductive Effects 2.2.1.6 Developmental Effects 2.2.1.7 Genotoxic Effects No studies were located regarding genotoxic effects in humans after inhalation exposure to MBOCA. The sex-linked recessive lethal (SLRL) test in Drosophila melanogaster was used to monitor the air in the vulcanizing, weighing, calendaring, and molding areas of a rubber plant (Donner et al. 1983). Male flies that were exposed in the molding area exhibited a small but significant increase (p<0.05) in SLRL genotoxicity. Exposure data were not provided, but high ambient exposures were expected to occur in the tested areas. Acute lethality to fruit flies in the molding area was 50%; sterility was not reported. Concomitant exposures to other chemicals may also have occurred. Other genotoxicity studies are discussed in Section 2.4. 2.2.1.8 Cancer MBOCA is suspected of being a human carcinogen because its chemical structure is similar to a known human bladder carcinogen, benzidine, and to that of a potent animal carcinogen, 3,3’-dichloro- benzidine (Osorio et al. 1990; Ward et al. 1990). A limited number of epidemiological studies were found that examined the incidence of cancer in workers exposed to MBOCA. Workers who were exposed to MBOCA for a median duration of employment of 3.2 months (between 1968 and 1981) were examined for bladder cancer (Ward et al. 1990). Of the 200 workers examined, 3 men were diagnosed with bladder tumors by cystoscopy: 1 with a papillary tumor, and 2 with low-grade papillary transitional cell tumors. Two of the men were less than 30 years old. The interval between the time of first exposure and diagnosis was 11.5 years; the latency period for most bladder carcinogens is about 20 years (Ward et al. 1990). Since bladder carcinoma in young men is very uncommon, this finding increases the concern that MBOCA may cause bladder tumors in humans. A limitation of this study is that there were no controls (there were no data on the incidence of bladder tumors diagnosed by cystoscopy in an unexposed population). The study did not provide statistical evidence for an association between bladder tumor incidence and MBOCA exposure in workers. The statistical significance of three cases of bladder tumors could not be evaluated. There is no information on the MBOCA 9 2. HEALTH EFFECTS daily amount of MBOCA to which workers were exposed, and a possibility exists of coexposure to other chemicals such as 4,4’-methylenedianiline, 4-chloro ortho-toluidine, aniline, and ortho-toluidine (Hogan 1993; Ward 1993). MBOCA may not be solely responsible for the observed carcinogenic effects. No studies were located regarding cancer in animals after inhalation exposure to MBOCA. 2.2.2 Oral Exposure Very little information on acute oral toxicity of MBOCA in humans or animals is available. Information on the long-term health effects of MBOCA in humans is also limited. However, several chronic oral exposure studies in mice (Russfield et al. 1975), rats (Kommineni et al. 1979; Stula et al. 1975), and dogs (Stula et al. 1977) clearly demonstrate the carcinogenic potential of MBOCA in these species. These studies are discussed in Section 2.2.2.8. 2.2.2.1 Death No studies were located regarding increased mortality in humans from any cause following exposure to MBOCA. Decreased lifespan has been noted in rats after chronic exposure to MBOCA (Kommineni et al. 1979). Male Sprague-Dawley rats were fed a standard diet (with 27% protein) containing 0, 12.5, 25, or 50 mg/kg/day of MBOCA or a protein-deficient diet (with 8% protein) containing 0, 6.25, 12.5, or 25 mg/kg/day of MBOCA for 18 months. Decreased lifespan was observed in the rats fed 25 mg/kg/day of MBOCA in either the standard or protein-deficient diet. Rats fed the standard diet containing the highest concentration of MBOCA, 50 mg/kg/day, also experienced a shortened lifespan. Similar decrease in lifespan was observed in Wistar II rats fed an average dose of 54 mg/kg/day of MBOCA for 500 days in a low-protein diet (Grundmann and Steinhoff 1970). The first death in females occurred on day 200 of treatment and in males on day 390 of treatment; the mean lifespan in control females was 535 days and in males 565 days. In another study, one of six female beagle dogs died after 3.4 years of oral administration of an average dose of 10 mg/kg/day of MBOCA (Stula et al. 1977). However, the report concludes that the death was not MBOCA-related, because the dog died from pyelonephritis. The report did not discuss any possible connection between MBOCA administration and pyelonephritis. No additional deaths were reported for the five remaining dogs that were part of the same 9-year study (Stula et al. 1977). The LOAEL value for death in rats for chronic exposure is reported in Table 2-1 and Figure 2-1. 2.2.2.2 Systemic Effects No studies were located regarding cardiovascular or musculoskeletal effects in humans or animals after oral exposure to MBOCA. The systemic effects observed after oral exposure are discussed below. The highest NOAEL values and all LOAEL values from each reliable study for systemic effects in cach species for chronic duration are presented in Table 2-1 and Figure 2-1. MBOCA 10 2. HEALTH EFFECTS Respiratory Effects. No studies were located regarding respiratory effects in humans after oral exposure to MBOCA. Lung adenomatosis was found in rats fed 25 (low dose) or 50 (high dose) mg/kg/day of MBOCA for 18 months. Three of 22 rats from the low-dose group and 4 of 19 from the high-dose group developed this preneoplastic condition (Russfield et al. 1975). Although this finding was not statistically significant, it is important because exposure to MBOCA induces preneoplastic lesions in the lungs. Gastrointestinal Effects. Extremely limited information is available regarding gastrointestinal effects of MBOCA after oral exposure. In a case study of an accidental occupational exposure, a worker complained of feeling ill in the stomach after his face was sprayed with molten MBOCA that also entered his mouth (Hosein and Van Roosmalen 1978). He wore overalls, gloves, and safety glasses but no respirator or face shield. He washed his face and eyes immediately after the exposure. No further information was given about his condition. As with other occupational exposure studies, there was no information on the dose to which he was exposed. Another limitation of this study is the possibility that some of the MBOCA was inhaled or dermally absorbed. No studies were located regarding gastrointestinal effects in animals following oral exposure to MBOCA. Hematological Effects. No studies were located regarding hematological effects in humans after oral exposure to MBOCA. No changes in hemoglobin, hematocrit, erythrocyte count, or mononuclear leukocyte count were noted in female beagle dogs after 9 years of exposure to MBOCA. Dogs were fed MBOCA at a concentration of 10 mg/kg/day, 3 days/week, for the first 6 weeks of the study and then an average of 10 mg/kg/day, 5S days/week, for the remaining 9 years (Stula et al. 1977). The analyses were performed only twice a year, so the reported observations may not be meaningful. Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to MBOCA. While no information is available on acute or intermediate exposure, evidence of adverse hepatic effects was seen in rats and dogs after chronic exposure to MBOCA. Several adaptive changes occurred in the livers of rats fed 50 mg/kg/day of MBOCA for 2 years. These changes included hepatomegaly, fatty change, necrosis, fibrosis, and bile duct proliferation (Stula et al. 1975). Similar changes were seen in dogs fed 10 mg/kg/day of MBOCA 3 days/week for the first 6 weeks and then an average of 10 mg/kg/day, 5 days/week, for 9 years (Stula et al. 1977). Histopathology revealed nodular hepatic hyperplasia and disruption of liver architecture in three of six MBOCA-treated dogs but not in controls. Another indication of liver damage was a statistically significant increase in serum glutamic-pyruvic transaminase (SGPT) in MBOCA-treated dogs. The highest levels of SGPT occurred during the first 2 years and after 7.5-8 years of treatment (Stula et al. 1977). Based on the occurrence of hepatic effects in dogs exposed to MBOCA, a chronic-duration MRL of 0.003 mg/kg/day has been calculated from the LOAEL of 10 mg/kg/day (Stula et al. 1977) as described in the footnote on Table 2-1. Renal Effects. There is very limited information regarding renal effects in humans after oral MBOCA 11 2. HEALTH EFFECTS exposure to MBOCA. Five hours after an acute exposure to molten MBOCA, in which MBOCA was accidentally sprayed over the worker’s face and some entered his mouth, a urine sample from the exposed worker contained 220 mg/L of protein indicating an immediate, transient effect on tubular reabsorption of low molecular weight proteins (Hosein and Van Roosmalen 1978). However, 11 hours after the accident, there was only a trace of protein in the urine of this worker. The urine specimens collected in the course of 24 hours after the accident had low specific gravity, indicating possible transient damage to the renal tubules and an attendant inability to concentrate urine. Dermal/Ocular Effects. There is very little information on dermal/ocular effects after oral exposure to MBOCA. In one occupational exposure study, a worker was accidentally sprayed with molten MBOCA, and some of the compound entered his mouth. The worker complained of burning face and eyes shortly after exposure (Hosein and Van Roosmalen 1978). At the time of the accident the worker was wearing gloves and safety glasses, but he had no respirator or face shield. It is therefore likely that exposure also occurred by inhalation and dermal absorption. This study is limited because only one exposed individual was described, and no further information was given regarding the worker’s condition, the dose of MBOCA, or the precise exposure route. No studies were located regarding dermal/ocular effects in animals after exposure to MBOCA. Other Systemic Effects. No studies were located regarding other systemic effects in humans after oral exposure to MBOCA. In rats fed 25 or 50 mg/kg/day of MBOCA-hydrochloride for 18 months, the average body weight was 50 g and 100 g lower, respectively, than the body weight of controls at the end of the treatment period. The study does not provide the body weights for experimental animals at either dose level. During the first 20-25 weeks of the experiment, however, there was no difference in food consumption between MBOCA-treated animals and control animals (Russfield et al. 1975). The major limitation of this study is that data on weight changes are incomplete, which makes the interpretation difficult. 2.2.2.3 Immunological Effects No studies were located regarding immunological effects in humans or animals after oral exposure to MBOCA. 2.2.2.4 Neurological Effects No studies were located regarding neurological effects in humans after oral exposure to MBOCA. Cystic hyperplasia of the pars intermedia of the anterior pituitary gland was found in one of five female beagle dogs after 8.3 years of treatment with an average dose of 10 mg/kg/day of MBOCA (Stula et al. 1977). This change was not present in other treated dogs or in controls and was not considered to be treatment related. The dog also had a papillary transitional cell carcinoma of the urinary bladder. The limitations of this study are the lack of correlation between pituitary hyperplasia and transitional cell bladder carcinoma and the small number of animals used. MBOCA 12 2. HEALTH EFFECTS 2.2.2.5 Reproductive Effects No studies were located regarding reproductive effects in humans or animals after oral exposure to MBOCA. 2.2.2.6 Developmental Effects No studies were located regarding developmental effects in humans or animals after oral exposure to MBOCA. 2.2.2.7 Genotoxic Effects No studies were located regarding genotoxic effects in humans after oral exposure to MBOCA. D. melanogaster larvae, heterozygous for visible wing mutations, were fed a medium made with a 5-millimolar aqueous solution of MBOCA for 48 hours (Kugler-Steigmeier et al. 1989) to determine whether MBOCA exposure could induce recombinations and somatic mutations in fruit flies. The resulting adults exhibited a small but statistically significant increase in small single wing spots. Higher concentrations could not be tested because of MBOCA'’s extremely limited solubility. Because the number of large single spots was not increased, the authors concluded that the somatic mutations occurred late in development. Similarly, the authors concluded, because of the absence of induced twin spots, that MBOCA did not exhibit strong recombinogenic activity. In an SLRL test, adult D. melanogaster were fed diets containing MBOCA at a concentration of 3.75- or 7.5-millimoles for 3 days (Vogel et al. 1981). A statistically significant increase in SLRL test values was observed at the 7.5-millimolar concentration in one of two tests. The experiment was incomplete because mating was attempted only once after exposure; therefore, germ cells were sampled at only one stage of development. The method of feeding makes concomitant dermal exposure possible in both experiments. However, MBOCA is expected to be too non-polar to penetrate through the larval integument. Radioactive deoxyribonucleic acid (DNA) adducts were identified in rats administered 75 mg/kg of MBOCA labeled with '*C at the methylene carbon by gavage (Cheever et al. 1990). The highest level of radioactive adducts was found in the liver (49.11 pmol/mg DNA at 24 hours). Covalent binding to bladder and lymphocyte DNA was also observed (about 7 and 0.9 pmol/mg DNA, respectively, at 24 hours). In a separate experiment, in which rats were exposed to 1.4-30 mg/kg [*C-methylene]-MBOCA by gavage. Significant quantities of DNA adducts were found in the liver and lung, marginal amounts were found in the kidney, and no adducts were detected in the bladder (Kugler-Steigmeier et al. 1989). Enzymatic hydrolysis of the DNA showed that two DNA adducts were generated in the liver and two different adducts were found in the lung. Kugler-Steigmeier et al. (1989) did not observe significant binding to kidney or bladder DNA because the quantity of DNA isolated from these tissues was too small to measure the amount of covalently bound MBOCA to DNA in these tissues. The research results suggest that oral exposures to MBOCA induce vascular and liver neoplasms in mice (Russfield et al. 1975), lung and liver tumors in rats (Kommineni et al. 1979; Russfield et al. 1975; Stula et al. 1975), and bladder tumors in dogs (Stula et al. 1977), as discussed in Section 2.2.2.8. Other genotoxicity studies are discussed in Section 2.4. MBOCA 13 2. HEALTH EFFECTS 2.2.2.8 Cancer MBOCA is suspected of being a human carcinogen because its chemical structure is similar to that of a known human bladder carcinogen, benzidine, and to that of a potent animal carcinogen, 3,3’-dichloro-benzidine (Osorio et al. 1990). One epidemiological study examined the incidence of cancer in workers exposed to MBOCA. Workers who were occupationally exposed to MBOCA for a median employment period of 3.2 months (between 1968 and 1981) were examined for bladder tumors (Ward et al. 1990). Although the route of exposure was not specified in the report, the most likely exposure pathways were inhalation and dermal. The oral route cannot be excluded, however, since some of the inhaled dust to the lungs could be subsequently swallowed. Low-grade papillary transitional cell tumor of the urinary bladder was diagnosed by cystoscopy in 2 out of 200 examined workers, and 1 was diagnosed with a full-blown papillary bladder tumor. Two of the men were less than 30 years old. The interval between the time of first exposure and the initiation of the study averaged 11.5 years, while the latency period for most bladder carcinogens is about 20 years (Ward et al. 1990). Since bladder carcinoma in young men is very uncommon, this finding increases the concern that MBOCA has a potential for causing bladder cancer in humans. A limitation of this study is that there were no controls (there are no data on the incidence of bladder tumors diagnosed by cystoscopy in an asymptomatic nonexposed population). There was also no evidence for statistical associations regarding bladder tumor incidence in exposed workers. There is also no information on the daily dose of MBOCA to which the workers were exposed or about the route of exposure. Further investigations of this occupational exposure revealed the possibility of coexposure to other chemicals such as 4,4’-methylenedianiline, 4-chloro ortho-toluidine, aniline, and ortho-toluidine (Hogan 1993; Ward 1993). The dose and duration of exposure to these chemicals is not known. It is therefore difficult to estimate the exact contribution of MBOCA to the observed carcinogenic effects in the occupationally exposed workers. No studies were located on cancer in animals after acute or intermediate oral exposure to MBOCA. Several studies in rats, mice, and dogs reported finding a carcinogenic response after chronic exposure to MBOCA. Lungs, liver, breast, and urinary bladder were the main target organs for cancer in these species. A dose-dependent increase in lung tumors was observed in Sprague-Dawley rats fed 12.5, 25, or 50 mg/kg/day of MBOCA for 18 months; the incidence of lung tumors was 23%, 37%, and 70%, respectively (Kommineni et al. 1979). A similar observation (presence of lung adenocarcinomas) was made in Charles River rats fed 50 mg/kg/day of MBOCA for 2 years (Stula et al. 1975). In another study, male Charles River CD-1 rats were fed 25 or 50 mg/kg/day of MBOCA for 18 months. The authors reported finding lung adenocarcinoma in 1/22 and 1/19 animals, respectively (Russfield et al. 1975), although this finding was not statistically significant. In addition, 3 of 22 and 4 of 19 rats receiving 25 or 50 mg/kg/day of MBOCA, respectively, developed adenomatosis, which is a prencoplastic lesion (Russfield et al. 1975). A limitation of this study is the small number of animals on which the tumor incidence was based. All these studies were done with animals fed standard- protein diets. In contrast, the incidence of lung tumors in rats was lower if animals were fed MBOCA in a protein-deficient diet. In rats fed 6.25, 12, or 25 mg/kg/day of MBOCA in a low- protein diet for 18 months, the incidences of lung tumors were 6%, 15%, and 26%, respectively, while no tumors were found in control animals (Kommineni et al 1979). These numbers are less than half of the incidences reported for comparable doses of MBOCA given to rats fed a standard diet. Similar observations were made in rats fed a higher dose of MBOCA (50 mg/kg/day for 2 years); the MBOCA 14 2. HEALTH EFFECTS incidence of lung tumors in rats fed a protein-deficient diet was roughly one-half of that observed in rats fed a standard-protein diet (Stula et al. 1975). In Wistar II rats fed an average of 54 mg/kg/day of MBOCA in a low-protein diet for 500 days, 32% of males and 12% of females developed lung tumors (Grundmann and Steinhoff 1970). These results indicate that, in general, rats given MBOCA in a low-protein diet, have a decreased incidence of lung adenocarcinomas when compared to rats given MBOCA in a standard-protein diet. Some exceptions to this generalization occur. Species, strain, and gender may also play a role. MBOCA also induces liver tumors in rats. An increase in the incidence of hepatomas was found in Charles River CD rats fed 25 or 50 mg/kg/day of MBOCA in a standard-protein diet for 18 months; the incidences were 5% and 21%, respectively (not statistically significant) (Russfield et al. 1975), compared to 4% and 36% found in Sprague-Dawley rats fed the same diet and the same amount of MBOCA (Kommineni et al. 1979). Similar findings were made in random-bred female albino mice (derived from HaM/ICR mice in 1959) fed 130 or 260 mg/kg/day of MBOCA for 18 months and then observed for an additional 6 months while on a control diet. They had a significantly increased incidence of hepatomas, 43% in the 130-mg/kg/day group and 50% in the 260-mg/kg/day group (Russfield et al. 1975). The incidence of hepatomas in treated random-bred male albino mice (derived from HaM/ICR mice in 1959) was not significantly different from that in controls. These results indicate that in mice there is a gender difference regarding MBOCA-induced hepatomas; female mice are affected, and male mice are not affected. The effect of standard- and low-protein diets on the incidence of MBOCA-induced hepatocellular carcinomas was investigated in Sprague- Dawley and Charles River rats (Kommineni et al. 1979; Stula et al. 1975). The results are inconclusive. In Sprague-Dawley rats fed 12.5 or 25 mg/kg/day of MBOCA in a standard-protein diet for 18 months, the incidences of hepatocellular carcinomas were 3% and 4%, respectively; in rats fed the same amounts of MBOCA in a protein-deficient diet, the incidences were 0% and 18%, respectively (Kommineni et al. 1979). These results indicate that a protein-deficient diet did not reduce the MBOCA-induced incidence of hepatocellular carcinoma in Sprague-Dawley rats. When male Charles River rats were fed 50 mg/kg/day of MBOCA for 2 years, however, the incidences of hepatocellular carcinomas were 7% in rats fed a standard-protein diet and 52% in rats fed a low- protein diet (Stula et al. 1975). This observation, that the protein-deficient diet accentuates the carcinogenic effects of MBOCA, was not seen in female rats that had 7% and 5% hepatocellular carcinomas when fed standard-protein and low-protein diets, respectively (Stula et al. 1975). However, in Wistar II rats fed an average dose of 54 mg/kg/day of MBOCA in a low-protein diet for 500 days, liver cancer was present in 88% of males and 72% of females (Grundmann and Steinhoff 1970). The results suggest that gender and strain of rats play a role in the effect of low-protein diet on MBOCA-induced incidence of hepatocellular carcinomas. Furthermore, the possible contrasting effects of a protein-deficient diet on MBOCA-induced lung and liver tumors suggests different induction mechanisms for the formation of these two tumors by MBOCA (Kommineni et al. 1979). Another target organ for MBOCA carcinogenesis is the urinary bladder. Six female beagle dogs were fed an average of 10 mg/kg/day of MBOCA for 9 years. One beagle died approximately 3.4 years of pyelonephritis unassociated with MBOCA exposure. Of the five surviving dogs, three developed papillary transitional cell carcinomas of the urinary bladder; and one dog had a combined urethral adenocarcinoma and transitional cell carcinoma (Stula et al. 1977). Despite the small number of animals used, this study demonstrates that ingestion of MBOCA over 9 years was associated with the appearance of carcinomas of the urinary bladder and urethra in dogs. A statistically significant increase of malignant mammary tumors was found in female Sprague-Dawley rats fed 50 mg/kg/day of MBOCA in a low-protein diet for 2 years (Stula et al. 1975). A similar MBOCA 15 2. HEALTH EFFECTS finding was made in male rats fed 25 mg/kg/day of MBOCA in a low-protein diet for 18 months; the incidence of mammary tumors was 6% (Kommineni et al. 1979). When rats were fed a standard diet, the incidence of mammary adenocarcinoma was 28% at 50 mg/kg/day of MBOCA and 11% at 25 mg/kg/day (Kommineni et al. 1979). Zymbal’s gland carcinomas were found in 12% of rats fed a low- protein diet and in 7% of rats fed a standard-protein diet; both diets contained 25 mg/kg/day MBOCA (Kommineni et al. 1979). The results in rats fed MBOCA in low- and standard-protein diets indicate that there is a dose-related increase in the incidence of lung and mammary carcinomas, while the incidence of hepatocellular carcinoma is not dose related (Kommineni et al. 1979). Other tumor types were also found after chronic oral administration of MBOCA. The incidence of hemangiosarcomas was 4% and 8% in Sprague-Dawley rats fed 25 mg/kg/day of MBOCA in standard- protein or low-protein diets, respectively (Kommineni et al. 1979). In another study, vascular tumors (generally subcutaneous hemangiomas and hemangiosarcomas) were present in randomly bred male albino mice (derived from the HaM/ICR strain in 1959) fed 130 or 260 mg/kg/day of MBOCA for 18 months; the incidences were 23% and 40%, respectively (Russfield et al. 1975). In female mice, vascular tumors (43%) were present only in the group treated with the high dose of 260 mg/kg/day of MBOCA. The incidence of pituitary adenomas (including adenocarcinomas) in Sprague-Dawley rats fed a standard-protein diet was reduced, especially in animals treated with high doses of MBOCA (Kommineni et al 1979). Rats fed 12.5, 25, or 50 mg/kg/day of MBOCA for 18 months had 36%, 25% (statistically significant), and 4% (statistically significant) incidences of pituitary adenomas, respectively. The incidence of pituitary adenomas in the control group was 42% (Kommineni et al. 1979). A statistically significant decrease in pituitary tumors was also observed in female, but not male, ChR-CD rats (Charles River Cesarean Delivered, Sprague-Dawley origin, random-bred albino rats) fed a standard-protein diet with 50 mg/kg/day of MBOCA for 2 years (Stula et al. 1975). This decrease in incidence of pituitary tumors was not as consistent in Sprague-Dawley rats fed a low- protein diet; the incidences were 16%, 12% (statistically significant), and 20% in animals fed 6.25, 12.5, and 25 mg/kg/day, respectively, for 18 months (Kommineni et al. 1979). In the control group, the incidence of pituitary adenomas was 23%. Neither the certainty of these findings nor the mechanism by which they could occur are known at the present time. The precise mechanism of chemical carcinogenesis is not known, but it is thought that in some instances it involves the formation of chemical adducts in the genetic material through covalent binding of nucleophilic sites by electrophilic parent compounds or their activated electrophilic metabolites (Cheever et al. 1991). Therefore, it seems that the capacity of MBOCA to form adducts with tissue DNA (Cheever et al. 1990), hemoglobin (Cheever et al. 1991; Sabbioni and Neumann 1990), and globin and serum albumin (Cheever et al. 1991) in rats may play a role in its toxicity and carcinogenicity. The lowest doses that produced tumors (CELs) are presented in Table 2-1 and plotted in Figure 2-1. TABLE 2-1. Levels of Significant Exposure to 4,4'-Methylenebis(2-chloroanaline) MBOCA - Oral Exposure LOAEL (effect) Key to duration/ NOAEL Less serious Serious figure? Species Route frequency System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference CHRONIC EXPOSURE Death 1 Rat (F) 18 mo 25 (23/50 males died Kommineni et al. 1 x/d after 72 weeks) 1979 Systemic 2 Rat 18 mo Resp 50 (adenomatosis) Russfield et al. Other 25 (6.5% lighter than 1975 control) 3 Rat (Fy 2yr Hepatic 50 (hepatocytomegaly, Stula et al. ad lib fatty change, 1975 necrosis, fibro- sis, bile duct proliferation) 4 Dog (Cc) 9 yr Hepatic 10° (increased GPT, Stula et al. 5d/wk nodular hepatic 1977 1x/d hyperplasia) S103443 H1TV3H 2 vOO8W gi TABLE 2-1. Levels of Significant Exposure to 4,4'-Methylenebis(2-chloroanaline) (continued) Exposure LOAEL (effect) Key to duration/ Less serious Serious figure® Species Route frequency (mg/kg/day) (mg/kg/day) Reference Cancer 5 Rat (F) 2 yr 50 (CEL-lung adenocar- Stula et al. ad lib cinoma, liver 1975 hepatocellular adenomas and carcinomas, mammary fibroadenomas and adenocarcinomas) 6 Rat (F) 500d 54 (CEL- liver hepato- Grundmann and 1x/d cellular carcinoma Steinhoff 1970 in 88% of males and 72% of females; primary lung adenomatosis in 32% of males and 12% of females) 7 Rat (F) 18 mo 6.25 (CEL-6% incidence of Kommineni et al. 1 x/d lung adeno- 1979 carcinomas) 12.5 (CEL-5% incidence of Zymbal's gland carcinoma, 15% lung adeno- carcinomas) 25 (CEL-26% incidence of lung adeno- carcinomas; 6% incidence of mammary adeno- carcinomas; 12% incidence of Zymbal's gland carcinoma; 18% incidence of hepatocellular carcinomas; 8% incidence of hemangiosarcomas) S103443 H1TVaH 2 YOO8W LL TABLE 2-1. Levels of Significant Exposure to &4,4'-Methylenebis(2-chloroanaline) (continued) Exposure LOAEL (effect) Key to duration/ NOAEL Less serious Serious figure® Species Route frequency System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 8 Rat (F) 18 mo 1 x/d 9 Rat 18 mo 12.5 (CEL-23% incidence of lung adeno- carcinoma tumors; 8% incidence of Zymbal's gland carcinoma) 25 (CEL-37% incidence of lung adeno- carcinomas; 11% incidence of mammary adenocar- cinomas) 50 (CEL-70% incidence of lung adeno- carcinomas; 28% incidence of mammary adenocarcinomas; 22% incidence of Zymbal's gland carcinomas; 36% incidence in hepatic carcino- mas; 8% incidence of skin hemangio- sarcomas) 25 (CEL-12% incidence of lung adeno- matosis; 4% incidence of bladder transition cell tumor) Kommineni et al. 1979 Russfield et al. 1975 S103443 H1TV3H 2 vYOOgW St TABLE 2-1. Levels of Significant Exposure to 4,4'-Methylenebis(2-chloroanaline) (continued) Exposure LOAEL (effect) Key to duration/ NOAEL Less serious Serious figure? Species Route frequency System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 10 Mouse (F) 18 mo 130 (CEL-hepatomas in Russfield et al. 43% of females) 1975 260 (CEL-hepatomas in 50% of females) 130 (CEL- subcutaneous hemangiomas and hemangiosarcomas in 23% of males) 260 (CEL-subcutaneous hemangiomas and hemangiosarcomas in 43% females and 40% males) 1" Dog (Cy 9yr 10 (papanicolaou IV Stula et al. 5d/wk and V - indicating 1977 1x/d neoplasia of genitourinary system) 10 (CEL- combined transitional cell carcinoma and adenocarcinoma of the urethra - 1/5 dogs; papillary transitional cell carcinoma of the urinary bladder - 3/5 dogs) .The number corresponds to entries in Figure 2-2. Used to derive a chronic oral Minimal Risk Level (MRL) of 0.003 mg/kg/day; dose divided by an uncertainty factor of 3,000 (10 for use of a LOAEL, 10 for interspecies extrapolation, 10 for human variability, and 3 for limitations in the database) ad lib = ad libitum; (C) = capsule; CEL = cancer effect level; d = days; (F) = feed; GPT = glutamate pyruvate transaminase; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; papanicolaou = cytologic evaluation for cancer detection and diagnosis using a multichromatic stain; wk = week(s); x = time(s); yr = year(s) S103443 H1TV3H 2 vOOgn Si FIGURE 2-1. Levels of Significant Exposure to 4,4’-Methylenebis (2-chloroaniline) (MBOCA) - Oral CHRONIC (> 365 Days) Systemic S © S & & & & FF 3 (mghkg/day) —— is 1,000 p= 1m @iom 100 F @iom @iom @®x @®3 @sr Gor Por @n ®2r On Ps Oo 10 Quad @11d ®11d or So 1 on 1 1 A 1 ' 1 01 1 1 1 I 0.01 | ! 1 Kh 0.001 I Key r Rat @ LOAEL for serious effects (animals) : Minimal risk level f m Mouse (DP LOAEL for less serious effects (animals) 1 or other than ar d Dog @ CEL - Cancer Effect Level (animals) 1 / The number next to each point corresponds to entries in Table 2-1. * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. S103443 H1TV3H ¢ vOOgaWN oc MBOCA 21 2. HEALTH EFFECTS 2.2.3 Dermal Exposure 2.2.3.1 Death No studies were located regarding death in humans or animals after dermal exposure to MBOCA. 2.2.3.2 Systemic Effects No studies were located regarding respiratory, cardiovascular, hematological, musculoskeletal, or hepatic effects in humans or animals after dermal exposure to MBOCA. The systemic effects observed after dermal exposure are discussed below. Gastrointestinal Effects. Extremely limited information is available regarding gastrointestinal ctfects of MBOCA after dermal exposure. In a case study of an accidental occupational exposure, a worker complained of feeling ill in the stomach after his face was sprayed with molten MBOCA that also entered his mouth (Hosein and Van Roosmalen 1978). He wore overalls, gloves, and safety glasses but no respirator or face shield. He washed his face and eyes immediately after the incident. No further information was given about his condition. As with other occupational exposure studies, there was no information on the dose to which he was exposed. Another limitation of this study is the likelihood that some of the MBOCA was inhaled or ingested. No information was located regarding gastrointestinal effects in animals after dermal exposure to MBOCA. Renal Effects. In a retrospective bladder cancer incidence study conducted among 532 workers exposed to MBOCA from 1968 to 1979 and 20 workers who were first employed in 1980 and 1981, 385 participated in the urine screening test (Ward et al. 1990). Exfoliative cytology revealed that 21 urine samples contained atypical cells. No cytology readings were positive for cancer. Sixteen urine samples were positive for heme (Ward et al. 1990). Almost 90% of the exposed workers were males, and the median duration of exposure was 3.2 months. This study is important because it gathered information on a relatively large group of workers exposed to MBOCA and alerted medical professionals to the limitations of follow-up procedures used after MBOCA exposure. In the course of the screening, one of the participants who had negative cytology and was negative for the presence of heme in the urine was diagnosed with a tumor of the urinary bladder. Because of this development, cystoscopy was introduced as a part of the follow-up procedure, and two additional low- grade papillary tumors of the bladder were detected. The analysis of data on free urinary MBOCA from workers in companies of the PMA confirmed the importance of biological monitoring (Lowry and Clapp 1992). Cumulative results for 1985-1990 show an increase in percentage of urinary samples containing below 25 pg/L of MBOCA (from 77% in 1985 to 86% in 1990) and a decrease in percentage of samples with over 5 pg/L of MBOCA (from 12% in 1985 to 8% in 1990). These findings indicate that wearing protective clothing, using MBOCA in pellets not powder, monitoring indoor air for MBOCA, and other protective measures may play an important role in controlling occupational exposures. Five hours after an acute exposure in which a worker was accidentally sprayed in the face with molten MBOCA, a urine sample contained 220 mg/L of protein, indicating possible damage to renal tubules (Hossein and Van Roosmalen 1978). There was only a trace amount of protein in the urine of this worker 11 hours after the accident. The two urine specimens collected over the course of 24 hours after the accident had low specific gravity indicating possible transient damage to the renal tubules. MBOCA 22 2. HEALTH EFFECTS No studies were located regarding renal effects in animals after dermal exposure to MBOCA. Dermal/Ocular Effects. Very limited information is available regarding dermal/ocular effects after dermal exposure to MBOCA. In a case study, one worker was accidentally sprayed in the face with molten MBOCA. Some of the compound entered his mouth. The worker complained of burning face and eyes shortly after exposure (Hossein and Van Roosmalen 1978). The worker was wearing gloves and safety glasses but no respirator or face shield. Therefore, it is likely that the exposure may have also occurred by inhalation or oral absorption. The limitations of this study are that no further information was provided regarding the worker’s condition, the dose of MBOCA, or the precise exposure route, and that only one exposed individual was described. In another case of accidental exposure to approximately 11.3 L of molten MBOCA, the worker described a burning sensation on his skin. He was sprayed over his chest, abdomen, and extremities (NIOSH 19864; Osorio et al. 1990). Some exposure by inhalation and ingestion was also likely in this case. No studies were located regarding dermal/ocular effects in animals after dermal exposure to MBOCA. No studies were located regarding the following health effects in humans or animals after dermal exposure to MBOCA: 2.2.3.3 Immunological 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 No studies were located regarding genotoxic effects in humans following dermal exposure to MBOCA. Radiolabeled MBOCA was applied to a 36- cm? shaved skin patch on the dorsal side of rats at a dose of 75 mg/kg. DNA adduct formation was determined for various tissues at intervals ranging from 1 to 29 days after exposure (Cheever et al. 1990). Throughout the study, less than 14% of the applied dose was absorbed. Adduct formation was about 100-fold less than after oral exposure to the same dose, but adducts were found in the same tissues with the same order of binding (liver > bladder > lymphocytes). This indicates that MBOCA was absorbed through the skin to react with DNA in target organs. Two studies of the genotoxicity of MBOCA in D. melanogaster also involved concomitant oral and dermal exposure and were discussed under oral exposure in Section 2.2.2.7. Other genotoxicity studies are discussed in Section 2.4. 2.2.3.8 Cancer A limited number of epidemiological studies were located that examined the incidence of cancer in workers exposed to MBOCA. Workers who were exposed to MBOCA for a median employment period of 3.2 months (between 1968 and 1981) were examined for bladder tumors (Ward et al. 1990). After cystoscopy revealed a papillary cell tumor in one worker, a low-grade papillary transitional cell tumor of the urinary bladder was diagnosed by cystoscopy in 2 of the remaining 200 workers examined. Two of the men were less than 30 years old. The interval between the time of first exposure and the initiation of the study averaged 11.5 years, while the latency period for most bladder carcinogens is about 20 years (Ward et al. 1990). Since bladder tumors in young men arc very uncommon, this finding increases the concern that MBOCA has a potential for causing bladder MBOCA 23 2. HEALTH EFFECTS cancer in humans. A limitation of this study is that there were no controls (there are no data on the incidence of bladder tumors diagnosed by cystoscopy in an asymptomatic nonexposed population). The daily dose of MBOCA to which workers were exposed, was only estimated by assigning an exposure score of 0-20. Further investigation of this occupational exposure raised a possibility of coexposure to other chemicals such as 4’4-methylenedianiline, 4-chloro o-toluidine, aniline, and o- toluidine (Hogan 1993; Ward 1993). The dose and duration of exposure to these chemicals could only be estimated on the basis worker recollection. It is therefore difficult to estimate the exact contribution of MBOCA to carcinogenic effects observed in occupationally exposed workers. No studies were located regarding cancer in animals after dermal exposure to MBOCA. 2.3 TOXICOKINETICS Limited information is available regarding absorption of MBOCA. Following acute inhalation/dermal exposure to very low concentrations of MBOCA, absorption appears to be rapid, based on the presence of MBOCA in the urine of occupationally exposed workers (Ichikawa et al. 1990). The urine of a worker accidentally exposed to molten MBOCA, contained 1,700 ppb of MBOCA 9 hours later indicating absorption and distribution following dermal and/or inhalation exposure (NIOSH 1986a; Osorio et al. 1990). The distribution of MBOCA in laboratory animals is relatively uniform. In rats, the highest amount of MBOCA and its metabolites (total 4C radioactivity) was found in the liver following an acute P.O. injection or intermediate, gavage, exposure (Cheever et al. 1991). The tissue distribution of radioactivity following the acute exposure, was as follows: liver > kidney > lung > spleen > urinary bladder > testes > brain > lymphocytes (Cheever et al. 1991). The highest concentration of radioactivity in dogs following acute dermal exposure to radiolabeled MBOCA was in the bile (Manis et al. 1984). Detectable concentrations were also found in the liver, kidney, and fat. These results suggest that the distribution of MBOCA is similar in rats and dogs following oral and dermal exposures. A distribution study in rats following a single intravenous exposure to MBOCA, further support to these observations (Tobes et al. 1983). MBOCA metabolism can proceed via several pathways: N-acetylation, N-hydroxylation, which may be followed by N-oxidation, and ring hydroxylation (see Figure 2-2). Some of these processes may be followed by conjugation. These are considered important pathways with regard to carcinogenicity (as cited in Cheever et al. 1991). Results of in vitro studies suggest that MBOCA rapidly penetrates the skin but is not metabolized in the process (Chin et al. 1983). After inhalation of low levels, excretion of MBOCA has been monitored by measuring the amount in urine. Excretion occurs rapidly during the first 48 hours (NIOSH 1986a). Following an accidental exposure to 11.34 L of molten MBOCA, 1,700 ppb of MBOCA was found in the worker’s urine four hours later (NIOSH 1986a; Osorio et al. 1990). This result suggests the rapid excretion of MBOCA after acute dermal and/or inhalation exposure. It is not clear, however, if urine is indeed the primary route of excretion in humans, since no information was presented on the amount of MBOCA contained in the feces. In rats that received a single oral dose of 10 mg/kg of radiolabeled MBOCA, 60% of the radioactivity was found in the feces (Farmer et al. 1981; Groth et al. 1984). Little information is available on the kinetics of urinary MBOCA excretion in humans. One study found that 23 hours after an acute occupational exposure to MBOCA, 50% of this material was excreted in the urine (Osorio et al. 1990). However, other work (NIOSH 1986b) indicates that FIGURE 2-2. Proposed Metabolic Pathway of 4,4’-Methylenebis (2-chloroaniline) (MBOCA)* MBOCA Cl Cl Cl QH | -— HaoN Oyore Owe CT RING oy a. N- TN MBOCA BENZHYDROL / /, wi} Sw Oyen oe yon & Leon war Soe N-ACETYL MBOCA, N-OH MBOCA ; 4 %/ MBOCA BENZOPHENONE © ORTHO-OH MBOCA ‘or h oc 56 i255 wae jr NL H3COC-N rye N- COCH3 H3N a Cl HoN Spin TN N,N'-DIACETYL MBOCA , MONONITROSO MBOCA 3-CHLORO-4-AMINOBENZYL AMINOCHLOROPHENOL | 56 ALCOHOL 56 > Cl \ N Or © r -Rat d -Dog gp - Guinea Pig h - Human “OH 3-CHLORO-4-AMINOBENZOIC ACID 4 * Derived from Butler et al. 1989 (1); Cheever et al. 1991 (2); Chen et al. 1989 (3); Farmer et al. 1981 (4); Kuslikis et al. 1991 (5); Morton et al. 1988 (6). S103443 H1TV3H 2 vYOO8Wn ve MBOCA 25 2. HEALTH EFFECTS clearance may be much slower, especially at higher exposures. The duration of exposure may also be a factor. The precise mechanism of carcinogenicity for MBOCA is not completely understood. Some evidence suggests, however, that the ability to form adducts with tissue DNA (Cheever et al. 1990; Sabbioni and Neumann 1990), hemoglobin (Cheever et al. 1988, 1990, 1991; Chen et al. 1991; Sabbioni and Neumann 1990), and globin and serum albumin (Cheever et al. 1991) in rats following MBOCA exposure may play a role in its carcinogenicity. 2.3.1 Absorption 2.3.1.1 Inhalation Exposure Studies that directly measure absorption in humans following inhalation exposure to known concentrations of MBOCA were not located. However, absorption was indirectly estimated in five male factory workers by measuring urinary MBOCA levels over a 5-day period (Ichikawa et al. 1990). Personal air exposure levels were obtained by continuously monitoring the breathing zone of each worker for 6-7 hours every other day. The air MBOCA levels ranged from 0.0002 to 0.0089 mg/m’. The results showed that the preshift and postshift urine MBOCA levels of the exposed workers, including the worker with the highest level of exposure, did not significantly differ. Forty-eight hours after the exposure, urinary MBOCA levels were still similar to preshift levels; the authors interpreted this finding as evidence that the biological half-life of urinary MBOCA is relatively long (Ichikawa et al. 1990). The amount of MBOCA measured in the urine was much higher than the amounts of inhaled MBOCA as estimated from personal exposure measurements. This observation suggests that a certain amount of MBOCA exposure occurs in the workplace by some additional route(s). The limitations of this study include the small sample size and the probability that dermal exposure occurred as well. In another study, 13 urine samples were collected from workers occupationally exposed to MBOCA on a Monday morning after 2 days away from work. The results showed that there was no detectable MBOCA in the urine of workers who had low MBOCA exposure(s) during the previous week. Only the urine from the worker with the highest MBOCA urine concentration (30 pg/L) the week before had detectable MBOCA. In most cases, this clearance of MBOCA after the weekend suggested that MBOCA had a relatively short retention time in the body (Clapp et al. 1991). As in the previous study, the exposure route(s) probably included inhalation and dermal. In another study of occupational exposure (combination of inhalation and dermal exposures), levels of MBOCA in the urine were determined in workers over a period of 22 months. The mean concentrations of MBOCA during that time ranged from 5.3 to 43.8 pg/L (NIOSH 1986b). Further analysis of the data revealed that urine samples with highest levels of MBOCA came from workers that are in direct daily contact with MBOCA, such as mixers and molders (NIOSH 1986b). One of the samples from a mixer was analyzed during the week (average urine MBOCA level was 29.9 pg/L) and after a 2-day weekend (average urine MBOCA level was 8.9 pg/L). This demonstrated that MBOCA was not cleared from the urine of a worker who had relatively high levels of MBOCA during the previous week (NIOSH 1986b). The reasons for the different clearance rates observed in the studies described (Clapp et al. 1991; Ichikawa et al. 1990; NIOSH 1986b) are not clear. Since relatively small numbers of workers were observed, the results may merely reflect individual differences. Alternatively, MBOCA may be excreted at different rates depending upon exposure conditions including both the rate and duration of exposure. This question needs further examination. MBOCA 26 2. HEALTH EFFECTS No studies were located regarding absorption in animals after inhalation exposure to MBOCA. 2.3.1.2 Oral Exposure No studies were located regarding absorption in humans after oral exposure to MBOCA. Several studies in animals following oral MBOCA exposure, detected MBOCA or its metabolites in body tissues, urine, and feces (Cheever et al. 1988; Farmer et al. 1981; Groth et al. 1984). When rats were given a single oral dose of *C-MBOCA by gavage, 16.5% of the dose was excreted in the urine within 72 hours, while 13.7% was retained in the tissues (Groth et al. 1984). These data indicate that some MBOCA is absorbed after oral exposure in animals. Approximately 60% remains unabsorbed in the feces (Groth et al. 1984). 2.3.1.3 Dermal Exposure Studies that directly measure MBOCA absorption in humans following dermal exposure to known concentrations of this compound were not located. However, absorption was measured in five male factory workers by measuring urinary MBOCA levels over a 5-day period (Ichikawa et al. 1990). Personal air exposure levels were obtained by continuously monitoring the breathing zone of each worker for 6-7 hours every other day. The air levels of MBOCA ranged from 0.0002 to 0.0089 mg/m>. The results showed that the preshift and postshift urine MBOCA levels did not significantly differ among exposed workers. Forty-eight hours after the exposure, the urinary MBOCA levels were similar to preshift levels suggesting that the biological half-time of MBOCA is relatively long (Ichikawa et al. 1990). The amount of MBOCA measured in urine was much higher than the amount(s) of inhaled MBOCA. The authors concluded that occupational exposures occur by other routes in addition to inhalation (in this case dermal exposure) and are important contributors to total body burdens of MBOCA. The limitations of this study include the small sample size and the probability that dermal exposure had occurred as well. In another study, 13 urine samples were collected from workers occupationally exposed to MBOCA on a Monday morning after 2 days away from work. The results showed that there was no detectable MBOCA in samples that had low MBOCA levels during the past week. Only the sample that came from the worker who had the highest average MBOCA urine concentration (30 pg/L) the week before had detectable MBOCA (Clapp et al. 1991). These results suggest that the higher initial dose of MBOCA the longer it takes to be excreted. In another occupational study the exposure resulted from a combination of inhalation and dermal routes. The levels of MBOCA in the urine of exposed workers were determined over a period of 22 months. The mean concentrations of MBOCA ranged from 5.3 to 43.8 pg/L (NIOSH 1986b). A detailed data analysis revealed that the urine samples with the highest MBOCA levels were obtained from mixers and molders, i.e., workers that are in direct daily contact with MBOCA (NIOSH 1986b). Urinary measurements of MBOCA were obtained for one worker a mixer over a 5-day work week and over a 2-day weekend. The average urine MBOCA level during the week was approximately 30 pg/L; the level dropped to 8.9 pg/mL over the weekend. This finding suggests that MBOCA was not completely excreted via urine in a worker who had relatively high levels of MBOCA during the preceeding week (NIOSH 1986b). The reasons for the different clearance rates reported in the studies described (Clapp et al. 1991; Ichikawa et al. 1990; NIOSH 1986b) are unclear. It is possible that due to the small sample size the results may merely reflect individual differences. It is also possible that MBOCA may be removed at different rates depending on the length and rate of exposure. MBOCA 27 2. HEALTH EFFECTS Differential absorption rates of 4C.MBOCA were investigated following dermal and intravenous exposures in beagle dogs (Manis et al. 1984). By comparing the levels of MBOCA excreted via the urinary and biliary systems, following percutaneous and intravenous (100% absorption) administration, the investigators calculated that, after 24 hours, only 2.4% of the applied MBOCA was absorbed through the skin. In another study in rats, 11.5-21.9% of MBOCA applied to the skin was calculated to have been absorbed within 72 hours of application (Groth et al. 1984). Using radiolabeled MBOCA and fresh human neonatal foreskin organ cultures, the absorption and penetration of MBOCA through a 7 mm x 7 mm area was evaluated over 4 hours (Chin et al. 1983). One hour after application, 46% of the radiolabeled MBOCA was detected on the skin and 0.5% was detected on the underlying membrane (the remaining radioactivity was unabsorbed and stayed on the coverglass). Four hours later, 61% was detected in the skin, 26% had passed through and was on the underlying membrane filter, while 12% remained unabsorbed. The absorption process was optimal at 37°C and decreased sharply at 0°C. These findings indicate that MBOCA penetrates the neonatal foreskin readily without being metabolized, and that the absorption process is temperature dependent (Chin et al. 1983). 2.3.2 Distribution 2.3.2.1 Inhalation Exposure No studies were located regarding distribution in humans or animals after inhalation exposure to MBOCA. 2.3.2.2 Oral Exposure No studies were located regarding distribution in humans after oral exposure to MBOCA. Twenty-four hours after oral administration of 28 pmol/kg of radioactive [methylene-4C-]MBOCA to male Sprague-Dawley rats, the highest level of radioactivity was in the liver. The tissue distribution of total radiolabels (i.e., MBOCA plus its metabolites) was as follows: liver > kidney > lung > spleen > urinary bladder > testes > brain > lymphocytes (Cheever et al. 1991). In another study, the distribution in female Wistar rats 24 hours after gavage with radioactive MBOCA was similar: liver > intestine > lung > kidney > blood > stomach > spleen > uterus (Sabbioni and Neumann 1990). The remaining radioactivity was recovered in urine and feces. After 28 consecutive oral doses of radioactive MBOCA, the radioactivity accumulated more rapidly in the liver than in the blood (1,455 femtomoles/mg versus 122 femtomoles/mg tissue, respectively) (Cheever et al. 1991). The results show that, after either acute or intermediate oral administration, the highest amount of MBOCA and its metabolites accumulates in the liver. The results also show that after 28 doses, accumulation in the liver increased more than 100-fold. Similar findings in terms of liver accumulation were found in a study in which two female LAC:Porton rats were fed 10 mg/kg of 4C-MBOCA, and MBOCA’s distribution was measured 48 hours later (Farmer et al. 1981). Again, liver had the highest amount of radioactivity, followed by white fat, blood, and kidney. This study is limited because it used only two animals. In another acute study, radiolabeled MBOCA was administered by gavage to rats by gavage (Morton et al. 1988). Forty-eight hours later, 64-87% of the administered radioactivity was recovered in the urine and feces. Excretion occurred primarily in the first 24 hours with feces containing 1.5-3 times the radioactivity of the MBOCA 28 2. HEALTH EFFECTS urine. These results are consistent with previous reports that in rats MBOCA is extensively metabolized and rapidly excreted. 2.3.2.3 Dermal Exposure Although no studies were located regarding distribution in humans after dermal exposure to MBOCA, it appears that it is rapidly eliminated from the body following dermal exposure. Nine hours after an accidental spill, the urine of a worker exposed to 11.34 L of molten MBOCA contained 1,700 ppb of the chemical (NIOSH 1986a; Osorio et al. 1990). The biological half-life of MBOCA in the human body based on the amount excreted into the urine was estimated to be 23 hours (Osorio et al. 1990). The authors assumed a one-compartment model and estimated that about 94% of the initial MBOCA would be eliminated within 4 days. They concluded that urinary excretion of MBOCA was rapid, a finding that is consistent with the observations made by Hosein and Roosmalen (1978). Although this study is limited because only one case was presented, it is important because it indirectly demonstrates that after dermal exposure of humans a large portion of MBOCA is distributed by the circulatory system to the kidneys (Osorio et al. 1990). In a study using beagle dogs dermally exposed to 0.4 mg of radioactive MBOCA/cm?, no measurable radioactivity was detected in blood or plasma up to 24 hours later (Manis et al. 1984). The highest concentration of radioactivity 24 hours post-exposure was found in the bile. No unmetabolized MBOCA was present in the bile. Detectable concentrations of radioactivity were found in the liver, kidney, fat, and lung. Urinary excretion of unmetabolized MBOCA was a small but consistent fraction, 0.4-0.5%, of the total urinary radioactivity, and the authors concluded that this may be a useful index of acute exposure. These results support the hypothesis that dermal absorption is a viable entry for MBOCA. They also demonstrate that the circulatory system distributes MBOCA to the liver, kidneys, fat, and lungs. In this study, intravenously exposed animals were used as controls in order to obtain a 100% absorbed dose baseline for comparison with the pharmacokinetic data obtained for dermal exposure. 2.3.2.4 Other Routes of Exposure One hour after a single intravenous dose of 0.49 mg/kg of radiolabeled MBOCA to rats, radioactivity was found in the tissues in the following order: small intestine > liver > fat tissue > lungs > kidneys > skin > adrenals (Tobes et al. 1983). Similar results in terms of liver accumulation were obtained in rats 48 hours after an intraperitoneal injection of 1 mg/kg of radioactive MBOCA (Farmer et al. 1981). However, only 1-2% of MBOCA and 95-97% of radiolabeled metabolites were found in the urine indicating rapid and extensive MBOCA metabolism in the rat. The finding that liver has the highest amount of radiolabel 24 hours after a single exposure is consistent with the findings from other intraperitoneal exposure studies (Cheever et al. 1991). Similar results were obtained in dogs after intravenous administration of MBOCA (Manis et al. 1984). 2.3.3 Metabolism MBOCA 29 2. HEALTH EFFECTS 2.3.3.1 Inhalation Exposure Analysis of 23 urine samples from workers occupationally exposed to MBOCA showed that all contained MBOCA, but only 10 of 23 contained N-acetyl MBOCA. The ratio of N-acetyl MBOCA to parent MBOCA ranged from 0.005 to 0.09 indicating that N-acetyl MBOCA is a relatively minor urine metabolite in humans (Cocker et al. 1988). Further analysis shows that the urine of MBOCA- exposed workers also contains a major hydrolysis-labile metabolite, probably B-N-glucuronide (Cocker et al. 1990). This metabolite, upon hydrolysis, yields two to three times more MBOCA than the amount of parent compound present in urine after exposure to MBOCA. Another metabolite detected in the urine is a heat-labile conjugate of N-acetyl MBOCA (Cocker et al. 1990). In another study of occupational exposure, 35% of the MBOCA metabolites were excreted as conjugates in the urine (Osorio et al. 1990). No studies were located regarding metabolism in animals after inhalation exposure to MBOCA. 2.3.3.2 Oral Exposure No studies were located regarding metabolism in humans after oral exposure to MBOCA. MBOCA is extensively metabolized in experimental animals (Morton et al. 1988), and its metabolism can follow several pathways: N-acetylation, N-hydroxylation/N-oxidation, and ring hydroxylation. These are important pathways with respect to the carcinogenicity of MBOCA (Cheever et al. 1991). Some of these metabolic conversions can be followed by conjugation. Carcinogenicity probably involves activation and adduct formation by MBOCA. A scheme of MBOCA metabolism and excretion in animals is presented in Figure 2-2. The microsomal P-450 enzyme system is involved in N-hydroxylation of MBOCA, which is considered to be an activation step related to adduct formation (Morton et al. 1988). Microsomes from phenobarbital-treated rats had an increased rate of MBOCA hydroxylation (Morton et al. 1988). Such effects were not obtained after treatment with methylcholanthrene. These findings were confirmed using human liver microsomes and purified rat liver cytochrome P-450 monooxygenases showing that the activation of MBOCA is catalyzed by the phenobarbital-inducible enzymes (Butler et al. 1989). More recently, it was shown that much higher binding of MBOCA to hemoglobin occurred in phenobarbital-treated rats (Cheever et al. 1991). These results suggest that enzymatic induction by phenobarbital leads to increased capacity of MBOCA and/or its metabolites to covalently bind to proteins and probably to DNA. After acute intragastric exposure of male rats to radiolabeled MBOCA, the levels of MBOCA and its metabolites were determined in the urine. The level of MBOCA was higher when urine was treated with exogenous glucuronidase or sulfatase, indicating the presence of conjugates (Morton et al. 1988). The conjugates, which are highly polar, can be digested with a sulphatase-glucuronidase mixture leading to 30-50% of deconjugation. When glucuronidase alone was tested it had a small effect on conjugates (Farmer et al. 1981). These findings suggest that a large proportion of the conjugates consists of sulfates (Farmer et al. 1981). The metabolism of MBOCA was also investigated in vitro by incubating human and rat liver microsomes with '*C-MBOCA. The formation of metabolites was quantified using appropriate chemically synthesized standards (Morton et al. 1988). The rate of N-hydroxylation of MBOCA, an obligatory step in metabolic activation of aromatic amines, was higher in rat than in human microsomes (Morton et al. 1988). Rat liver microsomes were also found to be more efficient in o- hydroxy-MBOCA formation when compared with human microsomes (see Figure 2-2). The same in MBOCA 30 2. HEALTH EFFECTS vitro microsomal system was used to elucidate the role of hepatic cytochrome P-450 monooxygenases in metabolic oxidation and detoxification of MBOCA (Butler et al. 1989). The analysis of 22 human liver microsome preparations showed that there was variation in N-oxidation of MBOCA by different preparations, and analysis of metabolism catalyzed by different rat isozymes showed that the process was catalyzed by phenobarbital-inducible cytochrome P-450 species. Since MBOCA is considered a potential human carcinogen, this result indicates that individual profiles of cytochromes P-450 may be important determinants of an individual's susceptibility to MBOCA carcinogenesis (Butler et al. 1989). Hydroxylation by liver microsomes results in two major metabolites, N-hydroxy- and o-hydroxy- MBOCA (Chen et al. 1989; Morton et al. 1988) (see Figure 2-2). The formation of these two metabolites was evaluated using canine, guinea pig, and rat liver microsomes (Chen et al. 1989). These results indicate that there are species differences in the oxidation of MBOCA. The major metabolite in the guinea pig liver microsome system was N-hydroxy MBOCA, while dog microsomes oxidized MBOCA to the o-hydroxylated metabolite with significant amounts of hydroxylamine. In the rat liver microsome system, other polar metabolites were predominant, while there were fewer N- and o-hydroxylated MBOCA derivatives (Chen et al. 1989). 2.3.3.3 Dermal Exposure MBOCA metabolites were investigated in urine samples from workers occupationally exposed to MBOCA (Cocker et al. 1988, 1990). Of 23 urine samples, small amounts of N-acetyl MBOCA were present in only 10 samples, even after heat treatment, while MBOCA was present in all of the samples (Cocker et al. 1988). A similar observation was made by Ducos et al. (1985) who found that the level of N-acetyl MBOCA in urine was less than 10% of the level of MBOCA recovered in urine of exposed workers. Skin absorption of MBOCA was considered an important factor in both these studies. In a further attempt to identify the heat-labile MBOCA urine metabolites, Cocker et al. (1990) compared them to the chemically synthesized glucuronide. The results indicate that the major heat-labile conjugate of MBOCA in the urine of exposed workers is probably the p-N-glucuronide of the unmetabolized compound. MBOCA glucuronide spontaneously decomposes at 37°C within 24 hours to yield the unmetabolized MBOCA (Cocker et al. 1990). Studies in dogs (Manis et al. 1984) and rats (Groth et al. 1984) indicate that MBOCA is rapidly metabolized following dermal exposure to '*C-MBOCA and that urinary levels of unmetabolized MBOCA represents only a small fraction of the total (MBOCA plus metabolites) excreted in urine. Twenty-four hours after exposure, 1.3% of the dose applied to the skin of dogs was excreted in the urine as total radioactivity, of which only 0.005% represented unmetabolized MBOCA. Similarly, in rats after 72 hours, 2.54% of the amount applied to the skin was excreted in the urine as total radioactivity, while only 0.008% represented unmetabolized MBOCA. The major DNA adduct of MBOCA was identified by a combination of in vivo and in vitro studies (Silk et al. 1989). Male rats derived from a Wistar strain were exposed intraperitoneally to 89 pmol/kg of radioactive MBOCA or 141 pmol/kg of acetyl-MBOCA. Twenty-four hours later, animals were sacrificed, and their liver DNA was extracted and analyzed for the amount of bound radioactivity and DNA adduct formation. Results showed that the cleavage of the bond between the methylene bridge and one aromatic nucleus of MBOCA resulted in the formation of N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol, the major in vivo adduct. Two other adducts were also formed but were not characterized. MBOCA 31 2. HEALTH EFFECTS 2.3.4 Excretion 2.3.4.1 Inhalation Exposure MBOCA has been analyzed and detected in the urine of exposed humans. Nine hours after an accidental spill, the urine of a worker exposed to 11.34 L of molten MBOCA contained 1,700 ppb of MBOCA (NIOSH 1986a; Osorio et al. 1990). The biological half-life of MBOCA in the urine was estimated to be approximately 23 hours (Osorio et al. 1990). This was determined by measuring the levels of MBOCA in urine 2 weeks after exposure ceased. Assuming a one-compartment model, about 94% of the initial dose of MBOCA would be eliminated within 4 days. In another report, after an acute occupational exposure to molten MBOCA, a worker complained of burning eyes and face. Hot, liquid MBOCA was sprayed over his face and some entered his mouth (Hosein and Van Roosmalen 1978). The worker was monitored and his urine was analyzed for MBOCA over a 3-week period. Five hours after the spill, the level of MBOCA in the urine was 3.6 mg/L (3,600 ppb). MBOCA was rapidly excreted during the first 18 hours after exposure. Twenty-four hours after exposure, urine MBOCA level was down to 0.03 mg/L (30 ppb), and 3 weeks later it was below that level (Hosein and Van Roosmalen 1978). It was unclear from this study whether urine was the primary route of MBOCA excretion in humans following dermal exposure since no information was presented on the amount of MBOCA measured in feces. Analysis of urine samples from 34 exposed workers showed that the urine levels of MBOCA were proportional to the exposure levels. Workers exposed to the highest levels of MBOCA (MBOCA mixers) had the highest level of the compound in the urine (NIOSH 1986b). Ten out of 10 mixers had a urinary MBOCA level of >5 pg/L and 5 of 10 had >50 pg/L of MBOCA in their urine. Urine samples from workers in the molding department (intermediate exposure) had levels of MBOCA ranging from <5 to 50 pg/L. All trimmers and supervisors (the lowest exposure group) had no detectable levels of MBOCA in the urine. The body clearance of MBOCA was tested in 13 urine samples from workers who were absent from exposure for 48 hours. Only one of the 13 workers, an MBOCA mixer, had a detectable MBOCA urine level (average: 8.9 pg/L) (NIOSH 1986b). The level of MBOCA in the air was <3 pg/m> most of the time, and the results of the wipe test ranged from 0.035 to 53 pg. The limitations of this study are that precise exposure levels and the exact routes of exposure were not given and that there were too few data points for any general conclusions to be made. In another study on five male workers exposed to MBOCA for 3-27 years, MBOCA levels in preshift (before starting work) and postshift (after work) urine samples were not significantly different (Ichikawa et al. 1990). Also, the MBOCA urine levels 48 hours after exposure were similar to preshift levels. The study authors concluded that the biological half-time of urinary MBOCA is relatively long. The breathing zone levels of MBOCA were 0.0002-0.089 mg/m”. Limitations of this study include a small sample size and lack of controls. The MBOCA levels in urine have been used to assess occupational exposure of workers engaged in the manufacture of polyurethane elastomers (Thomas and Wilson 1984). The objective of the study was also to identify areas that could be improved in order to reduce the exposure. Between 12 and 15 workers were followed over a 5-year period, and the exposure levels were reported as the monthly average amount of nmol MBOCA/mmol creatinine. The results of this biological monitoring program indicated that urine levels of MBOCA slowly but steadily decreased. Between July and September of 1978, the average level was 50 nmol MBOCA/mmol creatinine. Although the individual variations were present, by February 1980 the average level had fallen to 5 nmol MBOCA/mmol creatinine. Some of the measures that helped reduce the exposure to MBOCA were improved ventilation system, use of protective clothing, dry cleaning of clothing, improvement of the general hygiene in the factory, MBOCA 32 2. HEALTH EFFECTS and special attention to workers with consistently high urinary MBOCA levels (Thomas and Wilson 1984). N-acetyl MBOCA is a minor metabolite that was found in the urine of occupationally exposed workers (Cocker et al. 1988). Also present was a major labile metabolite, probably B-N-glucuronide (Cocker et al. 1990). After hydrolysis, this conjugate yields unmetabolized MBOCA. In an occupational exposure study of 122 workers from 19 different plants (one MBOCA manufacturer, 18 MBOCA users) urine MBOCA levels were determined in 215 samples collected mainly at the end of the shift (Ducos and Gaudin 1983). The highest urine levels of MBOCA were found in workers from the manufacturing plant: the highest mean urinary MBOCA level among the 12 workers was 600 pg/L or 450 pg/g relative to creatinine. This level was greatly reduced (to an average of 62 ng/g of creatinine) following manufacturing and processing modifications related to increased workers protection and good manufacturing practices. The levels of urinary MBOCA in workers of the other 18 businesses varied greatly and were highest in workers who had the greatest contact with MBOCA. The individual urinary MBOCA levels ranged from undetectable to a maximum of 940 pg/L (510 pg/g of creatinine). However, if the values outside of the detection limits (1-1400 p/L) are excluded from the calculation, the urinary MBOCA averages are low varying from 4 to 318 pg/L (2-174 pg/g of creatinine) (Ducos and Gaudin 1983). The results indicate that the urinary MBOCA levels greatly depended on the frequency of MBOCA use, the quantities used, the form of MBOCA (granules, solid, or in solution), methods of manufacturing, and from work practices in general. Individual factors also affect the excretion levels. The predominant metabolite in urine was monoacetylated MBOCA present at low concentration of <1/10 of the total. Another metabolite, N,N -diacetyl MBOCA was also identified in one of the workers. This was an unexpected finding because of the poor water solubility of diacetyl MBOCA. The identification of diacetyl MBOCA is important because it is possible that from N,N’-diacetyl MBOCA, N-hydoxy-N,N’-diacetyl MBOCA can be formed, using benzidine metabolism as a model. N-hydoxy-N,N’-diacetyl MBOCA is important because it can directly bind to nucleic acids. If the existence of N-hydoxy-N,N’-diacetyl MBOCA is confirmed it would provide a plausible biochemical basis for adduct formation that has been proposed as one of the mechanisms involved in carcinogenesis. One proposed mechanism of MBOCA excretion suggests that very little MBOCA is excreted into the bladder as the free amine since most of the MBOCA in the renal perfusate may be present in the form of the p-N-glucuronide conjugate (Cocker et al. 1990). Urine contains B-glucuronidase, and therefore, p-N-glucuronide may undergo some hydrolysis in the bladder. This is proposed as the source of free MBOCA in the urine. 2.3.4.2 Oral Exposure No detailed studies were located regarding excretion in humans following oral MBOCA exposure. After an acute occupational exposure to molten MBOCA, one worker complained of burning eyes and face. Hot, liquid MBOCA was sprayed over his face and some entered his mouth (Hosein and Van Roosmalen 1978). The worker’s urine was monitored for MBOCA excretion over a 3-week period. MBOCA was found to be rapidly excreted during the first 18 hours following exposure, reaching a maximum concentration of 3.6 mg/L; 6 hours later, (24 hours post-exposure), the urine MBOCA level was down to 0.03 mg/L. At 3 weeks, only trace amounts of MBOCA were detected in urine (Hosein and Van Roosmalen 1978). A single oral dose of 10 mg/kg of radiolabeled MBOCA was administered to female LAC:Porton rats. Forty-eight hours later, the majority of the radioactivity was excreted in the feces (60%), while the MBOCA 33 2. HEALTH EFFECTS liver retained the most radioactivity (2%) (Farmer et al. 1981). Similar observations were made in Sprague-Dawley rats given 12 mg/kg of radiolabeled MBOCA (Groth et al. 1984). When male Sprague-Dawley rats were exposed to a single oral dose of 50 mg/kg/day of MBOCA, urinary MBOCA excretion is maximal on the Ist day and steadily decreases up to day (Ducos and Gaudin 1983). The quantity of free urinary MBOCA is very small, approximately 0.5 parts per 1,000. The most abundant MBOCA metabolite in urine was identified as a monoacetylated compound in comparison to the N-acetyl-4,4-methylenebis(2-chloroaniline) and N,N-diacetyl-4,4-methylenebis(2- chloroaniline) forms (Ducos and Gaudin 1983). The biological half-life of MBOCA was 4.4 and 16.7 days in rat liver and blood, respectively, after a single oral exposure to 75 mg/kg of *C-MBOCA (Cheever et al. 1988). The biological half-lives of MBOCA in adducts with globin and DNA were 14.3 and 11.1 days, respectively (Cheever et al. 1988). After chronic exposure to MBOCA (in either standard-protein or low-protein diets), urine of tumor-bearing rats contained a significantly higher level of MBOCA than urine from animals without tumors (Kommineni et al. 1979). 2.3.4.3 Dermal Exposure Nine hours after an accidental spill, the urine of a worker exposed to 11.34 L of molten MBOCA contained 1,700 ppb of MBOCA (NIOSH 1986a; Osorio et al. 1990). The biological half-life of MBOCA in the urine was approximately 23 hours (Osorio et al. 1990). This was evaluated by monitoring the MBOCA urine levels for 2 weeks after exposure. Assuming a one-compartment model, about 94% of the initial dose of MBOCA would be eliminated within 4 days. In another report, after an acute occupational exposure to molten MBOCA, one worker complained of burning eyes and face. Hot, liquid MBOCA was sprayed over his face and some entered his mouth (Hosein and Van Roosmalen 1978). The worker was monitored and his urine analyzed for MBOCA over a 3-week period. MBOCA was rapidly excreted within the first 18 hours following exposure, reaching a maximum concentration of 3.6 mg/L. Six hours later, (24 hours post-exposure), the MBOCA level in urine was down to 0.03 mg/L, dropping to trace amounts 3 weeks later (Hosein and Van Roosmalen 1978). In another report on occupational exposure, 340 analyses were performed on urine samples from 150 workers from 19 factories (Ducos and Gaudin 1983; Ducos et al. 1985). Samples were usually collected at the end of the shift. The levels of MBOCA ranged from undetectable (<0.5 pg/L) to 1,600 pg/L. The highest urine MBOCA levels were present in 12 workers from a factory that handled crystallized MBOCA. In a similar study, pre- and post-work urine samples from 11 employees of a polyurethane manufacturing plant and 10 control subjects were evaluated for MBOCA and thioether levels (Edwards and Priestly 1992). There was no information on the levels of MBOCA that employees were exposed to, on the duration of exposure, or on the route. Based on the type of work, it is likely that the main route of exposure was dermal. The urinary output of thioether was evaluated because thioethers are metabolic end products of the pathway involving mercapturic acid. The thioethers, therefore, were thought to be potential, non- specific, markers of exposure to electrophilic xenobiotics such as rubber monomers or petroleum. They have not been used as markers for MBOCA exposure. The urinary thioether levels were similar in pre- and post-work samples (p>0.05) and did not correlate with urinary MBOCA levels, indicating that they are not useful for estimating occupational MBOCA exposure. Although there were no significant differences in urinary MBOCA levels between the three groups of employees (supervisors, laboratory personnel, process workers), the results reflect the potential exposure levels. The median values of MBOCA in the post-work urine of process workers and supervisors were 9.33 and 1.05 pmol mol"! of creatinine, respectively. There was no MBOCA in the urine samples from 10 control workers. The small amount of urinary MBOCA in occupationally exposed workers suggests that other major metabolites, such as B-N-glucuronide conjugate (Cocker et al. 1990), may be present in the urine. From the results of all of the exposure studies it is not clear, however, if urine is the MBOCA 34 2. HEALTH EFFECTS primary excretion route in humans after dermal exposure to MBOCA since no information was provided regarding the amount of MBOCA in the feces. Male Sprague-Dawley rats were treated with a single dermal dose of 2.5 mg MBOCA or I4C_MBOCA. within 72 hours, 2.54% of the administered radioactive MBOCA was excreted as 'C, while only 0.008% was excreted as the parent compound (Groth et al. 1984). Similar results were obtained in dogs. Twenty-four hours after a single dermal application of 0.4 mg/kg of 4C.MBOCA to beagle dogs, the highest concentration of radioactivity was found in the bile (Manis et al. 1984). No unmetabolized MBOCA was present in the bile. The results support the hypothesis that dermal absorption is a viable mode of entry and that MBOCA is rapidly metabolized and excreted after it enters the body regardless of the route of entry. Urinary excretion of unmetabolized MBOCA was a small but consistent fraction, comprising 0.4-0.5% of the total urinary excretion of radioactivity (Manis et al. 1984). 2.3.4.4 Other Routes of Exposure Feces and urine were major excretion routes in rats after acute exposure to radiolabeled MBOCA by stomach intubation (Morton et al. 1988) or intravenous injection (Tobes et al. 1983). At 48 hours after exposure to 44-58 mg/kg of radiolabeled MBOCA by intubation, 64-87% of radioactivity was recovered in the urine and feces (Morton et al. 1988). A similar observation was made in rats 48 hours after a single intravenous dose of 0.51 mg/kg; feces contained 73% of the total cumulative dose of radiolabeled MBOCA (Tobes et al. 1983). These results indicate that total elimination of '4C following intubation or intravenous injection of 14C_MBOCA to rats was similar to that observed following oral dosing, which also occurred primarily in the feces (Farmer et al. 1981; Groth et al. 1984). After a single intraperitoneal injection of 1, 13, or 100 mg/kg of MBOCA, the compound was excreted most rapidly within the first 24 hours. Rats receiving the highest concentration of MBOCA produced three times more urine during the first 24 hours than did controls (Farmer et al. 1981). Studies with intravenous administration of MBOCA in the dog found that the major route of excretion was through the urine (Manis et al. 1984). Within 24 hours after intravenous injection of 14C.MBOCA, 46% of the dose had been excreted in the urine and 32% in the bile, accounting for 78% of the total dose. This indicates that often substantial differences occur between species and that route of exposure determines the route of elimination of MBOCA. In an attempt to use thioether as a potential urinary indicator for MBOCA exposure, male Wistar rats were exposed intraperitoneally to 125 or 250 mg/kg/day of MBOCA in peanut oil (vehicle) daily for 5 days, while the control received peanut oil only (Edwards and Priestly 1992). Urine was collected for 24 hours prior to and following exposure. Urinary MBOCA levels were significantly higher in the treated groups compared to controls. There were no significant differences noted between the two experimental groups. As in the study with occupationally exposed workers, the urinary thioether levels were not affected by MBOCA treatment indicating that thioether was not a useful indicator for MBOCA exposure (Edwards and Priestly 1992). The in vivo formation of MBOCA-hemoglobin adducts was determined in blood samples of male Sprague-Dawley rats intravenously injected with 0.04, 0.2, or 1.0 pmol/kg of N-hydroxy-MBOCA (Chen et al. 1991). The injection of the lowest dose of N-hydroxy-MBOCA resulted in formation of 0.9 ng/50 mg of MBOCA-hemoglobin adducts 24 hours after the injection. This level remained almost unchanged (0.8 ng/50 mg) 1 week after exposure indicating a relatively long half life of this adduct. A similar observation was made in guinea pigs injected subcutaneously with 4, 20, or 100 mg/kg of MBOCA. Detectable levels of MBOCA-hemoglobin adducts were still present MBOCA 35 2. HEALTH EFFECTS 10 weeks after exposure to the lowest dose of MBOCA. In guinea pigs, the life span of MBOCA- hemoglobin adducts was similar to that of red blood cells, 60-80 days. 2.3.5 Mechanisms of Action The precise mechanism of action of MBOCA is not completely understood. However, the capacity of MBOCA to form adducts with hemoglobin (Cheever et al. 1988, 1990, 1991: Chen et al. 1991; Sabbioni and Neumann 1990), tissue DNA (Cheever et al. 1990), and globin and serum albumin (Cheever et al. 1991) in rats may play a role in its carcinogenicity. The adduct formation is especially important in elucidating the carcinogenicity of MBOCA. Two metabolites were identified in the urine of one occupationally exposed worker: N-acetyl MBOCA and N,N’-diacetyl MBOCA (Ducos and Gaudin 1983). The finding of N,N’-diacetyl MBOCA was unexpected (because of its poor water solubility) and important. It can be assumed, using benzidine metabolism as a model, that from N,N’- diacetyl MBOCA, N-hydroxy-N,N’-diacetyl MBOCA can be formed. N-hydroxy-N,N’-diacetyl MBOCA is important because it can directly bind to nucleic acids. Therefore, the confirmation of its existence would elucidate the biochemical basis for adduct formation. This finding is in agreement with a recently proposed mechanism for chemical carcinogenesis that may involve the formation of chemical adducts in DNA through covalent binding. This proposition was based on the fact that MBOCA produces DNA adducts in rat liver at levels characteristic of genotoxic carcinogens (Kugler- Steigmeier et al. 1989). Since it is known that MBOCA can bind to rat liver DNA (Cheever et al. 1990), and that liver is one of the primary target organs for development of tumors after exposure of rats to MBOCA (Kommineni et al. 1979; Russfield et al. 1975), it may be assumed that MBOCA- DNA adducts play a role in the development of tumors. The mechanism of hemoglobin adduct formation by N-oxidized MBOCA metabolites was recently investigated in vivo and in vitro using human and rat hemoglobin (Chen et al. 1991). Male Sprague-Dawley rats were injected intraperitoneally with 0.5-50 mg/kg of MBOCA, and male English short hair guinea pigs were injected subcutaneously with 4-100 mg/kg of MBOCA. In the same study, treatment of rats with either phenobarbital or B-naphthaloflavone (BNF) resulted in induction of cytochrome P-450 as evidenced by increased enzymatic activity of two P-450 isozymes. This induction caused increased ortho-hydroxylation of MBOCA that was not statistically significant and resulted in the formation of various metabolites. Two of those MBOCA metabolites, N-hydroxy MBOCA and mononitroso- MBOCA, formed measurable amounts of adducts with both human and rat hemoglobin, while the amount of adducts formed by the parent compound itself was very small. This in vitro binding occurred in a dose-related manner and could be inhibited by cysteine and glutathione but not by oxidized glutathione or methionine (Chen et al. 1991). Therefore, the administration of MBOCA to rats resulted in a dose-related formation of hemoglobin adducts that were detectable 24 days after treatment. Similar observations were made in guinea pigs; the life span of MBOCA-hemoglobin adducts followed that of red blood cells in the guinea pig and was approximately 60-80 days. \ One of the major DNA adducts of MBOCA was identified by a combination of in vivo and in vitro studies (Silk et al. 1989). Male rats derived from a Wistar strain were injected intraperitoneally with 89 pmol/kg of radioactive MBOCA or 141 pmol/kg of acety-MBOCA. Twenty-four hours later, animals were sacrificed, and their liver DNA was extracted and analyzed for the amount of bound radioactivity and nature of adducts. Results showed that the cleavage of the bond between the methylene bridge and one aromatic nucleus of MBOCA resulted in the formation of the major in vivo adduct N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol. Two other adducts also formed after intraperitoneal injection were not identified. In an attempt to further elucidate the DNA adduct formation, DNA was isolated from liver, lung, and kidney of 14C.MBOCA-treated Charles River CD rats and compared to in vitro N-hydoxy-MBOCA-treated rat DNA (Segerback and Kadlubar 1992). MBOCA 36 2. HEALTH EFFECTS Rats were orally treated with 95 pmol/kg body weight of radiolabeled MBOCA and sacrificed 1, 3, 7, and 21 days later. In both in vivo and in vitro experiments, two major peaks were identified containing two adducts: ~~ N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N- (deoxyadenosine-8-yl)-4-amino-3-chlorotoluene. These two major peaks were present in both liver and kidney DNA samples. The results confirmed the existence of one adduct found in a similar study (Silk et al. 1989) and identified the second one. The study found that the binding of N-hydroxy- MBOCA to DNA can be inhibited by ascorbic acid, glutathione, nitrosobenzene, and methyl viologen but not by nitromethane, p-nitrobenzylpyridine or methionine. The precise mechanism of single-ring MBOCA DNA adduct formation is still not completely understood because of the indication that there is an unstable intermediate formed prior to the formation of the two major identified DNA adducts and that single-ring MBOCA adducts were not readily detectable (Segerback and Kadlubar 1992). There is some evidence that MBOCA itself may be a tumor promoter. MBOCA caused a statistically significant dose-dependent inhibition of gap-junctional cell communication (GJC) at doses ranging from 7.5 nmol/mL (noncytotoxic) to 15.5 nmol/mL (cytotoxic) (Kuslikis et al. 1991). Since GJC is important in controlling cell proliferation, and many known tumor promoters inhibit GJC, GIC- inhibition assays have been proposed as short-term screens for promoters. 2.4 RELEVANCE TO PUBLIC HEALTH Exposure to MBOCA contaminated soil or water at hazardous waste sites is possible via the oral or dermal routes. Inhalation and dermal exposure to this compound in air are less likely because MBOCA is not volatile. There is no information regarding the level that would be of concern to a population living in the vicinity. Occupational or accidental exposure may occur in industries using MBOCA as a curing agent in manufacturing processes. It is not known if MBOCA and/or its metabolites can be released from polyurethane (MBOCA is used as a curing agent for castable liquid polyurethane elastomers) (HSDB 1991; IARC 1974; Sax and Lewis 1987). Acute-duration exposure to high levels of MBOCA may cause eye and skin irritation in humans (Hosein and Van Roosmalen 1978). Intermediate and chronic exposures to MBOCA may lead to tumors of the urinary bladder. Of 200 occupationally exposed workers, 3 were found to have urinary bladder tumors (Ward et al. 1990). This finding is supported by results obtained in dogs (Stula et al. 1977). Results in other animal species also show that MBOCA is a potential carcinogen and that other target organs include the lung, liver, breast, and Zymbal’s gland in rats (Kommineni et al. 1978; Russfield et al. 1975; Stula et al. 1975) and lung, liver, and vascular system in mice (Russfield et al. 1975). It is not possible to estimate if these tumors would be of concern to humans since there is no information available. Limited information indicates that MBOCA is rapidly absorbed in humans and that the amount absorbed through skin and inhalation is in proportion to the dose (Chin et al. 1983; Cocker et al 1988, 1990; Ichikawa et al. 1990; NIOSH 1986b). No information is available regarding distribution and rate of metabolism in humans. However, toxicokinetic data obtained in animals indicate that MBOCA is rapidly absorbed, distributed, and excreted within 24-48 hours (Farmer et al. 1981; Groth et al. 1984). The bioaccumulation of MBOCA is unlikely because of its rapid metabolism and urinary excretion. Of potential concern, however, is the fact that MBOCA readily forms adducts with body proteins and DNA. Studies in rats and guinea pigs suggest that MBOCA-hemoglobin adducts have a biological life similar to that of red blood cells and may remain in the body for many weeks. MBOCA 37 2. HEALTH EFFECTS No information was located regarding reproductive or developmental effects in humans or animals. There is no information in humans regarding MBOCA retention or any delayed effects. Information on the effects of exposure to MBOCA in humans comes from case reports of accidentally or occupationally exposed workers in manufacturing facilities. In one such study, low-grade papillary tumors of the urinary bladder were observed in 3 out of 200 workers more than 8 years following occupational exposure to MBOCA (Ward et al. 1990). Studies in progress include assessing the incidence of bladder carcinomas in workers occupationally exposed to MBOCA and the role of adducts in MBOCA-induced carcinogenicity (see Section 2.9.3). Animal studies support the hypothesis that MBOCA and/or its metabolites have carcinogenic potential. MBOCA metabolites bound to DNA were found following oral (Cheever et al. 1990; Kugler-Steigmeier et al. 1989) or dermal (Cheever et al. 1990) exposures in rats. No studies were located regarding the genotoxicity of MBOCA in humans in vivo. Results from in vivo animal studies support the conclusion that MBOCA is a mutagen; mixed results from some assays can probably be attributed to incomplete testing. Minimal Risk Levels for MBOCA Inhalation MRLs No inhalation MRLs have been derived for any duration of exposure because of lack of data. Oral MRLs No acute-duration oral MRL has been derived because of lack of data. No intermediate-duration oral MRL has been derived because of lack of data. An MRL of 0.003 mg/kg/day has been derived for chronic-duration oral exposure to MBOCA. This is based on a LOAEL of 10 mg/kg/day for hepatic effects in dogs (Stula et al. 1977). Death. No increased mortality from any cause has been reported in humans who have been exposed to MBOCA. Decreased lifespan was observed in rats following chronic oral exposure to MBOCA (Kommineni et al. 1979). Data are insufficient to assess the effects that prolonged exposure to MBOCA may have on populations that are potentially at risk. Systemic Effects Respiratory Effects. No data are available regarding adverse effects of MBOCA on the respiratory tract in humans. Lung adenomatosis, a preneoplastic lesion, was observed in rats following chronic oral exposure to 25 or 50 mg/kg/day of MBOCA (Russfield et al. 1975). MBOCA may induce changes in the lungs that can progress to cancerous lesions. Since information on respiratory effects following exposure to MBOCA is limited, the impact of findings in animals on public health is difficult to assess. For more information, see the discussion of Cancer, below, in this section. Gastrointestinal Effects. Extremely limited information is available regarding adverse gastrointestinal MBOCA 38 2. HEALTH EFFECTS effects in humans after exposure to MBOCA. A worker complained of feeling ill in the stomach after being sprayed in the face with molten MBOCA (Hosein and Van Roosmalen 1978). No further information was given on his condition or the level of exposure. Evidence is insufficient to assess the relevance of this finding to public health. Hematological Effects. No information is available regarding adverse hematological effects in humans after exposure to MBOCA. However, no changes in hemoglobin, hematocrit, erythrocyte, or mononuclear leukocyte counts were observed in beagle dogs following 9 years of exposure to MBOCA (Stula et al. 1977). This study is of limited usefulness because blood analysis was done only twice a year, so that changes between analyses were probably not recorded. The significance of this finding with regard to human health cannot be assessed. Hepatic Effects. No information was located in humans regarding adverse hepatic effects following exposure to MBOCA. Hepatic effects such as elevated liver enzyme levels, nodular hepatic hyperplasia, fatty changes, necrosis, fibrosis, and bile duct proliferation were observed in rats and dogs following chronic exposure to MBOCA (Stula et al. 1975, 1977). Chronic exposures to high levels of MBOCA at hazardous waste sites may induce similar adverse hepatic effects. Renal Effects. Following an acute exposure in which a worker was sprayed in the face with molten MBOCA, protein was detected in the urine within the first 5 hours after exposure, suggesting that damage to renal tubules occurred (Hosein and Van Roosmalen 1978). Damage to the renal tubules was also indicated by a decrease in specific gravity of the urine collected the next day. Although the amount of MBOCA to which the worker was exposed was not known, it may have been substantial, since the urine MBOCA level was 3.6 mg/L 5 and 11 hours after the incident. It is unknown whether exposure to these high levels would occur at hazardous waste sites. In another study, exfoliative cytology revealed that 21 out of 385 urine samples collected from workers exposed to MBOCA contained atypical cells in urine sediment (Ward et al. 1990). None of the cytology readings were positive for cancer. Sixteen out of the 385 urine samples were positive for heme (Ward et al. 1990). The median duration of employment was 3.2 months. Although this study is limited because it lacks information on exposure levels, it is important because it alerted the medical community to the inadequacies of the procedures normally used following MBOCA exposure. Since one of the participants (who had negative cytology and was negative for heme) was diagnosed with a low-grade papillary tumor of the bladder, cystoscopy was introduced as a part of the follow-up procedure for workers exposed to MBOCA, and two additional cases of bladder tumors were detected subsequently. Therefore, exposure to MBOCA at hazardous waste sites may cause renal damage that may lead to cancer. Studies on chronic exposure of dogs to MBOCA support the findings in humans (Stula et al. 1977). In dogs treated orally with an average of 10 mg/kg/day of MBOCA for 9 years, urine sediment cytology revealed dysplastic changes indicative of neoplasia of the genitourinary system. Dermal/Ocular Effects. Burning of the face and eyes developed shortly after a worker was accidentally sprayed with molten MBOCA (Hosein and Van Roosmalen 1978). He was wearing gloves and safety glasses but no respirator or face shield. A similar burning sensation was described by a worker who was also accidentally sprayed over his upper body and extremities with molten MBOCA (Osorio et MBOCA 39 2. HEALTH EFFECTS al. 1990). The general population is not likely to be involved in such exposures at hazardous waste sites. However, in cases of occupational accidents, exposure to MBOCA may cause adverse dermal/ocular effects. Immunological Effects. No studies were located in humans or animals regarding immunological effects following exposure to MBOCA. Neurological Effects. No studies were located regarding adverse neurological effects in humans after exposure to MBOCA by any route. Cystic hyperplasia of the pituitary gland was found in one out of six dogs after 8.3 years of MBOCA exposure (Stula et al. 1977). This change was considered to be unrelated to chronic MBOCA treatment. Itis difficult to predict if analogous effects would occur in individuals or populations living near hazardous waste sites. Reproductive Effects. No studies were located regarding reproductive effects in humans or animals following exposure to MBOCA. Developmental Effects. No studies were located regarding developmental effects in humans or animals after exposure to MBOCA. Genotoxic Effects. No studies were located regarding the genotoxicity of MBOCA in humans in Vivo. In vivo animal studies provide direct and indirect evidence that MBOCA is a mutagen; MBOCA metabolites were bound to DNA following oral (Cheever et al. 1990; Kugler-Steigmeier et al. 1989) or dermal (Cheever et al. 1990) exposure in rats. Small increases in the SLRL values were observed in D. melanogaster adults fed a 7.5-millimolar MBOCA solution for 3 days (Vogel et al. 1981). MBOCA induced gene mutations at the thymidine kinase (TK) locus in mouse lymphoma cells (Caspary et al. 1988; Myhr and Caspary 1988). Unscheduled DNA synthesis (UDS) was induced in HeLa cells (Martin and McDermid 1981), in rat primary hepatocytes at >10 pmol (McQueen et al. 1981; Mori et al. 1988; Williams et al. 1982), and in hamster (McQueen et al. 1981) and rabbit (McQueen and Williams 1987) hepatocytes. The concentration that tested positive in the mouse was 50 pmol (McQueen et al. 1981). Sensitivity to MBOCA showed species-specific variations: rat > mouse > hamster > rabbit (McQueen et al. 1981, 1983). Because hepatocytes have their own metabolic activation systems, no exogenous metabolic activation is needed. In assays using attachment independence as an end point, MBOCA, at concentrations near the LCs, transformed baby hamster kidney (Daniel and Dehnel 1981; Styles 1981), rat embryo (Dunkel et al. 1981; Traul et al. 1981), and Balb/3T3 cells (Dunkel et al. 1981). Transformation assays have not been evaluated as thoroughly as some other genotoxicity assays. In an interlaboratory comparison, one laboratory found equivocal evidence that nonactivated MBOCA induced sister chromatid exchange in Chinese hamster ovary cells at a dose of 5.0 pg/mL. The response is considered equivocal because the dose-response curve was inconsistent. The result was not confirmed by the second laboratory. In the presence of S9 activation, positive results were obtained by one laboratory at 50 pg/mL. The other laboratory observed the beginning of a dose-response curve, but the high-dose (30 p g/mL) results did not meet the testing laboratory's criterion for a positive response. In a chromosome aberration assay testing activated and nonactivated concentrations up to 5 and 30 pg/mL, respectively, neither laboratory found a positive result (Galloway et al. 1985). Another study (Perry and Thomson 1981) found no MBOCA 40 2. HEALTH EFFECTS evidence of sister chromatid exchange at up to cytotoxic doses (100 pg/mL), but no doses were tested in the probable sensitive range (>10 and <100 pg/mL). Thus, MBOCA is clastogenic in some systems but not others. In another study, MBOCA was classified as a clastogen on the basis of results from an in vivo micronucleus bone marrow assay (Katz et al. 1981). Evaluations using a two-phase micronucleus assay and intraperitoneal dosing of B6C3F, hybrid mice were incomplete, but the results suggested that MBOCA is an in vivo clastogen at doses of >32 mg/kg (Salamone et al. 1981) or 50% of the LDs, (Katz et al. 1981). Results were inconclusive because of toxicity during the first phase of testing in which mice were dosed at 0 and 24 hours with 80% of the LDs (51 mg/kg), and results were evaluated at 48 hours. In the second phase of testing, mice were dosed with 32 or 51 mg/kg and sampled 48 hours later, or in a separate test, dosed with 32 or 48 mg/kg and sampled 36 hours later. Results were negative at 32 mg/kg when sampled at 48 hours but positive at the same dose in a separate assay when sampled at 36 hours. Results were positive at the higher dose for both sampling times. Multiple sampling times were not performed in any assay (Salamone et al. 1981). In another study, results in the micronucleus test were negative at doses up to 50% of the LDs, (32 mg/kg) using CD-1 mice (dosing was done intraperitoneally at 0 and 24 hours and sampling at 30 hours) (Tsuchimoto and Matter 1981). Results from in vivo testing are summarized in Table 2-2. There is some evidence that MBOCA itself may be a tumor promoter. MBOCA caused a statistically significant dose-dependent inhibition of gap-junctional cell communication (GJC) at doses ranging from 7.5 nmol/mL (noncytotoxic) to 15.5 nmol/mL (cytotoxic) (Kuslikis et al. 1991). Since GJC is important in controlling cell proliferation, and many known tumor promoters inhibit GJC, GJC- inhibition assays have been proposed as short-term screens for promoters. There is some evidence that MBOCA itself may be a tumor promoter. MBOCA caused a statistically significant dose-dependent inhibition of gap-junctional communication (GJC) at doses ranging from 7.5 nmol/mL (noncytotoxic) to 15.5 nmol/mL (cytotoxic) (Kuslikis et al. 1991). Since GJIC is important in controlling cell proliferation, and known tumor promoters inhibit GJC, GJC-inhibition assays have been proposed as short-term screens for promoters. The use of GJC-inhibition assays in individuals living in the vicinity of hazardous waste sites would provide needed information for better understanding of potential control of the tumor initiation process. In vitro testing has provided clear and convincing evidence that MBOCA is mutagenic in the Salmonella typhimurium/mammalian microsome mutagenesis assay and that the mutagenic effect requires exogenous metabolic activation (Baker and Bonin 1981; Cocker et al. 1985; Dunkel et al. 1984; MacDonald 1981; Martire et al. 1981; McCann et al. 1975; Messerly et al. 1987; Rao et al. 1982). Although not all investigators used each tester strain, the general result is that MBOCA is mutagenic only in strains TA98 and TA100, at >50 pg/plate, with some inconsistency regarding strain TA9S. MBOCA and its metabolites are not mutagenic in S. typhimurium strains TA1535, TA1537, or TA1538. This suggests that the mutagenic effect of MBOCA metabolites in some bacteria is dependent on the plasmid pKM101; strains TA98 and TA100 contain this plasmid, but strains TA1535, TA1537, and TA1538 do not (Ames et al. 1975). This hypothesis is supported by the finding that S9-activated MBOCA is mutagenic in Escherichia coli strain WP2uvrA only in the presence of the plasmid pKM101 (Matsushima et al. 1981). The plasmid carries genes involved in an "error- prone" DNA repair system that introduces mutations as it removes DNA damage (Walker 1984). S9 derived from dog and human liver could activate MBOCA to a form mutagenic to strain TA100 but only in a protocol using a fluctuation assay (Cocker et al. 1985). These data are presented in Table 2-3. TABLE 2-2. Genotoxicity of 4,4’-Methylenebis(2-chloroaniline) (MBOCA) In Vivo Species (test system) End point Results Reference Mouse CD1 (intraperitoneal) Mouse B6C3F; (intraperitoneal) Mouse B6C3F,/BR (intraperitoneal) Drosophila melanogaster (oral, dermal) D. melanogaster (oral, dermal) D. melanogaster (inhalation, occupational) Rat (Sprague-Dawley; oral) Rat (Sprague-Dawley; oral) Rat (Sprague-Dawley; dermal) Rat (Wistar-derived strain; intraperitoneal) Micronucleus formation Micronucleus formation Micronucleus formation Wing spot test Sex-linked recessive lethal mutation Sex-linked recessive lethal mutation DNA adduct formation DNA adduct formation DNA adduct formation DNA adduct formation Tsuchimoto and Matter 1981 Salamone et al. 1981 Katz et al. 1981 Kugler-Steigmeier et al. 1989 Vogel et al. 1981 Donner et al. 1983 Cheever et al. 1990 Kugler-Steigmeier et al. 1989 Cheever et al. 1990 Silk et al. 1989 - = negative result; + = positive result; DNA = deoxyribonucleic acid S103443 H1IV3H 2 YOO8W 34 TABLE 2-3. Genotoxicity of 4,4’-Methylenebis(2-chloroaniline) (MBOCA) In Vitro Results With Without Species (test system) End point activation activation Reference Prokaryotic organisms: Salmonella typhimurium TA100? (reverse Gene mutation + No data Cocker et al. 1986 mutation) S. typhimurium TA100 (histidine reversion) ~~ Gene mutation + - Cocker et al. 1985 S. typhimurium TA100 (histidine reversion) Gene mutation + No data Hesbert ct al. 1985 S. typhimurium TA98, TA100 (histidine Gene mutation + - Dunkel et al. 1984 reversion) S. typhimurium TA100 (histidine reversion) Gene mutation + - Messerly et al. 1987 S. typhimurium TA100 (histidine reversion) ~~ Gene mutation + No data Ichinotsubo et al. 1981a S. typhimurium TA100 (histidine reversion) ~~ Gene mutation + - McCann ct al. 1975 S. typhimurium TA100 (histidine reversion) Gene mutation + No data Kugler-Steigmeier et al. 1989 S. typhimurium TA98 (histidine reversion) Gene mutation + No data Rao et al. 1982 S. typhimurium TA98, TA100 (histidine Gene mutation + - Brooks and Dean 1981 reversion) S. typhimurium TA98, TA100 (histidine Gene mutation + - Baker and Bonin 1981 reversion) S. typhimurium TA98, TA100 (histidine Gene mutation + - Martire et al. 1981 reversion) S. typhimurium TA98, TA100 (histidine Gene mutation + - Garner et al. 1981 reversion) S. typhimurium TA98, TA100 (histidine Gene mutation + - MacDonald et al. 1981 reversion) S. typhimurium TA98, TA100 (histidine Gene mutation + - Simmon and Shepherd reversion) 1981 S. typhimurium TA98, TA100 (histidine Gene mutation + - Nagao and Takahashi reversion) 1981 S. typhimurium TA1535, TA98, TA100 Gene mutation + No data Trueman 1981 (histidine reversion) S103443 H1TVaH 2 vYOOaW cv TABLE 2-3. Genotoxicity of 4,4’-Methylenebis(2-chloroaniline) (MBOCA) In Vitro (continued) Results With Without Species (test system) End point activation activation Reference S. typhimurium TA98, TA100, (histidine Gene mutation + - Venitt and Crofton- reversion) Sleigh 1981 S. typhimurium TA100 (histidine reversion) Gene mutation + - Hubbard ct al. 1981 S. typhimurium TA1538 (histidine reversion) Gene mutation + - Gatehouse 1981 S. typhimurium TA100 (histidine reversion) Gene mutation + - Rowland and Severn 1981 S. typhimurium TM677 (contains pKM101; ~~ Gene mutation + No data Skopek et al. 1981 8-azaguanine") Escherichia coli WP2 uvrA (tryptophan Gene mutation - - Gatehouse 1981 reversion) E. coli WP2 uvrA/pKM101 (tryptophan Gene mutation + - Matsushima et al. 1981 reversion) E. coli WP85, WP2 uvrA(P) (tryptophan Gene mutation + - Venitt and Crofton reversion) Sleigh 1981 E. coli 58-161 envA (lambda lysogen) Phage lambda induction + No data Thomson 1981 (SOS induction) E. coli P3478 (polA7)/W3110 (polA™) Differential killing - + Rosenkranz et al. 1981 E. coli JC2921 (recA), JC5519 (recBCY) Differential killing + No data Ichinotsubo et al. 1981b E. coli WP67, CM871 Differential killing - + Tweats 1981 Bacillus subtilis rec Differential killing + + Kada 1981 Eucaryotic organisms: Fungi: Saccharomyces cerevisiae XV185-14C Gene mutation - - Mechta and Von Borstel (auxotroph reversion) 1981 S. cerevisiae XII Mitotic recombination - - Kassinova et al. 1981 S. cerevisiae D4 Mitotic gene conversion - - Jagannath ct al. 1981 vYOOaW S103443 HITV3H 2 TABLE 2-3. Genotoxicity of 4,4’-Methylenebis(2-chloroaniline) (MBOCA) In Vitro (continued) ment independence) Results With Without Species (test system) End point activation activation Reference S. cerevisiae JD1 Mitotic gene conversion + + Sharp and Parry 1981 S. cerevisiae D6 Mitotic aneuploidy + + Parry and Sharp 1981 Mammalian cells: Chinese hamster ovary Chromosomal aberrations - - Galloway et al. 1985 Chinese hamster ovary Sister chromatid exchange + /- +/- Galloway et al. 1985 Chinese hamster ovary Sister chromatid exchange - - Perry and Thomson 1981 HeLa cells Unscheduled DNA + - Martin and McDermid synthesis 1981 Rat primary hepatocytes Unscheduled DNA No data + McQueen et al. 1981 synthesis Rat primary hepatocytes Unscheduled DNA No data + Williams ct al. 1982 synthesis Rat primary hepatocytes Unscheduled DNA No data + Mori et al. 1988 synthesis Mouse primary hepatocytes Unscheduled DNA No data + McQueen et al. 1981 synthesis Hamster primary hepatocytes Unscheduled DNA No data + McQueen et al. 1981 synthesis Rabbit primary hepatocytes Unscheduled DNA No data + McQueen and Williams synthesis 1987 Primary hamster embryo cells Single strand DNA breaks No data + Casto 1983 Human male embryonic lung cells Single strand DNA breaks No data + Casto 1983 Mouse lymphoma (L5178Y TK + /-) Forward gene mutation + - Myhr and Caspary 1988 Mouse lymphoma (L5178Y TK + /-) Forward gene mutation + - Caspary ct al. 1988 RLV-infected rat embryo (2FR,50) Transformation (attach- No data + Traul et al. 1981 S103443 H1IV3H 2 vYOOaW vv TABLE 2-3. Genotoxicity of 4,4’-Methylenebis(2-chloroaniline) (MBOCA) In Vitro (continued) Species (test system) End point Reference RLV-infected rat embryo (2FR,50) Balb/3T3 mouse cells Baby hamster kidney (BHK21 C13) Baby hamster kidney (BHK21) C3H2K cells/MLV Human bladder explant culture Dog bladder explant culture Transformation (attach- ment independence) Transformation (attach- ment independence) Transformation (attach- ment independence) Transformation (attach- ment independence) Viral integration enhancement DNA adduct formation DNA adduct formation Results With Without activation activation No data + No data + + + + No data No data - No data + No data + Dunkel et al. 1981 Dunkel et al. 1981 Daniel and Dehnel 1981 Styles 1981 Yoshikura and Matsushima 1981 Stoner et al. 1988 Stoner et al. 1988 “Strains listed are those in which there was a positive response; not all strains were tested in each assay. - = negative result; + = positive result; +/- = inconclusive results; 8-azaguanine” = 8-azaguanine resistance; DNA = deoxyribonucleic acid; MLV = Moloney mouse sarcoma-leukemia virus; RLV = Rauscher leukemia virus; SOS induction = induction of an error-prone repair system; TK = thymidine kinase S103443 H1IV3IH 2 YOOdW St MBOCA 46 2. HEALTH EFFECTS Most of MBOCA'’s mutagenic activity appears to be due to the N-hydroxy metabolite, which caused dose-dependent increases in mutations of S. typhimurium strains TA100 and TA98 in a pre-incubation assay using nonactivated doses of >5 pg/plate (Kuslikis et al. 1991). This metabolite is produced by several species, including dogs and humans (Butler et al. 1989; Chen et al. 1989; Morton et al. 1988). The mononitroso derivative appears to be direct-acting mutagen but is much less potent, causing a statistically significant revertant increase in the pre-incubation assay at the highest tested nontoxic dose (50 pg/plate). Neither the o-hydroxy nor the dinitroso derivatives were direct-acting mutagens at up to 50 or 500 pg/plate, respectively. Neither chemical was tested to cytotoxic levels (Kuslikis et al. 1991). N-acetylation is a deactivating step. Neither N-acetyl nor N,N’-diacetyl derivatives were mutagenic in S. typhimurium in the absence of activation (Hesbert et al. 1985). In the presence of S9 activation, the mutagenic activity of the acetylated derivatives is less than that of the parent compound (Cocker et al. 1986; Hesbert et al. 1985). DNA adducts have been found following oral (Cheever et al. 1990; Kugler-Steigmeier et al. 1989; Segerback and Kadlubar 1992) and dermal (Cheever et al. 1990) administration of radiolabeled MBOCA to rats, following the incubation of radiolabeled MBOCA with explants of dog and human bladder urothelium (transitional cell epithelium) (Stoner et al. 1988) and incubation of rat DNA and radiolabeled N-hydroxy-MBOCA (Segerback and Kadlubar 1992). The level of binding increased with dose, but the increase was not linear. Considerable individual variation in binding levels, varying over at least a 10-fold range, was found in both dogs and humans. At least six adducts were found in dog bladder epithelium; four adducts were found in human bladder epithelium, three of which appeared to be the same as those found in dogs. DNA adduct formation in dog bladder tissue is of particular note, since MBOCA has been found to cause bladder tumors in dogs (Stula et al. 1977). The in vivo and in vitro studies identified two other major rat DNA MBOCA adducts, N- (deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosin-8-yl)-4-amino-3- chlortoluene (Segerback and Kadlubar 1992). The finding of DNA adducts in human bladder tissue suggests similar processes may occur in humans. Intraperitoneal injection into rats of ring-labeled MBOCA, or ring-labeled N-acetylated MBOCA, resulted in the generation of three DNA adducts (Silk et al. 1989). Two of these adducts were also produced by the in vitro reaction of the N-hydroxy derivative of MBOCA with rat liver slices. The major product of this reaction was also formed following incubation of DNA with N-hydroxy-4-amino- 3-chlorobenzyl alcohol, the compound resulting from cleavage of the methylene bridge of N-hydroxy MBOCA. While the single ring species appears to be an intermediate in the formation of the DNA adduct, it is not known whether N-hydroxylation or bridge cleavage occurs first in the formation of the reactive species. The DNA adduct was analytically identified as N-(deoxyadenosin-8-yl)-4-amino- 3-chlorobenzyl alcohol. Cancer. Since MBOCA has been suspected of being a human carcinogen, epidemiological studies have examined the incidence of cancer in workers exposed to MBOCA. Three out of 200 examined workers exposed to MBOCA for an average of 3.2 months were found to have low-grade papillary tumors of the urinary bladder (Ward et al. 1990). The latency period for most bladder cancers is about 20 years; therefore, the detection of bladder tumors in this study 11.5 years (mean latency period for the entire cohort) after first exposure to MBOCA provides suggestive evidence that MBOCA is a suspected human carcinogen. Two of the men affected with bladder tumors were less than 30 years old. Since it is known that the occurrence of bladder tumors in young men is very uncommon, this finding increases the concern that MBOCA has a potential for causing bladder tumors in humans. However, there was no evidence of recurrent bladder lesions in the above cases after the initial discovery. It is also difficult to estimate the biological and statistical significance of MBOCA 47 2. HEALTH EFFECTS these bladder lesions because of the small sample size. One important outcome of this study is the introduction of cystoscopy as a part of the monitoring protocol for workers exposed to MBOCA. The limitation of the study is that there are no data on bladder cancer incidence diagnosed by cystoscopy in an asymptomatic nonexposed (control) population. The importance of biological monitoring programs was also evident in the occupational study/review of urinary free MBOCA levels in workers from PMA member companies (Lowry and Clapp 1992). The data have been collected for over 15 years, and the study summarized them for 1985-1990. The analysis of 7,844 urine samples confirmed that after the introduction of biological monitoring it was possible to reduce the urinary MBOCA levels by using effective exposure control and protection in the workplace. The reduced percentage of samples containing >50 pg/L of MBOCA (from 12% in 1985 to 8% in 1990) and increased percentage of urine samples with <25 pg/mL (from 77% in 1985 to 86% in 1990) further supported the importance of biological urinary MBOCA monitoring. The probable carcinogenicity of MBOCA has been a concern for some time. In 1972, the British rubber industry imposed upon itself a ban on the use of MBOCA because it was a suspected human carcinogen (Parkes 1979). The initiation of a biological monitoring program that would examine exposure levels and help establish environmental or other pertinent controls as preventive measures has also been suggested (Ward et al. 1986). This biomonitoring program would also maintain a system to follow the workers exposed to MBOCA in order to detect possible neoplastic changes and initiate treatment. Chronic exposure studies in animals support the findings in humans. The main target organs for cancer development were the urinary bladder, lungs, liver, and breasts. The urinary bladder was a target organ in beagle dogs fed MBOCA for 9 years; three of the five surviving dogs developed papillary transitional cell carcinoma of the urinary bladder; another dog had a combined urethral adenocarcinoma and transitional cell carcinoma (Stula et al. 1977). This study, in spite of the use of a small number of animals, shows that the ingestion of MBOCA over 9 years was associated with the incidence of carcinomas of the urinary bladder and urethra. Several other chronic exposure studies in rats demonstrate the ability of MBOCA to cause lung cancer (Kommineni et al. 1979; Stula et al. 1975). A significant increase in the incidence of liver tumors after chronic exposure to MBOCA was found in rats (Kommineni et al. 1979; Russfield et al. 1975) and in female mice (Russtield et al. 1975). The rats fed MBOCA for 2 years had an increased incidence of mammary adenocarcinomas (Kommineni et al. 1979). Other tumor types were also observed after chronic oral administration of MBOCA. The incidence of hemangiosarcomas was 8% in rats (Kommineni et al. 1979); vascular tumors were present in male random-bred albino mice (Russfield et al. 1975); and hemangiosarcomas were present in both male and female mice (Russfield et al. 1975). Tumor incidence was affected by gender in mice; no data regarding gender specificity are available for rats or dogs. The precise mechanism of action of MBOCA is not completely understood, but it is thought that in some instances it involves the formation of chemical adducts in the genetic material through covalent binding of nucleophilic sites by electrophilic compounds or activated metabolites (Cheever et al. 1991). Therefore, it seems that the capacity of MBOCA to form adducts with tissue DNA (Cheever et al. 1990), hemoglobin (Cheever et al. 1990, 1991; Sabbioni and Neumann 1990) and globin and serum albumin (Cheever et al. 1991) in rats may play a role in its toxicity and carcinogenicity. The adduct formation is especially important in elucidating the carcinogenicity of MBOCA. It was recently proposed that chemical carcinogenesis may involve the formation of chemical adducts in DNA through covalent binding based on the fact that MBOCA produces DNA adducts in rat liver at levels characteristic of genotoxic carcinogens (Kugler-Steigmeier et al. 1989). Since it is known that MBOCA can bind to rat liver DNA (Cheever et al. 1990), and that liver is one of the primary MBOCA 48 2. HEALTH EFFECTS target organs for development of tumors after exposure of rats to MBOCA (Kommineni et al. 1979; Russfield et al. 1975), it may be assumed that MBOCA-DNA adducts play a role in the development of tumors. Further support for the carcinogenic potential of MBOCA comes from the study showing that MBOCA binds to DNA from human and dog urinary bladder tissue (Shivapurkar et al. 1987). The mouse skin assay was used in one study to evaluate the carcinogenicity of MBOCA in the presence of 12-o-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter (Nesnow et al. 1985). SENCAR mice were exposed to 0, 0.1, 1, 10, 100, or 200 mg of MBOCA and 1 week later to 2 pg of TPA biweekly for 26 weeks. No mouse skin papillomas were observed at any dose level, but the authors stressed that this finding was based on the limited dose levels tested in the study. There are several limitations to this study; it was not clear if TPA was administered to control animals, if MBOCA was applied to shaved skin, or if was the area was protected after treatment; in addition, no other parameters aside from tumor initiation were examined during the 26-week period. There is some evidence that MBOCA itself may be a tumor promoter. MBOCA caused a statistically significant dose-dependent inhibition of GJC at doses ranging from 7.5 nmol/mL (noncytotoxic) to 15.5 nmol/mL (cytotoxic) under in vitro conditions (Kuslikis et al. 1991). Since GJC is important in controlling cell proliferation, and known tumor promoters inhibit GJC, GJC-inhibition assays have been proposed as short-term screens for promoters. The use of GJC-inhibition assays in individuals living in the vicinity of hazardous waste sites would provide needed information for better understanding of potential control of the tumor initiation process. 2.5 BIOMARKERS OF EXPOSURE AND EFFECT Biomarkers are indicators of events in biologic systems. They have been classified as markers of exposure, markers of effect, and markers of susceptibility (NAS/NRC 1989). A biomarker of exposure is a xenobiotic substance or its metabolite(s), or the product of an interaction between a xenobiotic agent and some target molecule(s) or cell(s) that is measured within a compartment of an organism (NAS/NRC 1989). Preferred exposure biomarkers are either the substance proper or substance-specific metabolites in readily obtainable body fluid(s) or excreta. Several factors may confound the use and interpretation of exposure biomarkers: (1) the body burden of a substance may represent more than one exposure; (2) the substance being measured may be the metabolite of another substance (e.g., high levels of phenol in urine can result from exposure to several different aromatic compounds). Depending on the substance’s properties (e.g., biologic half-life), and exposure conditions (e.g., duration and route of exposure), the substance and metabolites may have left the body by the time samples are collected. 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) may be difficult to identify. Biomarkers of exposure to MBOCA are discussed in Section 2.5.1. 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). Biochemical or cellular signs of tissue dysfunction and physiologic alterations are included within this definition. Note that these markers are often not substance specific. Biomarkers of effect(s) may indicate potential health impairment(s) (e.g., DNA adducts). Biomarkers of effects caused by MBOCA are discussed in Section 2.5.2. MBOCA 49 2. HEALTH EFFECTS A biomarker of susceptibility may indicate 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 either an intrinsic genetic characteristic, a preexisting disease that results in greater absorbed dose, a decrease in the biologically effective dose, or a target tissue response. If biomarkers of susceptibility exist, they are discussed in Section 2.7, Populations That Are Unusually Susceptible. 2.5.1 Biomarkers Used to Identify or Quantify Exposure to MBOCA Exposure to MBOCA has commonly been determined by measuring levels of MBOCA or its metabolites in urine. No information on quantifiable dose threshold levels in humans was located. Since MBOCA is rapidly metabolized, its presence in urine indicates recent exposure. MBOCA is rapidly biodegraded and converted to its glucuronide conjugates. Since varying levels of B-glucuronidase are present in urine, it may be difficult to determine accurate levels of MBOCA in the urine. Following administration of radiolabeled MBOCA to rats (Farmer et al., 191; Groth et al., 1984) and dogs (Manis et al. 1984b), less than 2% and 1%, respectively, of the radioactivity recovered in urine was unmetabolized MBOCA. The biologic half-life of MBOCA in blood following intravenous exposure was estimated to be 0.70 hours; the levels of MBOCA found in various organs 24 hours after dermal exposure were relatively small (Manis et al. 1984). Measuring the metabolites of MBOCA, therefore, would be useful surrogate exposure biomarkers. Male and female CD rats treated with 44 or 58 mg/kg of radiolabeled MBOCA by gavage had o-glucuronide and o-sulfate MBOCA conjugates in urine; the mono-N-glucuronide was the major biliary metabolite 24 hours after treatment (Morton et al. 1988). After a single oral dose of 75 mg/kg, MBOCA formed adducts with globin and liver DNA in Sprague-Dawley rats (Cheever et al. 1988). The half-lives for rat globin and liver DNA were estimated to be 14 and 11 days, respectively. An o-hydroxysulfate, identified as 5-hydroxy-3,3’-dichloro-4,4’-diamino-diphenylmethane-5-sulfate, was the major metabolite found in dog urine. This metabolite also formed adducts with DNA in vitro in a time-dependent manner (Manis and Braselton 1984). Studies in rats and guinca pigs have demonstrated MBOCA adducts to hemoglobin (Chen et al. 1991; Sabboioni and Newmann 1990), with a disappearance rate that approximates the life of a red blood cell. These study results suggest that MBOCA adducts may be useful biomarkers to monitor MBOCA exposure. The levels of MBOCA have not been determined in chronic animal studies. It is unknown if chronic sequestering and low-level release of MBOCA, resulting in steady state levels, occur. 2.5.2 Biomarkers Used to Characterize Effects Caused by MBOCA The available information suggests that the bladder is a target organ for MBOCA-induced carcinogenesis. Medical surveillance of occupationally exposed workers, currently in progress, may help to ascertain the incidence of MBOCA-induced bladder cancer. Cystoscopy may help identify new biomarkers and characterize early prencoplastic changes in the bladder. The ability of MBOCA to covalently bind to body proteins has been less frequently used to estimate exposure to MBOCA. MBOCA is known to bind to globin (Cheever et al. 1991). Therefore measurement of MBOCA-globin adducts could be used to monitor MBOCA exposure since the half- life of MBOCA-globin adducts is greater than the half-life of MBOCA. Section 2.2 contains more detailed information on adverse effects of MBOCA. Additional information can be obtained from the CDC/ATSDR (CDC/ATSDR 1990) and Office of Technology Assessment (OTA 1990) reports listed in Chapter 8. MBOCA 50 2. HEALTH EFFECTS 2.6 INTERACTIONS WITH OTHER CHEMICALS Limited information was located on the interactive effects of other chemicals on MBOCA toxicity. MBOCA metabolism is influenced by phenobarbital (Chen et al. 1991; Morton et al. 1988). In vivo treatment with phenobarbital induced cytochrome P-450 enzymes which resulted in a slight increase in MBOCA hydroxylation (Chen et al. 1991). Rats treated with phenobarbital had a 4-8-fold increased metabolic rate for MBOCA (Morton et al. 1988). Phenobarbital did not, however, affect adduct formation (Chen et al. 1991). In vivo treatment with B-naphthoflavone did increase the rate of MBOCA-hemoglobin adduct formation in rats treated subcutaneously with either 100 or 500 mg/kg/day (Chen et al. 1991). MBOCA metabolism is NADPH-dependent. Moreover, MBOCA hydroxylation is inhibited by 2,4-dichloro-6-phenylphenoxyethylamine, an inhibitor of microsomal mixed function oxidases (Chen et al. 1989). Cysteine and glutathione inhibit in vitro hemoglobin adduct formation by N-hydroxy-MBOCA and mononitroso-MBOCA (Chen et al. 1991). The most recent findings suggest that binding of N-hydroxy-MBOCA to DNA in rat tissues can be inhibited by ascorbic acid, glutathione, nitrosobenzene, and methyl viologen but not by nitromethane, p- nitrobenzylpyridine, or methionine (Segerback and Kadlubar 1992). 2.7 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE A susceptible population may have a heightened response to MBOCA compared with the general population. A population’s susceptibility may be determined by their genetic make-up, developmental stage, age, health and nutritional status (including dietary habits that may increase susceptibility, such as inconsistent diets or nutritional deficiencies), and substance exposure history (including smoking). These unique characteristics could result in a decreased ability to detoxify and excrete the xenobiotic material and metabolites (mainly hepatic, renal, and respiratory). We expect that the elderly, (with declining organ function), and infants and children with immature and developing organs, may be more susceptible to the effects of toxic substance exposure(s) than healthy adults. Populations at greater risk because of their atypically high exposures are discussed in Section 5.6, Populations With Potentially High Exposure. Differences in species sensitivity to MBOCA have been explored in in vitro and in vivo studies. In one study examining the DNA adduct formation in human bladder explant cultures, there was a distinct difference in the sensitivity to MBOCA (Stoner et al. 1988). Results showed that some cultures had much higher levels of MBOCA binding to bladder epithelium than did others. These findings suggest that some individuals are likely to develop more damage from MBOCA exposure than others. This could be an important consideration in cases of occupational exposure to MBOCA. Another observation concerns the cytochrome P-450 family of enzymes. It is known that profiles of cytochromes P-450, enzymes that play an essential role in detoxification of MBOCA, vary from one individual to another (Butler et al. 1989). Consequently, the rates of MBOCA metabolism may vary significantly among the population because of these differences in individual profiles of cytochrome P-450. The ability of a person to acetylate may also be of importance since it is well established for benzidine that this ability is directly related to the genotoxicity and carcinogenicity of the compound. An indication of DNA adduct formation following acute exposure to MBOCA was found in autoradiographs of postlabeled liver DNA from exposed rats (Endo and Hara 1991). Compared to control, the relative adduct level (RAL) was considerably higher in experimental samples than controls. However, further studies are needed to identify these rat liver DNA adducts. In an attempt to elucidate the DNA adduct formation, DNA was isolated from liver, lung, and kidney of 4c. MBOCA 51 2. HEALTH EFFECTS MBOCA-treated rats and compared to in vitro N-hydoxy-MBOCA-treated rat DNA (Segerback and Kadlubar 1992). In both cases, two major peaks were identified containing two adducts: N- (deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosine-8-yl)-4-amino-3- chlorotoluene. Although the study found that the binding of N-hydroxy-MBOCA to DNA can be inhibited by ascorbic acid, glutathione, nitrosobenzene, and methyl viologen (but not by nitromethane, p-nitrobenzylpyridine, or methionine), the precise mechanism of single-ring MBOCA DNA adduct formation is not completely understood. Gluthatione (GSH) has also been shown to protect against hemoglobin adduct formation with oxidized MBOCA metabolites (Chen et al. 1991). Individuals with lowered GSH levels brought about by oxidative stress or excessive exposure to GSH depleting xenobiotics (such as acetaminophen) could also be at increased risk following MBOCA exposure. More studies are needed to understand the mechanism and the intermediaries in the DNA adduct formation. In the case of MBOCA, acetylation was not related to genotoxicity (McQueen et al. 1983). In a study performed in rabbits, the genotoxic response to MBOCA varied considerably; the net number of radioactive grains per nucleus in individual rabbits ranged from 0 to 13.7. Factors such as these may partially influence individual susceptibilities to MBOCA-induced carcinogenicity. Other populations that may show increased sensitivity include very young children who have an immature hepatic detoxification system and individuals with impaired liver or kidney function. Workers in MBOCA manufacturing facilities have a higher risk because of their higher-level exposures to MBOCA. Worker exposure occurs primarily through inhalation and dermal contact. Other populations with higher risk are those that live near these facilities (if releases are not controlled) and family members of exposed workers (because contamination of the household occurs as a result of clothing) (Willams 1979). 2.8 METHODS FOR REDUCING TOXIC EFFECTS This section will describe clinical practice and research concerning methods for reducing toxic effects of exposure to MBOCA. However, because some of the treatments discussed may be experimental and unproven, this section should not be used as a guide for treatment of exposure to MBOCA. When specific exposures have occurred, poison control centers and medical toxicologists should be consulted for medical advice. 2.8.1 Reducing Peak Absorption Following Exposure No information has been located regarding specific treatment of humans or animals following exposure to MBOCA other than removal from the source of exposure. However, as in cases of most toxic exposures, supportive therapy dealing with symptoms is recommended. These supportive therapies include the monitoring of vital functions, as well as electrocardiographic monitoring, observation of vital signs, the intake and output of fluids, body temperature, and possibly pulmonary wedge pressure if indicated (HSDB 1991). Of special importance in minimizing absorption is the selection of protective clothing and gloves. The permeability of eight commercially available glove materials to methanolic solutions of aromatic amines was tested (Weeks and Dean 1977). The results indicate that there are considerable differences in the permeability of tested materials. Natural latex seemed to be most protective for 9.8 mg/mL MBOCA (1.0 weight percent) in methanol (Weeks and Dean 1977). The recommendation of how frequently the gloves should be changed will depend on the specific situation. The results of the study show that penetration of the glove material by the MBOCA solution was concentration dependent, and that highly concentrated MBOCA (9.8 mg/mL) MBOCA 52 2. HEALTH EFFECTS penetrated the gloves within 2 hours, while MBOCA at a relatively low concentration did not penetrate for 35 hours (Weeks and Dean 1977). Biological monitoring of workers occupationally exposed to MBOCA is a preventive measure that can reduce potential toxic and carcinogenic effects. Among the other recommended procedures are monitoring MBOCA air levels and urinary MBOCA levels, doing wipe tests to check surface contamination that can potentially lead to skin exposure (Clapp et al. 1991; Lowry and Clapp 1992), and considering the performance of cystoscopy in exposed workers for early detection of bladder tumors (Mason and Volger 1990; Ward et al. 1990). Continued education of the work force about potential work hazards is also important (NIOSH 1986b). 2.8.2 Reducing Body Burden MBOCA is not retained by the body since the majority of the compound is excreted within 24-48 hours after exposure (Hosein and Van Roosmalen 1978). The biological half-life of MBOCA in the urine was estimated at approximately 23 hours in a case of an accidental exposure (NIOSH 1986a). The same study assumed a one-compartment excretion model that would take 4 days to eliminate about 94% of MBOCA from the body. Similar results were obtained in rats after a single dose of radioactive MBOCA; 64-87% of the radioactivity was recovered in the urine and feces within 48 hours after exposure (Morton et al. 1988). It is not known if MBOCA metabolites are retained in the body and for how long. No information was located regarding reduction of body burden in humans or animals following exposure to MBOCA. Because phenobarbital induces the microsomal cytochrome P-450 enzyme system responsible for MBOCA hydroxylation (Chen et al. 1991), phenobarbital treatment might increase the breakdown of MBOCA and speed its removal from the body. However, because MBOCA’s metabolites are more toxic than the unmetabolized moiety, this intervention must be used with caution. Such treatment could also affect MBOCA adduct formation. While phenobarbital did not increase in vivo hemoglobin adduct formation in one study in rats (Chen et al. 1991), Cheever et al. (1991) found an increase in globin adduct formation (radioactivity) following phenobarbital induction. The major DNA adduct of MBOCA was identified in another study by a combination of in vivo and in vitro experiments (Silk et al 1989). Male rats derived from a Wistar strain were intraperitoneally exposed to either 89 pmol/kg of radioactive MBOCA or 141 pmol/kg of acetyl-MBOCA. Twenty-four hours later, animals were sacrificed and their liver DNA was extracted and analyzed for the amount of bound radioactivity and nature of adducts. Results showed that the cleavage of the bond between the methylene bridge and one aromatic nucleus of MBOCA resulted in the formation of the major in vivo adduct identified as N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol. Two other adducts also formed after intraperitoneal injection were not identified. These findings were confirmed and extended when DNA was isolated from liver, lung, and kidney of '*C-MBOCA-treated rats and compared to in vitro N-hydoxy-MBOCA-treated rat DNA, and two major adducts were identified: N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosine-8-yl)-4- amino-3-chlorotoluene (Segerback and Kadlubar 1992). Although the results showed that the adduct formation (binding of N-hydroxy-MBOCA to DNA) can be inhibited by ascorbic acid, glutathione, nitrosobenzene, and methyl viologen but not by nitromethane, p-nitrobenzylpyridine, or methionine, the precise mechanism of single-ring MBOCA DNA adduct formation is not completely understood. Therefore, more information on DNA adduct formation especially in human bladder, and possibly in liver, is needed since DNA adducts are considered to play a major role in MBOCA-induced carcinogenesis. MBOCA 53 2. HEALTH EFFECTS 2.8.3 Interfering with the Mechanism of Action for Toxic Effects Existence of an antidote capable of reducing or alleviating MBOCA-induced toxicity has not been reported. Available information indicates that most harmful effects of MBOCA in humans come from its metabolites. Once the detailed mechanism of action of MBOCA is known, and more information is gathered on the potential adduct inhibitors, it may be possible to interfere with DNA adduct formation and reduce the adverse effects. 2.9 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 MBOCA 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 MBOCA. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. 2.9.1 Existing Information on Health Effects of MBOCA The existing data on health effects of inhalation, oral, and dermal exposure of humans and animals to MBOCA are summarized in Figure 2-3. The purpose of this figure is to illustrate the existing information concerning the health effects of MBOCA. Each dot in the figure indicates that one or more studies provide information associated with that particular effect. The dot does not imply anything about the quality of the study or studies. Gaps in this figure should not be interpreted as "data needs." A data need, as defined in ATSDR’s Decision Guide for Identifying 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. With regard to human health effects of MBOCA, the few available studies were either case reports of acute occupational exposure or involved intermediate or chronic epidemiological studies. Acute exposures to MBOCA were by the inhalation, oral, or dermal routes, although in some studies it was difficult to clearly define the exposure route. Intermediate exposures were by either inhalation and/or dermal contact; no intermediate oral exposure studies were located. Chronic exposure in humans occurred by inhalation and/or dermal contact; no chronic oral studies were located. No information is available regarding immunological, neurologic, reproductive, developmental, or genotoxic effects in humans by any route of exposure. Studies on cancer incidence in humans after inhalation and/or dermal exposure to MBGCA were located. Virtually all of the data regarding the health effects of MBOCA in animals were obtained from studies in which MBOCA was administered orally. Extremely limited information is available regarding health effects in animals following inhalation or dermal exposure. MBOCA 54 2. HEALTH EFFECTS FIGURE 2-3. Existing Information on Health Effects of 4,4’-Methylenebis (2-chloroaniline) (MBOCA) SYSTEMIC > © & 2 & © & & $$ 5 & S/S SS ESS SSE Q v & Oo & > Q & & ¢] Inhalation @ ® Oral ® Dermal ® @ HUMAN SYSTEMIC “© 2 ° . &/ s/f & & © & © & o> K 4 & Y S/S SSS SS SS ES SS J ¥/ 8 J $/ IS) */ d Inhalation Oral & ® & @ Dermal ® ANIMAL @ Existing Studies MBOCA 55 2. HEALTH EFFECTS 2.9.2 Identification of Data Needs Acute-Duration Exposure. Populations close to hazardous waste sites that contain MBOCA would be exposed to MBOCA for short periods of time, most likely be through the dermis, although inhalation and ingestion exposure(s) cannot be excluded. Limited information on acute exposure in humans comes from reports on accidental exposure by skin absorption and inhalation (Hosein and Van Roosmalen 1978). In this case, eyes and skin were affected since the worker was sprayed on the face with molten, liquid MBOCA. A small amount of protein in his urine indicated that the kidney was a target organ for MBOCA toxicity. Insufficient information was available to calculate an acute- duration MRL for MBOCA. No information was located regarding toxic effects in animals following acute exposure to MBOCA. Studies addressing toxic effects of MBOCA in animals after skin application and inhalation (since these are the most likely routes of exposure for human populations in the vicinity of hazardous waste sites) could provide needed information for estimation of possible acute toxicity of MBOCA in humans. Intermediate-Duration Exposure. Limited information is available in humans regarding toxic effects of MBOCA following intermediate exposure by inhalation and/or dermal contact, and no information was located regarding oral exposure. In one study, exposure most likely occurred as a result of combined inhalation and dermal exposures, and the exposure duration was averaged at 3.2 months. The results showed that the urinary bladder is the major target organ in humans after intermediate-duration exposures to MBOCA. Papillary neoplasms (one carcinoma) of the urinary bladder were found in 3 out of 200 occupationally exposed workers (Ward et al. 1990). No studies of intermediate-duration exposure in animals were located to support this finding. However, results from chronic animal studies clearly support the findings in humans after intermediate exposure to MBOCA. Lung, liver, and mammary tumors were found in rats (Kommineni et al. 1979; Stula et al. 1975). Hepatomas and vascular tumors were present in mice (Russfield et al. 1975), while transitional cell carcinoma of the urinary bladder was found in dogs (Stula et al. 1977) after chronic exposure to MBOCA. Insufficient information is available to calculate an intermediate-duration MRL for MBOCA. Animal studies examining the effects of intermediate-duration exposure to MBOCA via the inhalation and dermal routes are necessary to evaluate the likelihood of urinary bladder carcinoma in humans after intermediate exposure to MBOCA. Chronic-Duration Exposure and Cancer. Epidemiological studies in humans following chronic exposure to MBOCA are very limited. No information is available regarding toxic effects in humans following chronic exposure to MBOCA. One study of chronic occupational exposure in humans found low-grade papillary tumors of the urinary bladder in 3 out of 200 workers (Ward et al. 1990). This result suggests that the urinary bladder is the major target organ in terms of tumor incidence in humans following chronic exposure. This finding is supported by results obtained in dogs (Stula et al. 1977). Data in other animal species following oral exposure also support the finding that MBOCA is a potential carcinogen and show that other target organs include lungs, liver, breast, and Zymbal’s gland in rats (Kommineni et al. 1979; Russfield et al. 1975; Stula et al. 1975) and lungs, liver, and vascular system in mice (Russficld et al. 1975). Although the animal data support those in humans following an occupational exposure (Ward et al. 1990), the report in humans is limited in that it is a case report without controls to allow the MBOCA 56 2. HEALTH EFFECTS determination of a dose-response relationship and statistical significance of data. There was also potential of co-exposure to other chemicals. Further occupational exposure studies are needed to establish the carcinogenic potential of MBOCA in humans. Currently, there are several studies in progress sponsored by NIOSH that are investigating different aspects of human exposure to MBOCA (FEDRIP 1991; also Section 2.9.3). One of the studies is involves the worker population exposed to MBOCA between 1969 and 1979. The principal investigator is E.M. Ward, and the objective of the study is to see if the occurrence of cancer can be linked to exposure to MBOCA. The second study concerns biomonitoring for populations occupationally exposed to aromatic amines (of which MBOCA is one). F.B. Daniel is the principal investigator for this study entitled "Biomonitoring for Populations Occupations Exposed to Aromatic Amines." Hemoglobin and DNA adduct formation by MBOCA will be analyzed in order to propose a methodology for monitoring workplace exposure. Also sponsored by NIOSH is a third study which involves on developing and evaluating biological monitoring methodologies for detecting the presence and uptake of MBOCA by urine. L.L. Lowry is the principal investigator for this project entitled "Biological Monitoring Methods Research and Evaluation." This investigator will also evaluate the effectiveness in predicting worker uptake of MBOCA and two other compounds. The NTP also has a study in progress on "Aryl Amine Adducts in Blood as Indicators of Exposure." In this study, blood samples from 100 workers will be analyzed for hemoglobin o-toluidine adducts. MBOCA will be used to develop a high-performance liquid chromatography (HPLC) method for separation and isolation of mitochondrial or total aryl amine-DNA adducts. In addition, the in vitro activation of potential carcinogens will be studied, and a mathematical model for MBOCA distribution, metabolism, and adduct formation will be prepared. The overall objective of the project is to develop a more sensitive adduct isolation procedure to be used for biological monitoring. The contact person for this project is K. Cheever (NTP 1991a). A grant was awarded in September 1991 to the Michigan Department of Public Health to conduct an epidemiologic study under the title "Analytic Epidemiologic Study of MBOCA" (ATSDR 1991). The objective of the study is to determine if there is an association between exposure to MBOCA and occurrence of bladder cancer in workers, their family members, and residents who live near three NPL sites. Cystoscopy will be used in all cases with positive screening results. The information on all MBOCA-exposed members of the cohort will be maintained in a registry. A case-control study will be conducted to assess the association of MBOCA exposure and bladder cancer incidence. The contact person for this project is H. Humphrey from the Michigan Department of Public Health. Thus, there are sufficient on-going studies on biomonitoring of humans exposed to MBOCA. Genotoxicity. There is considerable evidence that MBOCA metabolites are mutagenic in S. typhimurium strains TA98 and TA100 (Cocker et al. 1985; MacDonald 1981; Matsushima et al. 1981; Messerly et al. 1987; Kuslikis et al. 1991) and in mouse lymphoma cells (Caspary et al. 1988; Myhr and Caspary 1988), and induce UDS in HeLa cells (Martin and McDermid 1981) and in hepatocytes from rats (McQueen et al. 1981; Mori et al. 1988; Williams et al. 1982), mice, hamsters (McQueen et al. 1981), and rabbits (McQueen and Williams 1987). However, nonactivated MBOCA is not a mutagen. [In vitro evidence indicates that human liver (Cocker et al. 1985) and bladder epithelium (Stoner et al. 1988) contain enzymes that can activate MBOCA to genotoxic derivatives. It is less clear whether MBOCA is a clastogen. Mixed results have been obtained in the in vivo micronucleus assay (Katz et al. 1981; Salamone et al. 1981; Tsuchimoto and Matter 1981) and in the in vitro assays for sister chromatid exchange (Galloway et al. 1985; Perry and Thomson 1981), while data regarding chromosome aberrations were negative (Galloway et al. 1985; Perry and Thomson 1981). Micronucleus assays done with multiple sampling times, and in vivo and in vitro chromosome aberration assays performed near cytotoxic levels would be useful in assessing the clastogenic MBOCA 57 2. HEALTH EFFECTS potential of MBOCA. MBOCA metabolites bind to DNA in rat (Cheever et al. 1990; Kugler- Steigmeier et al. 1989), dog, and human (Stoner et al. 1988) bladder and liver tissues. Wide variations in the amount of material bound were observed in dog and human tissue explants (Stoner et al. 1988). Identifying the source of this variability might help in identifying sensitive populations. One study reported that MBOCA itself may be a promoter (Kuslikis et al. 1991); confirmation or negation of this result might be useful in evaluating the interactions of MBOCA with other chemicals. Reproductive Toxicity. No studies were found regarding reproductive effects in humans or animals following exposure to MBOCA by any route. Thus, studies examining the effects on reproduction by any route of exposure would be useful. Developmental Toxicity. No human or animal studies are available on developmental effects for any exposure route. Studies assessing postnatal survival after maternal exposure to MBOCA by all three routes would be very informative. Immunotoxicity. No information on immunotoxicity after exposure to MBOCA by any of the three routes is available in humans or animals. Studies in laboratory animals following acute inhalation, oral, and dermal exposure to MBOCA would help define possible effects on antibody production and cellular immunity. This information would be useful for determining sensitive populations. Neurotoxicity. No studies on neurotoxicity in humans following exposure to MBOCA were located. In one out of six female beagle dogs, cystic hyperplasia of the pars intermedia of the anterior pituitary gland was found after 8.3 years of treatment with MBOCA (Stula et al. 1977). Although this change was not present in any of the control dogs, it was not considered to be treatment related. Studies in animals following acute, intermediate, or chronic exposure to MBOCA by any route would be useful in establishing if the central nervous system is one of the target organs for MBOCA toxicity. These studies should include neurobehavioral screening tests such as a functional observation battery of tests, tests for motor activity, and schedule-controlled operant performance tests. Neuropathology data from MBOCA-exposed animals would also be helpful. Epidemiological and Human Dosimetry Studies. Human studies on MBOCA consist of either case reports of accidental exposure or follow-up studies of workers previously exposed to MBOCA. Exposures in both cases are mainly inhalation and dermal. Since MBOCA is a potential human carcinogen, the follow-up studies focused on monitoring the possible development of bladder carcinomas in workers exposed to MBOCA (Ward et al. 1990). At the present time, there are three studies in progress sponsored by NIOSH that are investigating different aspects of MBOCA exposure in humans (FEDRIP 1991) (see Section 2.9.3). One of the studies involves workers exposed to MBOCA; the second involves biomonitoring for populations occupationally exposed to aromatic amines (including MBOCA); and the third involves the development and evaluation of biological methodologies for monitoring the presence and uptake of MBOCA in urine. Thus, at the present time, there are sufficient studies on biomonitoring of humans exposed to MBOCA. Biomarkers of Exposure and Effect Exposure. Sensitive methods for evaluation of MBOCA in urine are available. Since MBOCA can bind to body proteins and DNA, the presence of MBOCA adducts is an indication of exposure. Although information on some MBOCA metabolites and adducts is available, the development of sensitive methods for their determination is needed. Further identification of those two classes of MBOCA 58 2. HEALTH EFFECTS biomarkers in humans would be helpful in assessing MBOCA exposure levels in high-risk populations. Studies in rats showed that intraperitoneal injection of ring-labeled MBOCA, or ring-labeled N-acetylated MBOCA, resulted in the generation of three DNA adducts (Silk et al. 1989). These adducts were also produced by the in vitro reaction of the N-hydroxy derivative of MBOCA with rat liver slices. The major product of this reaction was also formed following incubation of DNA with N-hydroxy-4-amino-3-chlorobenzyl alcohol, the compound resulting from cleavage of the methylene bridge of N-hydroxy MBOCA. While the single ring species appears to be an intermediate in the formation of the DNA adduct, it is not known whether N-hydroxylation or bridge cleavage occurs first in the formation of the reactive species. The DNA adduct was analytically identified as N- (deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol. Since MBOCA adducts can be used as biomarkers of exposure additional information regarding their characteristics such as half-life would be useful in estimating MBOCA exposures. Effect. The urinary bladder is a target organ for MBOCA-induced carcinogenicity (Ward et al. 1990). Retrospective biological monitoring using cystoscopy would help identify new biomarkers necessary to characterize the preneoplastic state of the urinary bladder. Absorption, Distribution, Metabolism, and Excretion. Quantitative data on the absorption of MBOCA in humans and animals following all routes of exposure are very limited. Human studies indicate that MBOCA is absorbed rapidly and that the amount absorbed is proportional to the dose for the inhalation (Cocker et al. 1988, 1990; Ichikawa et al. 1990; NIOSH 1986b) and/or dermal routes (Chin et al. 1983; NIOSH 1986b). Data on absorption rates for all three routes are needed. Additional quantitative absorption data in animals via all three routes would be useful because they could be used to estimate absorption in humans. No studies were located regarding distribution in humans following inhalation, oral, or dermal exposures to MBOCA. Animal kinetic studies (following intraperitoneal or intravenous exposure) in rats and dogs indicate that MBOCA is distributed in the blood to liver, bile, kidney, lung, and fat (Cheever et al. 1991; Farmer et al. 1981; Manis et al. 1984; Morton et al. 1988; Sabbioni and Neumann 1990). It is not known if MBOCA reaches a steady state after repeated exposures. Additional inhalation and dermal exposure studies regarding distribution would be useful because of the potential for human exposure via those two routes. No information was located regarding metabolism in humans after oral exposure to MBOCA. Limited information is available regarding metabolism in humans following inhalation or dermal exposure (Cocker 1988, 1990; Ducos et al. 1985; Osorio et al. 1990) to MBOCA. Metabolism has been partially characterized in animals following oral exposure. In vitro studies investigating the capacity of MBOCA to form adducts characterized one of its metabolites, a product of cleavage between the methylene bridge and one of the aromatic nuclei, as a DNA adduct-forming metabolite, N-hydroxy-MBOCA (MBOCA-NHOH) (Silk et al. 1989). Several MBOCA metabolites were identified following N- and o-hydroxylation of MBOCA by the canine, guinea pig, and rat liver mixed-function oxidase systems (Chen et al. 1989). Because differences in metabolism may occur with differences in the route of exposure, more data on metabolism following inhalation and dermal exposures would be useful. Also needed is information on MBOCA metabolites in terms of their potential carcinogenic capacities. No human data were located regarding excretion following oral exposure to MBOCA. There is, MBOCA 59 2. HEALTH EFFECTS however, limited information on excretion in humans after inhalation and/or dermal exposure showing that the metabolites (N-acetyl MBOCA, B-N-glucuronide of MBOCA) and very limited amounts of parent MBOCA are excreted in the urine (Cocker et al. 1988; Ichikawa et al. 1990; NIOSH 1986b). Studies in rats show that, after acute oral exposure to radioactive MBOCA, the majority of the label is in the feces (Farmer et al. 1981; Groth et al. 1984). More information is needed on excretion rate in animals after exposure to MBOCA via all three routes in order to establish which is the major route of excretion. Comparative Toxicokinetics. Studies using rats (Farmer et al 1981; Groth et al 1984; Morton et al 1988; Tobes et al 1983) and dogs (Manis et al 1984b) indicate that the kinetics of MBOCA do not differ significantly across species and that the differences are primarily quantitative. Since the kinetic data alone do not allow for the identification of target organs common to humans and animals, additional studies on the distribution and toxicity may allow for identification of similar target organs. Additional studies in dogs would be helpful since they are similar to humans in that they develop bladder cancer following exposure to MBOCA. No animal data on toxicokinetics were located regarding interspecies differences or sex-related differences. The limited amount of animal data as well as a relative lack of data across different routes of exposure indicate that it may be difficult to compare the kinetics of MBOCA in animals with that in humans. Additional studies using several species and all three exposure routes are needed in order to determine similarities and differences between humans and animals. Methods for Reducing Toxic Effects. Protocols need to be developed to abolish or alleviate adverse effects following exposure to MBOCA. As in most cases of toxic exposures, supportive therapy dealing with symptoms is recommended. Biological monitoring of workers occupationally exposed to MBOCA is a preventive measure that can reduce potential toxic and carcinogenic effects. Among the other recommended procedures are monitoring MBOCA air levels and urinary MBOCA levels, doing wipe tests to check surface contamination that can potentially lead to skin exposure (Clapp et al. 1991), and considering the performance of cystoscopy in exposed workers for early detection of bladder tumors (Mason and Volger 1990; Ward et al. 1990). Also important in minimizing absorption is the selection of protective clothing and gloves. The permeability of eight commercially available glove materials (with regard to methanolic solutions of aromatic amines) differed considerably, and the penetration of MBOCA was dependent on the concentration (Weeks and Dean 1977). Therefore, the recommendation for how frequently the gloves should be changed depends on the specific situation. Continued education of the work force about potential work hazards is also important (NIOSH 1986b). 2.9.3 On-going Studies On-going studies regarding the health effects of MBOCA were reported in the Federal Research in Progress (FEDRIP 1991) database. The National Institute for Occupational Safety and Health (NIOSH) is the sponsoring institution of three studies involving MBOCA currently in progress. One project is entitled "Investigation of Workers Exposed to MBOCA" and the principal investigator is E.M. Ward from NIOSH. The main objective of this study is to see if the occurrence of cancer can be linked to exposure to MBOCA. To achieve this, the study will use biological markers (MBOCA in the urine and urinary bladder cytology) to develop more effective, sensitive, and specific early markers for occupationally related health conditions. F.B. Daniel from NIOSH is the principal investigator of the study on "Biomonitoring for Populations Occupations Exposed to Aromatic Amines." The investigator will analyze hemoglobin and DNA adduct formation by MBOCA in order to propose a methodology for monitoring workplace exposure. L.L. Lowry from NIOSH is the MBOCA 60 2. HEALTH EFFECTS principal investigator of the project entitled "Biological Monitoring Methods Research and Evaluation." This study will develop and evaluate biological monitoring methods in urine for their effectiveness in predicting worker uptake of MBOCA and two other compounds. The NTP has a study in progress on "Aryl Amine Adducts in Blood as Indicators of Exposure.” In this study, blood samples from 100 workers will be analyzed for hemoglobin o-toluidine adducts. MBOCA will be used to develop a high-performance liquid chromatography (HPLC) method for separation and isolation of mitochondrial or total aryl amine-DNA adducts. In addition, the in vitro activation of potential carcinogens will be studied, and a mathematical model for MBOCA distribution, metabolism, and adduct formation will be prepared. The overall objective of the project is to develop a more sensitive adduct isolation procedure to be used for biological monitoring. The contact person for this project is K. Cheever (NTP 1991a). A grant was awarded in September 1991 to the Michigan Department of Public Health to conduct an epidemiologic study under the title "Analytic Epidemiologic Study of MBOCA" (ATSDR 1991). The objective of the study is to determine if there is an association between exposure to MBOCA and occurrence of bladder cancer in workers, their family members, and residents who live near three NPL sites. The screening regimen includes urine dipstick self- testing (using Hemastix kits) and collection of specimens for cytology. Cystoscopy will be used in all cases with positive screening results. The information on all MBOCA-exposed members of the cohort will be maintained in a registry. A case-control study will be conducted to assess the association of MBOCA exposure and bladder cancer incidence. The contact person for this project is H. Humphrey from the Michigan Department of Public Health. MBOCA 3. CHEMICAL AND PHYSICAL INFORMATION 3.1 CHEMICAL IDENTITY Information regarding the chemical identity of MBOCA is located in Table 3-1. 3.2 PHYSICAL AND CHEMICAL PROPERTIES Information regarding the physical and chemical properties of MBOCA is located in Table 3-2. 61 MBOCA 3. CHEMICAL AND PHYSICAL INFORMATION 62 TABLE 3-1. Chemical Identity of 4,4’-Methylenebis(2-chloroaniline) (MBOCA) Characteristic Information Reference Chemical name 4,4'-Methylenebis(2-chloroaniline) Synonym(s) MBOCA,; 3,3'-dichloro-4,4'- CIS 1992; diaminodiphenylmethane; HSDB 1991; 4.4'-methylene(bis)-chloro- NRC 1981; aniline; 4,4’ -methylenebis OHM /TADS 1985; (o-chloroaniline); 4,4'-methylene- Smith and Registered trade name(s)? Chemical formula Chemical structure Identification numbers: CAS registry NIOSH RTECS EPA hazardous waste OHM/TADS DOT/UN/NA/IMCO shipping HSDB NCI bis[2-chlorobenzenamine]; bis(3-chloro-4-aminopropyl) methane; aniline, 4,4'-methylene- bis|2-chloro-; bis-(4-amino-3- chlorophenyl) methane; di(4-amino- 3-chlorophenyl) methane; bis amine; MCA; CL-MDA; DACPM and others Cuamine-M; Activator-M; CA-800; DAC; Bis-Amine A; Curene 442; MOCA C3H,CLN, DoD 101-14-4 CY 1050000 U158 8300209 No data 2629 No data Woodward 1983 CIS 1992 HSDB 1991; OHM /TADS 1985 IARC 1974 NRC 1981 HSDB 1991 HSDB 1991 HSDB 1991 OHM /TADS 1985 HSDB 1991 aMBOCA trade names that are not in use: Curalin M, Curalon M, Cyanaset, and LD 813 CAS = Chemical Abstracts Services; CIS = Chemical Information Systems; DOT/UN/NA/IMCO = Department of Transportation/United Nations/North America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency; HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NIOSH = National Institute for Occupational Safety and Health; NRC = National Research Council (Great Britain); OHM/TADS = Oil and Hazardous Materials / Technical Assistance Data System; RTECS = Registry of Toxic Effects of Chemical Substances MBOCA 63 3. CHEMICAL AND PHYSICAL INFORMATION TABLE 3-2. Physical and Chemical Properties of 4,4’-Methylenebis (2-chloroaniline) (MBOCA) Property Information Reference Molecular weight 267 IARC 1974 Color Pure form Colorless crystals IARC 1974 Technical form Yellow, tan, or brown pellets Smith and Physical state Melting point Boiling point Density at 24°C Odor Odor threshold Solubility: Water at 24°C Organic solvent(s) Partition coefficients: Log K,, Log K,. Vapor pressure at 25°C at 60°C at 100°C at 120°C Henry's law constant: Autoignition temperature Flashpoint Flammability limits Conversion factors Explosive limits Solid 110°C No data 1.44 g/mL Nearly odorless No data 13.9 mg/L Soluble in hot methyl ethyl ketone, alcohol, acetones, trichloroethylene, toluene, ether, esters, and lipids 3.94% 4,810 1.0x10° mmHg 1.3x10° mmHg 3.5x10° mmHg 54x10 mmHg 4x10 atm m /mole No data No data No data No data No data Woodward 1983; NRC 1981 HSDB 1991 HSDB 1991 NRC 1981; Sax and Lewis 1987 NRC 1981 Voorman and Penner 1986a HSDB 1991; OHM/TADS 1983; Smith and Woodward 1983; HSDB 1991 HSDB 1991 Smith and Woodward 1983 NRC 1981 Smith and Woodward 1983 NRC 1981 HSDB 1991 4Estimated value MBOCA 65 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 4.1 PRODUCTION MBOCA is a man-made chemical and has not been found in nature (IARC 1974). It is produced commercially by reacting formaldehyde with o-chloraniline (HSDB 1991; IARC 1974). Pure MBOCA is a colorless crystalline solid (Smith and Woodward 1983). The technical grade of MBOCA that is available in the United States comes mainly from Japan in the form of tan/yellow fused prills or pastilles. The diamine purity is 99.8%, typically with 0.2% free o-chloroaniline (monomer). Isomers are produced as side reactions such as trimers and tetrameres-diamines with three- and four-ring structures joined by methylene groups. Isomers constitute up to 8-10% of MBOCA. The dimer makes up to 90-92% of the MBOCA produced today for coatings and cast polyurethanes. There is no commercial use for pure dimer MBOCA other than for laboratory work. MBOCA has been produced commercially in the United States for some time. The first reported production was in 1956 (IARC 1974). U.S. production of MBOCA was estimated to be 3.3-5.5 million pounds in 1970 and 7.7 million pounds in 1972 (IARC 1974). In 1982, production of MBOCA in the United States was reported to have ceased (HSDB 1991). MBOCA has been manufactured in the United States by two companies E.I. Du Pont de Nemours and Company (Deepwater, New Jersey) and Anderson Development Company (Adrian, Michigan). However, EI. Du Pont de Nemours and Company ceased MBOCA production in 1978, and Anderson Development Company ceased production in 1979. Presently, all MBOCA used in the United States is imported. As of 1985, there were at least four production sites in the United States that use imported MBOCA: Polyester Corporation (Southampton, New York), American Cyanamid Company (Bound Brook, New Jersey), E.I. Du Pont De Nemours and Company (Deepwater, New Jersey), and Anderson Development Company (Adrian, Michigan) (OHM/TADS 1985). However, in 1992, Allchem Industries, Inc. (Gainesville, Florida), Maypro Industries, Inc. (Harrison, New York), and Miki Sangyo (USA), Inc. (New York, New York), were also reported to produce MBOCA for commercial sale (Van et al. 1992). Eighteen industrial sites (Table 4-1) were listed in the 1990 Toxics Release Inventory (TRI) as producers and/or users of MBOCA (TRI90 1991). However, since not all producers of MBOCA are required to report to TRI, the companies listed on the inventory cannot be considered the exclusive producers of MBOCA in the United States. This is not an exhaustive list. 4.2 IMPORT/EXPORT In 1978, approximately 0.4 million pounds of MBOCA were imported into the United States (HSDB 1991). The amount of MBOCA imported into the United States increased in 1983 to 1.51 million pounds. In 1991, approximately 2.0 million pounds of MBOCA were imported into the United States. The MBOCA was manufactured by two Japanese producers and a Taiwanese producer. 4.3 USE The majority of MBOCA consumed in the United States has been used as a curing agent for isocyanate-containing polymers, and only about 1% is used in epoxy/epoxy-urethane resin blends TABLE 4-1. Facilities That Manufacture or Process 4,4’-Methylenebis(2-Chloroaniline) (MBOCA)® Range of b maximum amounts Facility Location on site in pounds Activities AND uses PERMA-FLEX ROLLERS INC. NEWARK, DE 1,000-9,999 As a reactant; as an article component TOWNLEY MFG. CO. INC. CANDLER, FL 10,000-99,999 As a formulation component GALLAGHER CORP. GURNEE, IL 1,000-9,999 As a reactant MARTIN ENGINEERING CO. NEPONSET, IL 1,000-9,999 As a reactant ANDERSON DEVELOPMENT CO. GARY, IN 100,000-999,999 Import; for on-site use/processing; GATES RUBBER CO. POLYFLEX PLANT PERMATHANE CUSTOM MOLDED URETHANES VAIL RUBBER WORKS INC. POLYURETHANE SPECIALTIES CO. INC. DICAR INC. CONAP INC. MONARCH INDUSTRIAL TIRE CORP. D. S. BROWN CO. GRIFFITH POLYMERS INC. BELOIT CORP. MANHATTAN DIV. BAILEY -PARKS URETHANE TRCW INDUSTRIAL WHEELS INC. DICAR INC. TROSTEL POLYURETHANE ELIZABETHTOWN, KY WESTBROOK, ME SAINT JOSEPH, MI LYNDHURST, NJ PINE BROOK, NJ OLEAN, NY AKRON, OH NORTH BALTIMORE, OH HILLSBORO, OR AIKEN, SC MEMPHIS, TN NASHVILLE, TN TOMBALL, TX LAKE GENEVA, WI 1,000-9,999 1,000-9,999 1,000-9,999 10,000-99,999 10, 000-99, 999 10,000-99,999 1,000-9,999 1,000-9,999 10,000-99,999 1,000-9,999 1,000-9,999 1,000-9,999 1,000-9,999 1,000-9,999 for sale/distribution; as a formulation component As a formulation component As an article component As a reactant; as a formulation component For sale/distribution As a reactant As a formulation component; in re- packaging As a reactant In re-packaging As an article component As a reactant As a reactant As a reactant As a reactant As an article component 3perived from TRI91 (1993); MBOCA used in these facilities is imported States since 1979. Post office state abbreviations used from Japan because MBOCA has not been produced in the United Iv¥SOdSId ANY ‘ISN 'LHOJdWI ‘NOILONAOHd ‘+ vOOaW 99 MBOCA 67 4. PRODUCTION, IMPORT, USE, AND DISPOSAL (IARC 1974). These cured polymers have many commercial and military uses. MBOCA was reported to be the most widely used agent for curing castable liquid polyurethane elastomers (HSDB 1991; IARC 1974; Sax and Lewis 1987). Commercially, these MBOCA-cured polyurethanes have been used to produce shoe soles, rolls for postage stamp machines, cutting bars in plywood manufacturing, rolls and belt drives in cameras, computers, and reproducing equipment, and wheels and pulleys for escalators and elevators (NRC 1981). MBOCA has also been reported to be formulated with other aromatic diamines and sold under trade names as a curing agent (IARC 1974). MBOCA has also been used in the manufacture of gun mounts, jet engine turbine blades, radar systems, components in home appliances (HSDB 1991), and as a wiring patting and curing agent (Cowles 1978). Military applications of MBOCA-cured polyurethanes include ball seals on nuclear submarines, positioning strips in Poseidon missiles, and encapsulation of electric components (NRC 1981). 4.4 DISPOSAL Because MBOCA is defined as a "hazardous waste," companies that generate wastes containing 100 kg or more of MBOCA are required to conform with EPA regulations (EPA 1989; HSDB 1991). For more information on the regulations and guidelines that apply to MBOCA, see Chapter 7. No universal method exists for the disposal of carcinogenic compounds such as MBOCA (HSDB 1991). Product residues and sorbent media containing MBOCA have been packaged in epoxy-lined drums and disposed of at EPA-approved sites (OHM/TADS 1985). Destruction via chemical reaction is another method that has been used to dispose of small amounts of MBOCA (HSDB 1991). This method, in which MBOCA is oxidized with potassium permanganate, is generally used for laboratory wastes containing small amounts of MBOCA. Incineration technologies have been investigated for the disposal of MBOCA. MBOCA has been considered a good candidate for rotary kiln incineration at a temperature range of 820-1,600°C, with residence times of seconds for liquids and gases and hours for solids (EPA 1981b; HSDB 1991). MBOCA is also listed as a good candidate for fluidized bed incineration at temperatures ranging from 450°C to 980°C, and residence times similar to those for rotary kiln incineration (EPA 1981). Disposal of MBOCA contained in waste waters using activated carbon adsorption has been studied (HSDB 1991). Saturated filters used to remove MBOCA from waste water via carbon absorption can subsequently be destroyed by rotary kiln or fluidized bed incineration (EPA 1979). Biodegradation treatment of MBOCA using continuous flow reactors that are designed to remove potential hazardous chemicals from water and waste water may be also useful in clean-up operations. Similarly, activated carbon processes and ozone oxidation provide effective disposal treatment (EPA 1979). There is, however, no information on the availability of MBOCA residues from polyurethanes and other plastics. MBOCA 69 5. POTENTIAL FOR HUMAN EXPOSURE 5. POTENTIAL FOR HUMAN EXPOSURE 5.1 OVERVIEW MBOCA may be discharged to the environment as a result of an uncontained, open cycle, manufacturing process. Such a discharge could constitute a release to the atmosphere as a fugitive dust or as a spill of MBOCA pellets or heated liquid MBOCA. Otherwise non-hazardous solid wastes may become contaminated by MBOCA in the manufacturing process thus, making such wastes hazardous. The dust can settle to soil or surface waters where it will be strongly adsorbed to the organic matter in the soil or water column; therefore, it is unlikely to contaminate groundwater. Microbial degradation is a potentially significant degradation process and may be quite rapid if appropriate organisms are present in the soil or water. In air or surface waters, MBOCA may undergo photooxidation by alkoxyradicals. Members of the general population are unlikely to be exposed to MBOCA unless they live in an area that has been contaminated. Workers in plants that manufacture or use MBOCA have the potential to be highly exposed by inhalation or dermal contact. MBOCA has been identified in at least 4 of the 1,350 NPL hazardous waste sites (HAZDAT 1993). However, the number of sites analyzed for MBOCA is not known. The frequency of these sites within the United States can be seen in Figure 5-1. 5.2 RELEASES TO THE ENVIRONMENT 5.2.1 Air MBOCA may be released to the atmosphere in the exhaust emissions of facilities that manufacture it or use as a curing agent in the manufacture of diisocyanate-based polymers (Keeslar 1986) (Table 5-1). According to TRI91 (1993), a total of 1,362 pounds of MBOCA was released to the atmosphere in 1991, from manufacturing and processing facilities in the United States (see Table 5-1). The TRI data should be used with caution, however, since only certain types of facilities are required to report. 5.2.2 Water Of the 1,362 pounds of MBOCA released to the environment from TRI facilities in 1991, no discharges to surface waters were reported. One pound was reported to be transferred to a publicly owned treatment facility works (TRI91 1993). The TRI data should be used with caution since only certain types of facilities are required to report. 5.2.3 Soil Of the 1,362 pounds of MBOCA released to the atmosphere in 1991 from TRI facilities, no releases of MBOCA to land or through underground injection were reported. An additional 5,228 pounds were reported to have been transferred to off-site waste treatment, storage, and FIGURE 5—1. FREQUENCY OF NPL SITES WITH MBOCA CONTAMINATION * FREQUENCY BEER 1 sITE I 2 syes #Derived from HazDat 1993 3IHNSOdX3 NVWNH HO4 TVILN3LOd 'S vOoOoaw 0s TABLE 5-1. Releases to the Environment from Facilities That Manufacture or Process 4,4’ -Methylenebis(2-Chloroaniline) (MBOCA)® Reported amounts released in pounds Off-site b Underground Total . POTW waste Facility Location Air injection Water Land environment transfer transfer PERMA-FLEX ROLLERS INC. NEWARK, DE 0 0 0 0 0 0 0 TOWNLEY MFG. CO. INC. CANDLER, FL 750 0 0 0 750 0 0 GALLAGHER CORP. GURNEE, IL 0 0 0 0 0 0 0 MARTIN ENGINEERING CO. NEPONSET, IL 0 0 0 0 0 0 0 ANDERSON DEVELOPMENT CO. GARY, IN 0 0 0 0 0 0 0 * GATES RUBBER CO. ELIZABETHTOWN, KY 0 1] 0 0 0 0 255 POLYFLEX PLANT PERMATHANE CUSTOM MOLDED WESTBROOK, ME 5 0 0 0 5 0 3,606 URETHANES VAIL RUBBER WORKS INC. SAINT JOSEPH, MI 10 0 0 10 1 250 POLYURETHANE SPECIALTIES LYNDHURST, NJ 0 0 0 0 0 0 0 CO. INC. DICAR INC. PINE BROOK, NJ 0 0 0 0 0 0 0 CONAP INC. OLEAN, NY 92 0 0 0 92 0 367 MONARCH INDUSTRIAL TIRE AKRON, OH 500 0 0 0 500 0 0 CORP. D. S. BROWN CO. NORTH BALTIMOR, OH 0 0 0 0 0 0 GRIFFITH POLYMERS INC. HILLSBORO, OR 0 0 0 0 0 0 750 BELOIT CORP. MANHATTAN AIKEN, SC 0 0 0 0 0 0 0 DIV. BAILEY-PARKS URETHANE MEMPHIS, TN 5 0 0 0 5 0 0 TRCW INDUSTRIAL WHEELS NASHVILLE, TN 0 0 0 0 0 0 0 INC. DICAR INC. TOMBALL, TX 0 0 0 0 0 0 0 TROSTEL POLYURETHANE LAKE GENEVA, WI 0 0 0 0 0 0 0 Totals 1,362 0 0 0 1,362 1 5,228 Derived from TRI91 (1993); since 1979. MBOCA used in these facilities is imported from Japan because MBOCA has not been produced in the United States Post office state abbreviations used “The sum of all releases of the chemical to air, land, water Corrections regarding the amounts of released MBOCA have be POTW = publicly owned treatment works , and underground injection wells by a given facility en filed with EPA (Form R). 3HNSOdX3 NVANH HOA TVIINILOd 'S YOO8aW LL MBOCA 72 5. POTENTIAL FOR HUMAN EXPOSURE disposal facilities (TRI91 1993). The TRI data should be used with caution since only certain types of facilities are required to report. 5.3 ENVIRONMENTAL FATE 5.3.1 Transport and Partitioning Volatilization of MBOCA from soil or surface waters is unlikely to be a major factor for environmental fate because of its very low vapor pressure (1x107 mmHg at 25°C) (Keeslar 1986; NIOSH 1978b) and its strong adsorption to organic matter. MBOCA partitions to soil rather than water as a result of its relatively low solubility in water (13.9 mg/L) and its amine groups, which have an affinity for soil organic matter. This binding is rapid and very tight and results in virtually no movement of MBOCA through soil (Voorman and Penner 19864). The partitioning of MBOCA in the soil affects the uptake of the compound by plants grown in contaminated soil and its subsequent ingestion by humans. MBOCA is bioaccumulated by food plants (e.g., carrots, orchard grass, beans, cabbage, beet, sorghum, cucumber), but movement of the compound within the plant is extremely limited. MBOCA applied to leaf surfaces resulted in adsorption to the leaf cuticle but no movement beyond the application site. Exposure of roots of bean, sorghum, and carrots to aqueous solutions of 5 mg/L of MBOCA for 8 days resulted in relatively high concentrations on the root surfaces of these plants (37 mg/kg, 2,000 mg/kg, and 20 mg/kg, respectively), demonstrating bioconcentration at that site but limited translocation to plant shoots (1.7 mg/kg, 2-5 mg/kg, and virtually undetectable, respectively). MBOCA applied to soils at a concentration of 5 mg/kg again showed an uptake in the roots of cucumbers and beans (up to 17 mg/kg MBOCA); the shoots of these plants contained less than 0.2 mg/kg. This limited translocation may be due to the low water solubility of MBOCA (Voorman and Penner 1986b). This may be of concern in cases of accidents (or even during routine operations) in which MBOCA is released to the air. 5.3.2 Transformation and Degradation 5.3.2.1 Air The photooxidation half-life of MBOCA in air is estimated to be between 0.290 and 2.90 hours based on reactions with hydroxyl radicals (Howard et al. 1991), suggesting that this may be a significant fate process. 5.3.2.2 Water Studies examining the biodegradation of MBOCA, using activated sludge microorganisms, suggested that MBOCA was readily degraded (from 2.02 mg/L to 0.09 mg/L) in a continuous biological reactor within 24 hours, but not during a 7-day static incubation test (EPA 1979; Tabak et al. 1981). Other degradation processes were also effective in reducing the concentrations of MBOCA present in simulated waste water. Ozone oxidation reduced an initial concentration of 1.52 mg/L MBOCA to nondetectable levels within 5 minutes. Between 21 and 35 mg of carbon per liter, depending on the type of carbon, were required to reduce 1.0 mg/L MBOCA to 0.1 mg/L (EPA 1979). MBOCA was not susceptible to oxygen stripping (EPA 1979). MBOCA 73 5. POTENTIAL FOR HUMAN EXPOSURE The estimated photooxidation half-life of MBOCA in surface water is between 1.3 and 72 days; while in groundwaters, MBOCA may have a half-life of 8 weeks to 1 year (Howard et al. 1991). The estimated hydrolysis half-life of MBOCA in water at 25°C and pH 7 is more than 800 years (EPA 1988c). Studies of microbial degradation of MBOCA showed several biodegradation products, including N- monoacetyl MBOCA and N,N’-diacetyl MBOCA (Yoneyama and Matsumura 1984). 4,4’-Diamino- 3,3’-dichlorobenzophenone was produced from metabolic conversion of MBOCA by soil microorganisms (Voorman and Penner 1986a). 5.3.2.3 Sediment and Soil Carbon dioxide production from soil samples treated with MBOCA was less than 1% of the total applied, suggesting that aromatic rings are resistant to microbial degradation and oxidation (Voorman and Penner 1986a). These investigators did detect a metabolite with the methylene carbon oxidized to a carbonyl. Microbial degradation of MBOCA has been shown to occur using Bacillus megaterium and Nocardiopsis sp. isolated from soil. These microorganisms readily metabolize MBOCA with 39% and 24%, respectively, of the original concentration remaining after 3 hours of incubation. The major degradation pathways were: (1) acetylation of MBOCA to N-monoacetyl MBOCA and then to N,N'-diacetyl MBOCA, and (2) hydroxylation of N-monoacetyl MBOCA to N-hydroxy-N-acetyl MBOCA with the final metabolite being N-hydroxy-N,N-diacetyl MBOCA (Yoneyama and Matsumura 1984). Also present was a metabolite with the methylene carbon oxidized to a carbonyl (Voorman and Penner 1986a). The estimated half-life of MBOCA in soil based on aerobic biodegradation may range between 1 and 6 months (Howard et al. 1991). 5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT 5.4.1 Air Monitoring of MBOCA dust and vapor in the ambient air of a production facility in 1969 showed that the maximum 8-hour average concentrations were 0.32 mg/m® and 0.25 mg/m? respectively. Significant levels were detected only in areas adjacent to the pelletizing unit--although even these levels were only intermittently high. Skin absorption was the major source of exposure and could be effectively controlled with appropriate protective clothing and engineering controls (Linch et al. 1971). Ambient air and personal air monitoring was conducted at a plastics factory where MBOCA was used in the production of urethane. The results obtained from 10 ambient air samples (6 in the general work area, 4 in the area where MBOCA was melted) indicated that MBOCA was not present in the general area above the level of detection (0.015 pg/filter), and was present in the air near the MBOCA melting pot at levels up to 92 pg/m>. Personal air monitoring indicated that only those employed as mixers and molders were exposed to detectable levels of MBOCA, ranging from 0.06 to 0.70 pg/m>. Wipe samples of surfaces that workers were most likely to be exposed to contained low levels of MBOCA throughout. Surface wipe samples showed MBOCA contamination ranging from 0.1 pg/100 cm? near the trimmers work table to 19.1 pg/100 cm? adjacent to the MBOCA melting pot. Surfaces that were rarely wiped clean, but were not near the melting pot (such as the tops of storage cabinets), contained an average of 4.7 pg/100 cm? (Clapp et al. 1991). MBOCA air MBOCA 74 5. POTENTIAL FOR HUMAN EXPOSURE levels were also evaluated in a polyurethane elastomer factory, and the air exposure levels ranged from 0.2 to 8.9 pgm’ (Ichikawa et al. 1990). MBOCA levels were measured at 0.001-0.042 mg/m’ in the air of a factory producing rubber ski boots (Smith and Woodward 1983). MBOCA was present at only trace concentrations in air samples of dust taken 15 feet above ground level in a residential area known to be contaminated with MBOCA (Keeslar 1986). 5.4.2 Water A specialty chemical manufacturing plant in Adrian, Michigan, that produced more than 1 million pounds per year of MBOCA during the late 1970s was found to have released significant quantities of it in its waste water discharges. Water sampling surveys found the following concentrations of MBOCA associated with the facility (Parris et al. 1980): Industrial lagoon sediment >1,600 ppm Industrial lagoon effluent water 250 ppb Industrial site deep well water 1.5 ppb Surface runoff water from site 1 ppb Sewage treatment plant, influent water <0.5 ppb Sewage treatment plant, effluent water <0.5 ppb Sewage treatment plant, activated sludge 18 ppm (estimated) Raisin River water <0.1 ppb Samples of well water from a residential area adjacent to the manufacturing plant, however, did not contain detectable levels of MBOCA, suggesting that groundwater contamination had not occurred (Keeslar 1986). 5.4.3 Sediment and Soil Soil samples taken on the site of a manufacturing plant using MBOCA contained levels as high as 1,146 ppm, while concentrations along public roads near the site ranged from 4.6 to 590 ppm (Keeslar 1986). Soil from the yards of residences adjacent to the site (within a 1 kilometer radius) typically had 1.74 mg/kg MBOCA in the top 2 inches of soil and 0.02 mg/kg in the next 4 inches. 5.4.4 Other Environmental Media No studies were located on the levels of MBOCA found in other environmental media. However, it has been shown that MBOCA binds to and penetrates the roots of plants grown in contaminated soil. Once in the plant, MBOCA stays very close to the root surface and is not distributed throughout the plant (Voorman and Penner 1986b). 5.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE MBOCA is commercially used as a curing agent for isocyanate polymers by specialty manufacturers of industrial and commercial polyurethane products (e.g., as gears, gaskets, sport boots, and roller skate wheels). The form of MBOCA to which workers could be exposed is likely to be either a liquid emulsion, dust, or solid pellets (NIOSH 1986b; Schulte et al. 1988). Occupational exposures may occur at several stages of polymer production, especially where prepolymers are mixed with molten curing agent before molding (Edwards and Priestly 1992). In most cases, dermal absorption is the MBOCA 75 5. POTENTIAL FOR HUMAN EXPOSURE most important occupational exposure pathway (Edwards and Priestly 1992; Lowry and Clapp 1992). The National Occupational Health Survey estimated that 2,094 workers were potentially exposed to MBOCA in the workplace in 1980 (Schulte et al. 1988). A urinary monitoring program can determine aggregate worker exposure to MBOCA (Ward et al. 1986). Workers in a plastics plant that mixed or molded urethane products containing MBOCA were found to have detectable levels of MBOCA in their urine. Concentrations for the mixers ranged from 5 to greater than 100 pg/L urine (average concentration, 61.9 pg/L), whereas concentrations for the molders were considerably lower, nondetectable to 50 pg/L urine (average concentration, 14.8 pg/L). The greatest exposure route was inferred to be direct skin contact with MBOCA, despite the fact that the mixers wore gloves while transferring dry MBOCA, melting it, or dispensing the molten fluid (Clapp et al. 1991). After 2 days away from work, only 1 of 13 workers in the plastics plant had detectable levels of MBOCA in his urine. This worker also had the highest peak urinary MBOCA levels during the preceeding week (Clapp et al. 1991). Another investigation of workers in a polyurethane elastomer factory reported that preshift and postshift urinary levels were not significantly different in all exposed workers and that levels measured 48 hours after cessation of work were not always the lowest (Ichikawa et al. 1990). The difference may partially be explained by the actual levels of MBOCA in the workplace; workers that had MBOCA in the urine after 2 days away from work had the highest levels of the compound when last measured, suggesting that they were exposed to higher MBOCA levels than workers without any MBOCA in urine after the weekend. The reported findings may also reflect differences in metabolic rates between workers, or that different depots of MBOCA are excreted over different time frames from the body. Workers in a manufacturing plant using MBOCA had urine concentrations of MBOCA ranging from 13 to 458 ppb (mean 145 ppb). Their immediate families were found to have had exposures to MBOCA also--urine levels of MBOCA ranged from 0 to 15 ppb (Keeslar 1986). These findings suggest that direct exposure to MBOCA itself in an occupational setting or at a hazardous waste site may not be necessary for exposure, and that people can also be exposed to MBOCA by contact with an MBOCA-exposed individual. Monitoring of workers at seven facilities in Australia that used MBOCA in the manufacture of polyurethane polymers showed that average MBOCA levels in the urine of the workers dropped from 29.6 to 10.4 mg/L within 8-9 months after the implementation of an exposure prevention program (Wan et al. 1989). Another study of 150 workers in 19 factories with industrial exposure to MBOCA showed that, at the end of the workshift, excretion levels ranged from less than 0.5 pg/L to 1,600 pg/L of MBOCA, with the highest average urine concentrations (600 pg/L) in workers directly involved in MBOCA manufacture or use; urine MBOCA levels dropped after exposure controls were implemented in the plant (Ducos et al. 1985). Similar decreases in urine MBOCA concentrations were observed following improvements in ventilation and with the use of protective clothing by workers exposed to MBOCA (Thomas and Wilson 1984). Results of a voluntary biological monitoring program implemented by the PMA suggest that exposure to MBOCA among users of the compound decreased between 1985 to 1990. Following implementation of a number of engineering controls to limit exposure, including the use of closed transfer systems and the use of a fused, hardened MBOCA pellet, worker urine specimens containing less than 25 pg MBOCA/L increased from 77% to 86% of the total amount collected. Over this same time period, urine samples containing greater than 50 pg MBOCA/L decreased from 12% to 8% of the total number of samples collected (Lowry and Clapp 1992). MBOCA 76 5. POTENTIAL FOR HUMAN EXPOSURE Workers in shipyards where MBOCA is used as a potting and molding agent for wiring may also have potentially high exposures to MBOCA (Cowles 1978). Members of the general population may be exposed to MBOCA if they consume certain types of plants (e.g., root crops) grown in MBOCA-contaminated soil. MBOCA has been found to adhere to the leaves and roots of plants, and the compound is not removed by rinsing with water (Voorman and Penner 1986b). 5.6 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES Populations living near areas known to be contaminated with MBOCA can be considered to have high potentials for exposure. Although adults living in a contaminated area of Adrian, Michigan, did not have detectable levels of MBOCA in their urine, young children (all under the age of 6 years) from the area had urine concentrations of 0.3 to 1.0 ppb. The levels in children were believed to be a direct consequence of their frequent contact with contaminated soils. MBOCA was detected in the residences of this area, primarily on floors, carpeting, and vacuum cleaner bags; however, other household surfaces did not have significant concentrations of MBOCA (Keeslar 1986). 5.7 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 MBOCA 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 MBOCA. 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. 5.7.1 Identification of Data Needs Physical and Chemical Properties. The physical and chemical properties of MBOCA are sufficiently defined to allow assessments of the environmental fate of the compound to be made (see Chapter 3 and Tables 3-1 and 3-2). No further information is needed. Production, Import/Export, Use, Release, and Disposal. Presently, conflicting information exists on the number of people that have been or are being exposed to MBOCA in the workplace; the numbers range from 114 (NOES 1992) to 2,094 (Schulte et al. 1988). The general population is not likely to be exposed to MBOCA. Information is unavailable on current or historical MBOCA production in the United States. MBOCA is only used in the workplace in 18 facilities in the United States (TRI90 1992). Information on potential food contamination with MBOCA would also be useful in reducing risks associated with general population exposures. MBOCA may be released to the environment in waste waters or fugitive emissions from plants. Additional information is needed on atmospheric releases MBOCA 77 5. POTENTIAL FOR HUMAN EXPOSURE of MBOCA from manufacturing facilities to assess the potential for general population exposure. At the present time, there is no information on the amounts of MBOCA disposed by different methods except for TRI data on the amounts released into different media (see Tables 5-1 and 5-2). Additional information on currently used disposal methods would allow the determination of their efficiency. Also needed is information on the availability of MBOCA residues from polyurethanes and other plastics. According to the Emergency Planning and Community Right-to-Know Act of 1986, 42 U.S.C. Section 11023, industries are required to submit chemical release and off-site transfer information to the EPA. The Toxics Release Inventory (TRI), which contains this information for 1990, became available in May of 1992. This database will be updated yearly and should provide a list of industrial production facilities and emissions. Environmental Fate. The fate of MBOCA in soil has been described (EPA 1979; Voorman and Penner 1986a; Yoneyama and Matsumura 1984), and some information is available on the spread and transport of MBOCA in surface waters (Parris et al. 1980). Additional data on the aquatic fate of MBOCA, its residence time in the water column, and its absorption to sediment or organic matter in the water would assist in assessing drinking water contamination. Information on the fate of MBOCA adsorbed to sediment would be useful in assessing uptake by aquatic organisms and reentry of MBOCA into the water column. Information on the half-life of MBOCA in the environment would also be useful for assessing the risk for human exposure. Bioavailability from Environmental Media. Available pharmacokinetic data suggest that MBOCA is absorbed by humans following dermal and inhalation exposures (Chin et al. 1983; Cocker et al. 1988, 1990; Ichikawa et al. 1990; NIOSH 1986b). MBOCA has been measured in the urine of workers following dermal and/or inhalation exposures, suggesting rapid absorption and excretion. Information on the absorption of MBOCA by humans as a result of ingestion of contaminated water or food has not been found and would be useful in assessing the uptake of MBOCA from contaminated foods. Further information on the uptake of MBOCA by all three exposure routes, particularly the differentiation of dermal and inhalation exposure in workers, would be helpful in determining potential uptake of MBOCA as a result of exposure to contaminated air, water, or foods, or contact with contaminated surfaces. Food Chain Bioaccumulation. The bioconcentration factor of MBOCA has been estimated to be 5.75 in aquatic organisms (HSDB 1991). In addition, it has been shown that MBOCA binds to and penetrates the roots of plants grown in contaminated soil and is not easily removed by rinsing. However, MBOCA stays very close to the root surface and is not distributed throughout the plant, and the roots bioaccumulate the chemical (Voorman and Penner 1986b). This information suggests that there is a potential for food chain bioaccumulation both from aquatic organisms and the root systems of terrestrial plants. Actual data on the potential for aquatic organisms to bioaccumulate MBOCA would be useful in determining potential food chain concentrations. Exposure Levels in Environmental Media. Some information exists on levels of MBOCA found in the workplace and the environment around facilities that manufacture or use MBOCA (Keeslar 1986; Parris et al. 1980). Further information on atmospheric levels of MBOCA in areas other than the workplace would be helpful for estimating general population exposure. Reliable monitoring data for the levels of MBOCA at hazardous waste sites are also needed. The MBOCA 78 5. POTENTIAL FOR HUMAN EXPOSURE information collected on levels of MBOCA in the environment could be combined with the information on body burden to assess the potential risk of adverse health effects in populations living near hazardous waste sites. Exposure Levels in Humans. Certain population groups are known to have a higher risk of exposure to MBOCA than others. The highest exposures are found in workers at manufacturing facilities that use MBOCA in the production of polyurethane plastics (Clapp et al. 1991; Ichikawa et al. 1990; Schulte et al. 1988; Ward et al. 1987). The next highest levels are found in populations that live near facilities where uncontrolled MBOCA releases occur (Keeslar 1986). Specific information on where such releases occur, on the populations living near such facilities, and on the levels to which they may be exposed was not found. This information is needed to assess whether health studies on these populations need to be conducted. Exposure Registries. No exposure registries for MBOCA 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. 5.7.2 On-going Studies The NIOSH is conducting a long-term study of former MBOCA workers in Adrian, Michigan. The health of the workers will be followed for 10-15 years to help assess the risk of MBOCA as a potential human carcinogen (FEDRIP 1991; Keeslar 1986). MBOCA 79 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 MBOCA, its metabolites, and other biomarkers of exposure and effect to MBOCA. 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 MATERIALS Very few biological materials have been analyzed for the presence of MBOCA or its metabolites. MBOCA and its metabolites have been measured in urine of exposed humans and experimental animals. Hemoglobin adducts have also been measured in the blood of exposed animals. The most frequently used techniques are gas chromatography (GC) with electron capture detection (ECD) and high-performance liquid chromatography (HPLC) with electrochemical detection (ED). Detailed methodologies from selected studies are presented in Table 6-1. Only a small amount of absorbed MBOCA is excreted in urine as MBOCA in animals, and probably in humans as well (see Section 2.3). The methods used to detect MBOCA in urine are somewhat limited since they commonly measure unmetabolized MBOCA--not its metabolic by-products. Some methods have been developed to directly measure major MBOCA metabolites in urine. Other analytical methods pretreat urine samples with acid, base and/or heat to release MBOCA from its various conjugates. Another approach is to analyze the longer-lived complexes, such as MBOCA- hemoglobin adducts. The specific methods used are noted in the following text and in Table 6-1. Hemoglobin adducts of MBOCA and its metabolites have been detected in animals dosed with the chemical (Chen et al. 1991; Sabbioni and Neumann 1990). The methods used were HPLC/ED, gas chromatography/mass spectrometry (GC/MS), and GC/ECD. Sample preparation for all three methods required hemoglobin to be isolated from the blood of the test animals and hydrolyzed to release the bound MBOCA. Insufficient data were provided to compare the different methods, but MBOCA was detected and quantified in the blood of dosed rats by all three methods. GC/MS in the negative chemical ionization mode, with a detection limit of 2 pg, appeared to be the most sensitive of the methods tested (Sabbioni and Neumann 1990). Both GC and HPLC have been used to separate MBOCA and its metabolites from urine. Most recently, HPLC has become the method of choice to selectively detect MBOCA and its metabolites in urine. The most sensitive and specific detection methods for HPLC are ED (Ichikawa et al. 1990; NIOSH 1986b; Okayama et al. 1988; Trippel-Schulte et al. 1986; Vantulder et al. 1981) and photoconductivity detection (PCD) (Ducos et al. 1985). Of these, ED has been the most frequently used detection method. Ultraviolet detection (UV) has also been paired with HPLC (McKerrell et al. 1987; Angerer and Schaller 1985; Trippel-Schulte et al. 1986) but is less sensitive and less selective than either ED or PCD (Trippel-Schulte et al. 1986). TABLE 6-1. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Biological Materials Sample detection Percent Sample matrix Preparation method Analytical method limit recovery Reference Blood Centrifuge sample to ex- HPLC/ED 25 pg 84 Sabbioni and (hemoglobin tract red blood cells; lyse (MBOCA) (MBOCA) Neumann 1990 adducts) with EDTA; centrifuge to remove debris and GC/MS (EI, SIM) 50 pg 80 (Ac- precipitate hemoglobin (Ac-MBOCA) MBOCA) with ethanol; centrifuge and retain precipitate; GC/MS (NCI) 30 pg NR hydrolyze with NaOH and SDS; extract with 2 pg NR ether; evaporate; recon- stitute in methanol Blood Centrifuge sample to ex- GC/ECD NR NR Chen et al. 1991 (hemoglobin tract red blood cells; lyse adducts) with EDTA; precipitate hemoglobin with ace- tone/HCI; centrifuge and retain precipitate; wash with acetone/HCI, acetone, and ether; re- constitute in SDS and add HCI to hydrolyze; extract with hexane; eva- porate; derivatize with heptafluorobutyric anhy- dride in isooctane; ter- minate reaction with NH,OH SAOHL3N TVYOILATVYNY 9 vYOOo8n 08 TABLE 6-1. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Biological Materials (continued) Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Urine Urine Urine Acidify sample with GC/FID citric acid; adjust to pH 9.5 with NaOH; extract with diethyl ether; wash with sodium bicarbonate; dry with anhydrous so- dium sulfate; evaporate and redissolve in ace- tone; evaporate; frac- tionate using TLC; re- move region of silica gel containing MBOCA and extract with acetone; evaporate; derivatize with TFA; evaporate and reconstitute in triphenyl- amine in carbon disulfide Add internal standard to GC/ECD sample; add 1 mL of 1 M NaOH; extract with diethyl ether; evaporate; derivatize with HFBC Extract with diethyl GC/ECD ether; derivatize with HFBC 1 ug/L 2.7 ug/L 24 g/g creatinine 70-78 85-90 NR Van Roosmalen et al. 1979, 1981 (IARC Method 8) Gristwood et al. 1984 Thomas and Wilson 1984 SAOHL13IW TVOILATVYNY 9 vYOOoaW 18 TABLE 6-1. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Biological Materials (continued) Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Urine Urine (N-acetyl- MBOCA) Hydrolyze sample with HRGC/ECD NaOH; extract with methanol and diethyl ether/hexane; concentrate; add TEA, HFBA, and MDA; extract derivative with hexane in KH,PO, buffer; centrifuge; cleanup organic phase on Florisil® if needed Add internal standard to GC/MS (EI) sample; hydrolyze with NaOH; extract with diethyl ether; evaporate; derivatize with PFPA; evaporate and redissolve in toluene 1 pg/L 6.7 ug/L 79-89 NR NIOSH 1984 Cocker et al. 1988 SAOHLIN TVYOILATVYNY 9 vOOaW z8 TABLE 6-1. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Biological Materials (continued) Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Urine Urine Hydrolyze sample with acid and heat; adjust pH to >9 with NaOH; extract sequentially with diethyl ether and HCI, adjust pH with NaOH; extract with diethyl ether; dry with Na,SO,; evaporate and redissolve in acetonitrile /water; elute from reverse-phase column with acetoni- trile /water Hydrolyze sample with NaOH and heat; extract with hexane; centrifuge; evaporate; reconstitute with ammonium acetate/ acetonitrile; clute from reverse-phase column with ammonium acetate/ acetonitrile HPLC/UV HPLC/UV 3 pg/L 10 pg/L 101 94-98 Angerer and Schaller 1985 McKerrell et al. 1987 SAOHL3N TVYOILATVYNY 9 vYOO8g £8 TABLE 6-1. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Biological Materials (continued) Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Urine Urine Urine (human) Precipitate proteins from sample with TCA; cen- trifuge; extract with ether; concentrate using HPLC precolumn and elute from analytical column with ammonium acetate/acetonitrile mixture Stabilize sample with citric acid; elute from HPLC column with ammonium phosphate- buffered acetonitrile Stabilize sample with citric acid; add ethanol and sodium bicarbonate; extract with diethyl ether; concentrate organic layer; dilute with acetonitrile /sodium ace- tate; elute from reverse- phase column with ace- tonitrile /sodium acetate HPLC/UV HPLC/ED HPLC/ED HPLC/ED 51.5 ng 12.1 ng 5 pg/L 1-10 pg/L 79-80 42-44 97-111 NR Trippel-Schulte et al. 1986 NIOSH 1986b Vantulder et al. 1981 SAOHL3W TVOILATVYNY 9 vOOgn v8 TABLE 6-1. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Biological Materials (continued) Sample detection Percent Sample matrix Preparation method Analytical method limit recovery Reference Urine Mix sample with metha- HPLC/ED 1 ug/L 97-100 Okayama et al. nol/heptane sulfonate / 1988 acetic acid; centrifuge; cleanup supernatant on ODS® column; elute from HPLC column with methanol/water Urine Saturate citric acid- HPLC/PCD <1 pg/L 85-86 Ducos et al. 1985 preserved sample with sodium bicarbonate; cleanup on Extrelut cartridge; extract with methylene chloride; evaporate; redissolve in methylene chloride; elute from HPLC column with isooctane (or hexane)- isopropanol-methanol Ac-MBOCA = N-acetyl-4,4'-methylenebis-(2-chloroaniline); ECD = electron capture detection; ED = electrochemical detection; EDTA = ethylene diamine tetraacetic acid; EI = electron impact; FID = flame ionization detection; GC = gas chromatography; HCI = hydrochloric acid; HFBA = hepta-fluorobutyric anhydride; HFBC = heptafluorobutyryl chloride; HPLC = high-performance liquid chromatography; HRGC = high-resolution gas chromatography; KH,PO, = monopotassium phosphate; M = molar; MDA = 4,4'-methylenediamine; MS = mass spectrometry; NaOH = sodium hydroxide; Na,SO, = sodium sulfate; NCI = negative chemical ionization; NH;OH = ammonium hydroxide; NR = not reported; PCD = photoconductivity detection; PFPA = pentafluoropropionic anhydride; SDS = sodium dodecyl sulfate; SIM = selected ion monitoring; TCA = trichloroacetic acid; TEA = triethylamine; TFA = trifluoroacetic anhydride; TLC = thin layer chromatography; UV = ultraviolet detection SAOHL3N TVYOILATYNY ‘9 YOO8W G8 MBOCA 86 6. ANALYTICAL METHODS Although not the preferred method, GC may still be used to measure MBOCA and its metabolites in urine. Analysis of a heptatluorobutyryl derivative of MBOCA using GC/ECD has been frequently used (Gristwood et al. 1984; NIOSH 1984; Thomas and Wilson 1984), but GC/flame ionization detection (FID) has also been used to analyze a trifluoroacetyl derivative (Van Roosmalen et al. 1979, 1981). The sensitivity of GC with either ECD or FID is in the low-ppb (pg/L) range, but the electron capture detector is more selective and gave higher recovery of analyte. GC and MS are frequently used to confirm the identity of isolated MBOCA compounds (Chen et al. 1991), and a method for quantifying the metabolite N-acetyl-MBOCA in urine using GC/MS has been developed (Cocker et al. 1988). The sensitivity of this method is also in the low-ppb range. 6.2 ENVIRONMENTAL SAMPLES As with biological materials, the two primary methods used to separate MBOCA from other compounds in environmental samples are HPLC and GC. Most of the analytical methods found describe detection and measurement of MBOCA in air, the medium of primary interest for monitoring potential worker exposures in facilities manufacturing or using the chemical. Table 6-2 presents details on selected analytical methods for determining MBOCA in environmental samples. Air samples have been collected either in impingers containing a sorbent liquid (Ebell et al. 1980; Nieminen et al. 1983; Skarping et al. 1985) or on solid sorbent cartridges (Ichikawa et al. 1990; James et al. 1985; Sawicki et al. 1975; Yasuda 1975). Air samples have also been collected on glass fiber filters designed to trap and stabilize MBOCA in both particulate and vapor form (NIOSH 1985, 1986b; Purnell and Warwick 1981; Rappaport and Morales 1979). MBOCA is desorbed from the collection medium with an organic solvent. For GC analysis, the extract is usually reacted with a perfluoro fatty acid anhydride to make the corresponding MBOCA derivative that is then analyzed. The most commonly used detectors are FID, ECD, and thermionic nitrogen-specific detection (TS D), but TSD and ECD are preferred because of their increased sensitivity (sub- to low-ppb [pg/m”] range) and selectivity compared to FID (low-ppb range) (Ichikawa et al. 1990; Sawicki et al. 1975; Skarping et al. 1985; Yasuda 1975). Sample preparation for HPLC generally requires only desorption from the collection medium prior to separation on the HPLC column. Two detection methods, UV and ED, have been paired with HPLC for the analysis of air samples containing MBOCA (James et al. 1985; Nieminen et al. 1983; NIOSH 1986b; Purnell and Warwick 1980, 1981; Rappaport and Morales 1979). Of these, ED is clearly the most sensitive (ppt [ng/m’ | compared to sub to low ppb for UV) and selective detection method, and HPLC/ED is the method recommended by NIOSH for the detection of low levels of MBOCA in air (NIOSH 1986b; Purnell and Warwick 1980, 1981). Both the HPLC and GC methods gave high recoveries and precision making them reliable methods for determination of MBOCA in air samples. HPLC/UV and GC/FID can be used to determine MBOCA in incinerator effluents indicating that even highly contaminated air samples can be analyzed with these methods (James et al. 1985). While no specific information was located on the analysis of similar materials using the other methods, it is likely that they too can be used for highly complex samples. HPLC/ED can be used to measure MBOCA in surface water, groundwater, soil, and sludge and is the IARC-recommended method for these media (Rice and Kissinger 1981, 1982). Liquid samples require only filtering prior to concentration on a reverse-phase HPLC column and separation on an analytical column. Solid samples must be mixed with water and filtered prior to the concentration step. Sensitivity is in the low-ppt range and precision is excellent. No recovery data were reported. An alternative EPA-recommended method of determining MBOCA in waste water requires more extensive sample handling prior to analysis by GC/TSD (EPA 1981a). This method has a sensitivity TABLE 6-2. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Environmental Samples Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Air Air Air Air Collect sample on Gas- chrom® § cartridge; desorb with acetone Collect sample on silica gel; desorb with methanol; evaporate; derivatize with HFBA Collect sample in impinger containing HCl/acetic acid/water solution; add NaOH; extract with chloroform; derivatize with TFAA; evaporate; redissolve in toluene Collect sample in impinger containing alkaline ethanol; add phosphoric acid; evaporate; add phosphate buffer and toluene to extract; derivatize with PFPA; analyze toluene layer GC/FID GC/ECD GC/ECD; GC/TSD HRGC/TSD 2 pg/m’ 0.1 pg/m3 40 g/m’ 1 ng/m> 92 NR NR 100 Sawicki et al. 1975; Yasuda 1975 Ichikawa et a 1990 Ebell et al. 1980 Skarping et al. 1985 SAOHL3N TVOILATVNY ‘9 YOOaW .8 TABLE 6-2. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Environmental Samples (continued) Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Air Air Air Collect sample on glass ~~ HPLC/UV fiber filter /silica gel cartridge; extract with methanol; centrifuge; elute from reverse-phase column with aceto- nitrile /water Collect sample on HPLC/UV impinger containing KOH /ethanol; add HCI; filter out KCl precipitate; evaporate; redissolve in ethanol /water; elute from HPLC column with acetate-buffered tetrahydrofuran/aceto- nitrile /water Collect sample on a HPLC/UV glass fiber filter; desorb ~~ HPLC/ED with methanol; elute from reverse-phase column with phosphate- buffered methanol ~3 pg/m’ 1-5 pg/m’ 7.5 pug/m> 0.1 pg/m> 86-97 NR 86-98 86-98 Rappaport and Morales 1979 Nieminen et al. 1983 Purnell and Warwick 1980), 1981 SAOHL3N TVYOILATYNY 9 vOOgn 88 TABLE 6-2. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Environmental Samples (continued) Sample detection Percent Sample matrix Preparation method Analytical method limit recovery Reference Air Collect sample on glass HPLC/UV 0.015 pg/filter NR NIOSH 1986b fiber filter; desorb with KOH /methanol; elute HPLC/ED <0.05 pug/filter NR from HPLC column with acetonitrile /water Waste water Extract sample with GC/TSD 1 pg/L 44-93 EPA 1981a methylene chloride; dry over anyhdrous NaSO,; concentrate; reconstitute with hexane Surface water, Mix solid samples with HPLC/ED 25 ng/L NR Rice and Kissinger groundwater, soil water; filter water or water from soil sample; concentrate on reverse- phase HPLC sampling column; elute to analytical column with methanol/ammonium acetate 1981, 1982 (IARC Method 4) SAOHL13N TVYOILATYNY ‘9 vOO8Wn 68 TABLE 6-2. Analytical Methods for Determining 4,4’-Methylenebis(2-chloroaniline) (MBOCA) in Environmental Samples (continued) Sample matrix Preparation method Analytical method Reference Laboratory wastes Surface wipe Add ascorbic acid to waste solution; adjust pH to 8 with NaOH; centrifuge; add methanol to supernatant and centrifuge; elute from HPLC column with ammonium acetate /methanol Collect samples using Whatman filter tabs; desorb in 0.1N KOH in methanol; elute from HPLC column with acetonitrile /water HPLC/UV/ED HPLC/ED Sample detection Percent limit recovery 5 ng (UV); NR 4.6 ng (ED) 0.008 pg/wipe NR Barek et al. 1985 NIOSH 1986b ECD = electron capture detection; ED = electrochemical detection; FID = flame ionization detection; GC = gas chromatography; HCI = hydrochloric acid; HFBA = heptafluorobutyric anhydride; HPLC = high-performance liquid chromatography; HRGC = high- resolution gas chromatography; KCI = potassium chloride; KOH = potassium hydroxide; N = normal; NaOH = sodium hydroxide; NaSO, = sodium sulfate; NR = not reported; PFPA = pentafluoropropionic anhydride; TFAA = trifluoroacetic anhydride; TSD = thermionic nitrogen-specific detection; UV = ultraviolet detection SAOHL3N TVYOILATYNY 9 vOO8W 06 MBOCA 91 6. ANALYTICAL METHODS in the low-ppb range, but analyte recovery varies widely. Both methods are selective for MBOCA. MBOCA has been detected in laboratory wastes using HPLC/UV/ED (Barek et al. 1985). While ED was found to be more sensitive than UV for many chemicals in the wastes, it was comparable for detection of MBOCA. Either detector could be used to monitor the destruction of aromatic amines in laboratory wastes. No details on recovery or precision were provided. Surface wipe and hand monitoring samples taken from areas in a factory in which MBOCA was used were analyzed for the chemical using HPLC/UV and HPLC/ED (NIOSH 1986b). Because of its increased sensitivity, HPLC/ED was used for all samples containing low levels (<2 pg/mL) of MBOCA and HPLC/UV was used to analyze those with higher levels. Recovery and precision were both high using these methods. 6.3 ADEQUACY OF THE DATABASE Section 104(i)(S) 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 MBOCA 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 MBOCA. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. 6.3.1 Identification of Data Needs Methods for Determining Biomarkers of Exposure and Effect. The measurement of MBOCA in urine has been used to monitor populations exposed occupationally to the chemical. The most frequently used methods are HPLC/ED and GC/ECD (Gristwood et al. 1984; Ichikawa et al. 1990; NIOSH 1984, 1986b; Okayama et al. 1988; Thomas and Wilson 1984; Trippel-Schulte et al. 1986; Vantulder et al. 1981). These methods are sensitive and have produced reliable results in several monitoring studies to estimate exposure of workers who have contact with MBOCA during manufacturing operations. There are no known adverse health effects associated with exposure to MBOCA other than its weak association with bladder cancer. While hemoglobin adduct formation has been demonstrated in exposed animals (Chen et al. 1991; Sabbioni and Neumann 1990), the relevance of these adducts with bladder cancer or with other adverse health effects is not known. The methods that have been tested for measuring hemoglobin adducts include HPLC/ED, GC/ECD, and GC/MS (Chen et al. 1991; Sabbioni and Neumann 1990). The limited information on these methods suggests that they are sensitive and reliable. However, since there is no clear relationship between the presence of these adducts in blood and adverse health consequences, no additional methods development is recommended at this time. Methods for Determining Parent Compounds and Degradation Products in Environmental Media. Numerous methods have been developed for the measurement of MBOCA in air (Ebell et MBOCA 92 6. ANALYTICAL METHODS al. 1980; Ichikawa et al. 1990; Nieminen et al. 1983; NIOSH 1986b; Purnell and Warwick 1980, 1981; Rappaport and Morales 1979; Sawicki et al. 1975). The most sensitive and reliable of these are GC with TSD or ECD and HPLC with ED (Ebell et al. 1980; Ichikawa et al. 1990; NIOSH 1986b; Purnell and Warwick 1980, 1981). These methods are useful for the measurement of low concentrations (ppt) of MBOCA in air, and no additional methods development is needed. HPLC/ED and GC/TSD can be used to determine the presence of MBOCA in water and soil (EPA 1981a; Rice and Kissinger 1981, 1982), surface wipe and hand monitoring samples (NIOSH 1986b), and laboratory waste (Barek et al. 1985). While these methods are sensitive for measuring MBOCA in environment media, they are of unknown reliability. Some additional data on the accuracy and precision of measurements in liquid and solid media would be useful. 6.3.2 On-going Studies No on-going studies focused on analytical method development for MBOCA were located. However, several of the on-going studies on MBOCA (FEDRIP 1991) involve the development of methods to better identify MBOCA adducts, which should make the process of monitoring exposed individuals casier. NIOSH is the sponsoring institution of three studies involving MBOCA that are currently in progress. Two of the three studies involve the development of more sensitive methods to monitor and evaluate the effects following MBOCA exposure. F.B. Daniel from NIOSH is the principal investigator of the study on "Biomonitoring for Populations Occupations Exposed to Aromatic Amines." The investigator will analyze hemoglobin and DNA adduct formation by MBOCA in order to propose a methodology for monitoring workplace exposure. For the second project, L.L. Lowry from NIOSH is the principal investigator of the study entitled "Biological Monitoring Methods Research and Evaluation." This study involves the development and evaluation of biological monitoring methods in urine to determine their effectiveness in predicting worker exposure to MBOCA. The NTP has a study in progress on "Aryl Amine Adducts in Blood as Indicators of Exposure" (NTP 1991a). In this study, blood samples from 100 workers will be analyzed for hemoglobin o-toluidine adducts. MBOCA will be used to develop an HPLC method for separation and isolation of mitochondrial or total aryl amine-DNA adducts. In addition, the in vitro activation of potential carcinogens will be studied, and a mathematical model for MBOCA distribution, metabolism, and adduct formation will be prepared. The overall objective of the project is to develop a more sensitive adduct isolation procedure to be used for biological monitoring. The contact person for this project is K. Cheever (NTP 1991a). MBOCA 93 7. REGULATIONS AND ADVISORIES The international, national, and state regulations and guidelines regarding MBOCA in air, water, and other media are summarized in Table 7-1. ATSDR has derived one MRL value for MBOCA. A chronic-duration oral MRL of 0.003 mg/kg/day was derived for MBOCA based on its ability to cause hepatic effects in dogs (Stula et al. 1977). EPA has not assigned a reference dose or concentration for MBOCA. MBOCA is on the list of chemicals appearing in "Toxic Chemicals Subject to Section 313 of the Emergency Planning and Community Right-to-Know Act of 1986" (EPA 1988d, 1988e). MBOCA is designated as a hazardous substance (EPA 1985, 1989) and is subject to reporting and record-keeping requirements (EPA 1986, 1988f). The Department of Health and Human Services has determined that MBOCA may reasonably be anticipated to be a carcinogen (NTP 1991b). MBOCA 94 7. REGULATIONS AND ADVISORIES TABLE 7-1. Regulations and Guidelines Applicable to 4,4’-Methylenebis(2-chloroaniline) (MBOCA) Agency Description Information References INTERNATIONAL IARC Carcinogenic classification Group 2A8 IARC 1987 NATIONAL Regulations: a. Air OSHA PEL TWA (skin designation) 0.02 ppm OSHA 1989a (29 CFR (0.22 mg/m3) 1910.1000): OSHA 1989b b. Other: EPA OERR Reportable quantity 10 pounds EPA 1985 (40 CFR 302.4); EPA 1989 EPA OSW Designation of hazardous substances Yes EPA 1985 (40 CFR 302.4): EPA 1989 Listing as a hazardous waste; Yes EPA 1988a (40 CFR commercial chemical products, 261.33), EPA 19880 manufacturing chemical intermediates, or off-specification commercial chemical products Listing as a hazardous constituent Yes EPA 1988a (40 CFR 261, Appendix VIII); EPA 1988b Land disposal restriction; treatment standard EPA 1991a (40 CFR Waste water 0.5 mg/L 268); EPA 1991b Non-waste water 35 mg/kg EPA OTS Toxic Release Reporting; Community Yes EPA 1988d (40 CFR Right to Know 372); EPA 1988e TSCA Comprehensive assessment Yes EPA 1986: EPA 1988f information rule; reporting and (40 CFR 704) record keeping requirement Guidelines: a. Air ACGIH TLV TWA (skin designation) 0.02 ppm (0.22 mg/m3) ACGIH 1991 - Carcinogenic classification Group A2 NIOSH REL TWA 3 pg/m3 NIOSH 1992 b. Other: EPA Carcinogenic classification Group B2¢.d EPA 1990 Slope factor (q1*) (mg/kg/day)” 13x10" 1d NTP Carcinogenic classification Yes® NTP 1990 MBOCA 95 7. REGULATIONS AND ADVISORIES TABLE 7-1. Regulations and Guidelines Applicable to 4,4’-Methylenebis(2-chloroaniline) (MBOCA) (Continued) Agency Description Information References STATE Regulations and Guidelines: a. Air: Acceptable Ambient Air Concentrations Arizona (1 hour) 6.40 g/m NATICH 1992 Arizona (24 hours) 1.70 pg/m3 NATICH 1992 California (8 hours) 0.01 ppm GISO 1985 Connnecticut (8 hours) 1.50x1072 pg/m3 NATICH 1992 Florida-Pinellas (Annual) 2.10x1072 pg/m3 NATICH 1992 Kansas (1 year) 2.12¢1072 pg/m3 NATICH 1992 Maryiand 0.00 Nevada (8 hours) 0.00 North Dakota 0.00 Oklahoma (24 hours) 2.20 pg/m3 NATICH 1992 Pennsylvania-Phila- (1 year) 5.50x10" 1 pg/m3 NATICH 1992 delphia Rhode Island (24 hours) 1.00 pg/m3 NATICH 1992 Texas (30 minutes) 2.20 pg/m3 NATICH 1992 Texas (24 hours) 220x107" p/m’ NATICH 1992 Virginia (24 hour) 2.20 pg/m NATICH 1992 Wisconsin Best available control technology 250 pounds/year WAC 1988 for hazardous air contaminants 3Group 2A: probably carcinogenic in humans BGroup A2: suspected human carcinogen CGroup B2: probable human carcinogen interim data, not verified by the CRAVE group €May reasonably be anticipated to be a carcinogen ACGIH = American Conference of Governmental Industrial Hygienists: CRAVE = Carcinogenic Risk Assessment Validation Effort: EPA = Environmental Protection Agency; IARC = International Agency for Research on Cancer; NIOSH = National Institute for Occupational Safety and Health; NTP = National Toxicology Program;OERR = Office of Emergency and Remedial Response: OSHA = Occupational Safety and Health Administration; OSW = Office of Solid Wastes; OTS = Office of Toxic Substances: PEL = Permissable Exposure Limit; q1* = an upper-bound estimate of the low-dose slope of the dose-response curve as determined by the multistage procedure; REL = Recommended Exposure Limit; TLV = Time Weighted Average; TSCA = Toxic Substance; Control Act; TWA = Time Weighted Average MBOCA 97 8. 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Efficacy of urinary monitoring for 4,4’-methylenebis-(2- chloroaniline). J Occup Med 28(8):637-642. *Ward E, Smith AB, Halperin W. 1987. 4,4’-Methylenebis(2-chloroaniline): An unregulated carcinogen. Am J Ind Med 12(5):537-549. Ward E, Halperin W, Thun M, et al. 1988. Bladder tumors in two young males occupationally exposed to MBOCA. Am J Ind Med 14(3):267-272. “Ward E, Halperin W, Thun M, et al. 1990. Screening workers exposed to 4,4-methylenebis-(2- chloroaniline) for bladder cancer by cystoscopy. In: International Conference on Bladder Cancer Screening in High-Risk Groups, September 13-14, 1989. J Occup Med 32(9):865-868. *Ward E. 1993. Response to "Case study "Carcinogenesis: The MBOCA TLV example". Am Ind Hyg Assoc J 54(8):461-463. *Weeks RW Jr, Dean BJ. 1977. Permeation of methanolic aromatic amine solutions through commercially available glove materials. J Am Ind Hyg Assoc 38(12):721-725. Weigel WW, Savage RE Jr. 1990. Induction of ornithine decarboxylase activity of 4,4’-methylene- bis(2-chloroaniline) (MOCA) in the rat. In: Society of Toxicology 29th Annual Meeting, Miami MBOCA 116 8. REFERENCES Beach, FL, February 12-16, 1990. Washington, D.C.: Society of Toxicology, 760. Williams DE. 1979. Progress report on the analysis and control of exposure to 4,4'methylene bis (2 chloroaniline. Lansing, MI: Michigan Department of Public Health, Division of Occupational Health, Division of Environmental Epidemiology. *Williams GM, Laspia MF, Dunkel VC. 1982. Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens. Mutat Res 97(5):359-370. Wilson JD. 1978. Review of in vitro systems with potential for use in teratogenicity screening. J Environ Pathol Toxicol 2(1):149. Wu K, Leslie CL, Stacy NH. 1989. Effects of mutagenic and non-mutagenic aniline derivatives on rat liver drug-metabolizing enzymes. Xenobiotica 19(11):1275-1283. *Yasuda SK. 1975. Determination of 3,3’-dichloro-4,4’-diaminodiphenylmethane in air. J Chromatogr 104(2):283-290. *Yoneyama K, Matsumura F. 1984. Microbial metabolism of 4,4’-methylenebis(2-chloroaniline). Arch Environ Contam Toxicol 13(4):501-507. *Yoshikura H, Matsushima T. 1981. MLV test (integration enhancement test) of 42 coded compounds in mouse kidney cells. In: deSerres FJ, Ashby J, eds. Evaluation of short-term tests for carcinogens: Report of the International Collaborative Program. Prog Mutat Res 1:647-650. Yun C, Shimada T, Guengerich FP, et al. 1991. Purification and characterization of human liver microsomal cytochrome P-450 2A6. Mol Pharmacol 40(5):679-685. Yun CH, Shimada T, Guengerich FP. 1992. Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4-methylene-bis(2-chloroaniline). Carcinogenesis 13:217-222. Zimmermann FK, Scheel I. 1981. Induction of mitotic gene conversion in strain D7 of saccharomyces cerevisiae by 42 coded chemicals. In: deSerres FJ, Ashby J, eds. Evaluation of short- term tests for carcinogens: Report of the International Collaborative Program. Prog Mutat Res 1:481-490. MBOCA v7 9. GLOSSARY Acute Exposure — Exposure to a chemical for a duration of 14 days or less, as specified in the Toxicological Profiles. Adsorption Coefficient (K,.) — 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 absorbed per gram of soil or sediment. Bioconcentration Factor (BCF) — The quotient of the concentration of a chemical in aquatic organisms at a specific time or during a discrete time period of exposure divided by the concentration in the surrounding water at the same time or during the same period. Cancer Effect Level (CEL) — The lowest dose of chemical in a study, or group of studies, that produces significant increases in the incidence of cancer (or tumors) between the exposed population and its appropriate control. Carcinogen — A chemical capable of inducing cancer. Ceiling Value — A concentration of a substance that should not be exceeded, even instantaneously. Chronic Exposure — Exposure to a chemical for 365 days or more, as specified in the Toxicological Profiles. Developmental Toxicity — The occurrence of adverse effects on the developing organism that may result from exposure to a chemical prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation. Adverse developmental effects may be detected at any point in the life span of the organism. Embryotoxicity and Fetotoxicity — Any toxic effect on the conceptus as a result of prenatal exposure to a chemical; the distinguishing feature between the two terms is the stage of development during which the insult occurred. The terms, as used here, include malformations and variations, altered growth, and in utero death. EPA Health Advisory — An estimate of acceptable drinking water levels for a chemical substance based on health effects information. A health advisory is not a legally enforceable federal standard, but serves as technical guidance to assist federal, state, and local officials. Immediately Dangerous to Life or Health (IDLH) — The maximum environmental concentration of a contaminant from which one could escape within 30 min without any escape-impairing symptoms or irreversible health effects. Intermediate Exposure — Exposure to a chemical for a duration of 15-364 days, as specified in the Toxicological Profiles. MBOCA 118 9. GLOSSARY Immunologic Toxicity — The occurrence of adverse effects on the immune system that may result from exposure to environmental agents such as chemicals. In Vitro — Isolated from the living organism and artificially maintained, as in a test tube. In Vivo — Occurring within the living organism. Lethal Concentration; (LCpro) — The lowest concentration of a chemical in air which has been reported to have caused death in humans or animals. Lethal Concentrations (LCs) — 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 (LD; ,) — The lowest dose of a chemical introduced by a route other than inhalation that is expected to have caused death in humans or animals. Lethal Doses) (LDgy) — The dose of a chemical which has been calculated to cause death in 50% of a defined experimental animal population. Lethal Time sq, (LT5)) — A calculated period of time within which a specific concentration of a chemical is expected to cause death in 50% of a defined experimental animal population. Lowest-Observed-Adverse-Effect Level (LOAEL) — The lowest dose of chemical in a study, or group of studies, that produces statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. Malformations — Permanent structural changes that may adversely affect survival, development, or function. Minimal Risk Level — An estimate of daily human exposure to a dose of a chemical that is likely to be without an appreciable risk of adverse noncancerous effects over a specified duration of exposure. Mutagen — A substance that causes mutations. A mutation is a change in the genetic material in a body cell. Mutations can lead to birth defects, miscarriages, or cancer. Neurotoxicity — The occurrence of adverse effects on the nervous system following exposure to chemical. No-Observed-Adverse-Effect Level (NOAEL) — The dose of chemical at which there were no statistically or biologically significant increases in frequency or severity of adverse effects seen between the exposed population and its appropriate control. Effects may be produced at this dose, but they are not considered to be adverse. Octanol-Water Partition Coefficient (K,,) — The equilibrium ratio of the concentrations of a chemical in n-octanol and water, in dilute solution. Permissible Exposure Limit (PEL) — An allowable exposure level in workplace air averaged over MBOCA 119 9. GLOSSARY an 8-hour shift. q;* — The upper-bound estimate of the low-dose slope of the dose-response curve as determined by the multistage procedure. The q;* can be used to calculate an estimate of carcinogenic potency, the incremental excess cancer risk per unit of exposure (usually pg/L for water, mg/kg/day for food, and pg/m’ for air). Reference Dose (RfD) — An estimate (with uncertainty spanning perhaps an order of magnitude) of the daily exposure of the human population to a potential hazard that is likely to be without risk of deleterious effects during a lifetime. The RfD is operationally derived from the NOAEL (from animal and human studies) by a consistent application of uncertainty factors that reflect various types of data used to estimate RfDs and an additional modifying factor, which is based on a professional judgment of the entire database on the chemical. The RfDs are not applicable to nonthreshold effects such as cancer. Reportable Quantity (RQ) — The quantity of a hazardous substance that is considered reportable under CERCLA. Reportable quantities are (1) 1 pound or greater or (2) for selected substances, an amount established by regulation either under CERCLA or under Sect. 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. Short-Term Exposure Limit (STEL) — The maximum concentration to which workers can be exposed for up to 15 min continually. No more than four excursions are allowed per day, and there must be at least 60 min between exposure periods. The daily TLV-TWA may not be exceeded. Target Organ Toxicity — This term covers a broad range of adverse effects on target organs or physiological systems (e.g., renal, cardiovascular) extending from those arising through a single limited exposure to those assumed over a lifetime of exposure to a chemical. Teratogen — A chemical that causes structural defects that affect the development of an organism. Threshold Limit Value (TLV) — A concentration of a substance to which most workers can be exposed without adverse effect. The TLV may be expressed as a TWA, as a STEL, or as a CL. Time-Weighted Average (TWA) — An allowable exposure concentration averaged over a normal 8- hour workday or 40-hour workweek. Toxic Dose (TDs) — A calculated dose of a chemical, introduced by a route other than inhalation, which is expected to cause a specific toxic effect in 50% of a defined experimental animal population. Uncertainty Factor (UF) — A factor used in operationally deriving the RfD from experimental data. UFs are intended to account for (1) the variation in sensitivity among the members of the human population, (2) the uncertainty in extrapolating animal data to the case of human, (3) the uncertainty in extrapolating from data obtained in a study that is of less than lifetime exposure, and (4) the MBOCA 120 9. GLOSSARY uncertainty in using LOAEL data rather than NOAEL data. Usually each of these factors is set equal to 10. MBOCA A-1 APPENDIX A USER’S GUIDE Chapter 1 Public Health Statement This chapter of the profile is a health effects summary written in nontechnical language. Its intended audience is the general public especially people living in the vicinity of a hazardous waste site or substance 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 substance. The major headings in the Public Health Statement are useful to find specific topics of concem. 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 (2-1 and 2-2) are used to summarize health effects by duration of exposure and end point and to illustrate graphically levels of exposure associated with those effects. All entries in these tables and figures represent studies that provide reliable, quantitative estimates of No-Observed-Adverse-Effect Levels (NOAELSs), Lowest-Observed-Adverse-Effect Levels (LOAELS) for Less Serious and Serious health effects, or Cancer Effect Levels (CELs). In addition, these tables and figures illustrate 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. The LSE tables and figures can be used 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. The legends presented below demonstrate the application of these tables and figures. A representative example of LSE Table 2-1 and Figure 2-1 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 (I). 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 exist, three LSE tables and two LSE figures are presented in the document. The three LSE tables present data on the three principal routes of exposure, i.e., inhalation, oral, and dermal (LSE Table 2-1, 2-2, and 2-3, respectively). LSE figures are limited to the inhalation (LSE Figure 2-1) and oral (LSE Figure 2-2) routes. (2). Exposure Duration Three exposure periods: acute (14 days or less); intermediate (15 to 364 days); and chronic (365 days or more) are presented within each route of exposure. In this example, an inhalation study of intermediate duration exposure is reported. MBOCA 3). 4. (5). (6). . (8). 9). (10). (11). (12). A-2 APPENDIX A 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. 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 define a NOAEL and a Less Serious LOAEL (also see the two "18r" data points in Figure 2-1). Species The test species, whether animal or human, are identified in this column. 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 LOAELSs from different studies. In this case (key number 18), rats were exposed to [substance x] via inhalation for 13 weeks, 5 days per week, for 6 hours per day. System This column further defines the systemic effects. These systems include: respiratory, cardiovascular, gastrointestinal, hematological, musculoskeletal, hepatic, renal, and dermal/ocular. "Other" refers to any systemic effect (e.g., a decrease in body weight) not covered in these systems. In the example of key number 18, one systemic effect (respiratory) was investigated in this study. 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"). LOAEL A Lowest-Observed-Adverse-Effect Level (LOAEL) is the lowest exposure level used in the study that caused a harmful health effect. LOAELSs have been classified into "Less Serious" and "Serious" effects. These distinctions help readers identify the levels of exposure at which adverse health effects first appear and the gradation of effects with increasing dose. A brief description of the specific end point used to quantify the adverse effect accompanies the LOAEL. The "Less Serious" respiratory effect reported in key number 18 (hyperplasia) occurred at a LOAEL of 10 ppm. Reference The complete reference citation is given in Chapter 8 of the profile. CEL A Cancer Effect Level (CEL) is the lowest exposure level associated with the onset of carcinogenesis in experimental or epidemiological studies. CELs are always considered serious effects. The LSE tables and figures do not contain NOAELSs for cancer, but the text may report doses which did not cause a measurable increase in cancer. Footnotes Explanations of abbreviations or reference notes for data in the LSE tables are found in the footmotes. Footnote "b" indicates the NOAEL of 3 ppm in key number 18 was used to derive an MRL of 0.005 ppm. LEGEND See LSE 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 levels for particular exposure duration. » TABLE 2-1. Levels of Significant Exposure to [Chemical x] - Inhalation Exposure LOAEL (effect) Key to frequency / NOAEL Less serious Serious figure* Species duration System (ppm) (ppm) (ppm) Reference INTERMEDIATE EXPOSURE Systemic 18 Rat 13 wk Resp 3° 10 (hyperplasia) Nitschke et al. 5d/wk 1981 6hr/d CHRONIC EXPOSURE Cancer 38 Rat 18 mo 20 (CEL, multiple Wong et al. 1982 Sd/wk organs) Thr/d 39 Rat 89-104 wk 10 (CEL, lung tumors, NTP 1982 5d/wk nasal tumors) 6hr/d 40 Mouse 79-103 wk 10 (CEL, lung tumors, NTP 1982 5d/wk hemangiosarcomas) 6hr/d * The number corresponds to entries in Figure 2-1. ® Used to derive an intermediate inhalation Minimal Risk Level (MRL) of 5 x 10° ppm; dose adjusted for intermittent exposure and divided by an uncertainty factor of 100 (10 for extrapolation from animal to humans, 10 for human variability). CEL = cancer effect level; d = day(s); hr = hour(s); LOAEL = lowest-observed-adverse-effect level; mo = month(s); NOAEL = no-observed-adverse-effect level; Resp = respiratory; wk = week(s) V XION3ddVY VvOO8W ev FIGURE 2-1. Levels of Significant Exposure to [Chemical X] - Inhalation INTERMEDIATE (15-364 Days) Systemic CHRONIC (> 365 Days) Systemic & & s & & . Bs -. 8 z 5 4 & s F&F FH oe 5 $f F of & & F & FF & F&F FSF (ppm) 10,000 = 1,000 [~ 00 | @1or @17r 25m 1 ® D2eg PD o O2ir M24g P21r O22h ®x Osom Bos Basr Bose Br “ 19r 38r wo k O25m (Par @33m Os2r (P3sm Das Oszar Osar Osa O34 Parr @aom Pasr Q1erOitor L 1 ! 1 1 L | °! 1 104 1 0.01 | : 10-6 - Estimated Upper- <=— 1 Bound Human 0.001 | ! Cancer Risk ~ 10-¢ 1 Levels 0.0001 |= Key 107 r Rat @ LOAEL for serious effects (animals) ’ 0.00001 = m Mouse ( LOAEL for less serious effects (animals) : Minimal risk level for h Rabbit oO NOAEL (animals) . effects other than cancer g Guinea pig @ CEL - Cancer Effect Level (animals) ~r h Monkey The number next to each point corresponds to entries in Table 2-1. * Doses represent the lowest dose tested per study that produced a tumorigenic response and do notimply the existence of a threshold for the cancer end point. 139017-1 V XIAN3ddV vOOan vv MBOCA A-5 APPENDIX A (13). Exposure Duration 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 exist. The same health effects appear in the LSE table. (15). Levels of Exposure Exposure levels for each health effect in the LSE tables are graphically displayed in the LSE figures. Exposure levels are reported on the log scale "y" axis. Inhalation exposure is reported in mg/m’ or ppm and oral exposure is reported in mg/kg/day. (16). NOAEL In this example, 18r NOAEL is the critical end point for which an intermediate inhalation exposure MRL is based. As you can see from the LSE figure key, the open-circle symbol indicates 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 one of three studies for which Cancer Effect Levels (CELs) were derived. The diamond symbol refers to a CEL for the test species (rat). 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 EPA’s Human Health Assessment Group’s upper-bound estimates of the slope of the cancer dose response curve at low dose levels (q,"). (19). Key to LSE Figure The Key explains the abbreviations and symbols used in the figure. Chapter 2 (Section 2.4) Relevance to Public Health The Relevance to Public Health section provides a health effects summary based on evaluations of existing toxicological, epidemiological, 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 discusses health effects by end point. Human data are presented first, then animal data. Both are organized by route of exposure (inhalation, oral, and dermal) and by duration (acute, intermediate, and 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. MBOCA A-6 APPENDIX A 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. MRLs for noncancer end points if derived, and the end points from which they were derived are indicated and discussed in the appropriate section(s). Limitations to existing scientific literature that prevent a satisfactory evaluation of the relevance to public health are identified in the Identification of Data Needs section. Interpretation of Minimal Risk Levels Where sufficient toxicologic information was available, MRLs were derived. MRLs are specific for route (inhalation or oral) and duration (acute, intermediate, or chronic) of exposure. Ideally, MRLs can be derived from all six exposure scenarios (e.g., Inhalation - acute, -intermediate, -chronic; Oral - acute, -intermediate, - 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 substance emission, given the concentration of a contaminant in air or the estimated daily dose received via food or water. MRLs are based largely on toxicological studies in animals and on reports of human occupational exposure. MRL users should be familiar with the toxicological information on which the number is based. Section 2.4, "Relevance to Public Health," contains basic information known about the substance. Other sections such as 2.6, "Interactions with Other Chemicals" and 2.7, "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 used by the Environmental Protection Agency (EPA) (Bames and Dourson 1988; EPA 1989a) to derive reference doses (RfDs) for lifetime exposure. To derive an MRL, ATSDR generally selects the end point which, in its best judgement, represents the most sensitive human health effect for a given exposure route and duration. ATSDR cannot make this judgement or derive an MRL unless information (quantitative or qualitative) is available for all poten- tial effects (e.g., systemic, neurological, and developmental). In order to compare NOAELs and LOAELSs for specific end points, all inhalation exposure levels are adjusted for 24hr exposures and all intermittent exposures for inhalation and oral routes of intermediate and chronic duration are adjusted for continuous exposure (i.e., 7 days/week). If the information and reliable quantitative data on the chosen end point are available, ATSDR derives an MRL using the most sensitive species (when infor- mation from multiple species is available) with the highest NOAEL that does not exceed any adverse effect levels. The NOAEL is the most suitable end point for deriving an MRL. When a NOAEL is not available, a Less Serious LOAEL can be used to derive an MRL, and an uncertainty factor of (1, 3, or 10) is employed. MRLs are not derived from Serious LOAELs. Additional uncertainty factors of (1, 3, or 10 ) are used for human variability to protect sensitive subpopulations (people who are most susceptible to the health effects caused by the substance) and (1, 3, or 10) are used for inter- species variability (extrapolation from animals to humans). In deriving an MRL, these individual uncertainty factors are multiplied together. Generally an uncertainty factor of 10 is used; however, the MRL workgroup reserves the right to use uncertainty factors of (1, 3, or 10) based on scientific judgement. The product is then divided into the adjusted 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. ACGIH ADME atm ATSDR BCF BSC C CDC CEL CERCLA CFR CLP cm CNS d DHEW DHHS DOL ECG EEG EPA EKG F F| FAO FEMA FIFRA fpm ft FR £ GC gen HPLC hr IDLH IARC ILO in Kd kg kkg K K oC ow B-1 APPENDIX B ACRONYMS, ABBREVIATIONS, AND SYMBOLS American Conference of Governmental Industrial Hygienists Absorption, Distribution, Metabolism, and Excretion atmosphere Agency for Toxic Substances and Disease Registry bioconcentration factor Board of Scientific Counselors Centigrade Centers for Disease Control Cancer Effect Level Comprehensive Environmental Response, Compensation, and Liability Act Code of Federal Regulations Contract Laboratory Program centimeter central nervous system day Department of Health, Education, and Welfare Department of Health and Human Services Department of Labor electrocardiogram electroencephalogram Environmental Protection Agency see ECG Fahrenheit first filial generation Food and Agricultural Organization of the United Nations Federal Emergency Management Agency Federal Insecticide, Fungicide, and Rodenticide Act feet per minute foot Federal Register gram gas chromatography generation high-performance liquid chromatography hour Immediately Dangerous to Life and Health International Agency for Research on Cancer International Labor Organization inch adsorption ratio kilogram metric ton organic carbon partition coefficient octanol-water partition coefficient L LC LC, LCs LD Lo LDs, LOAEL LSE m MBOCA mg min mL mm mmHg mmol mo mppcf MRL MS NIEHS NIOSH NIOSHTIC ng nm NHANES nmol NOAEL NOES NOHS NPL NRC NTIS NTP OSHA PEL pg pmol PHS PMR ppb ppm ppt REL RfD RTECS ysec SCE SIC B-2 APPENDIX B liter liquid chromatography lethal concentration, low lethal concentration, 50% kill lethal dose, low lethal dose, 50% kill lowest-observed-adverse-effect level Levels of Significant Exposure meter 4,4 -methylenebis(2-chloroaniline) milligram minute milliliter millimeter millimeters of mercury millimole month millions of particles per cubic foot Minimal Risk Level mass spectrometry National Institute of Environmental Health Sciences National Institute for Occupational Safety and Health NIOSH’s Computerized Information Retrieval System nanogram nanometer National Health and Nutrition Examination Survey nanomole no-observed-adverse-effect level National Occupational Exposure Survey National Occupational Hazard Survey National Priorities List National Research Council National Technical Information Service National Toxicology Program Occupational Safety and Health Administration permissible exposure limit picogram picomole Public Health Service proportionate mortality ratio parts per billion parts per million parts per trillion recommended exposure limit Reference Dose Registry of Toxic Effects of Chemical Substances second sister chromatid exchange Standard Industrial Classification SMR STEL STORET TLV TSCA TRI TWA U.S. UF yr WHO wk = PT®R QA A VV TT ug B-3 APPENDIX B standard mortality ratio short term exposure limit STORAGE and RETRIEVAL threshold limit value Toxic Substances Control Act Toxics Release Inventory time-weighted average United States uncertainty factor year World Health Organization week greater than greater than or equal to equal to less than less than or equal to percent alpha beta delta gamma micron microgram % U.S. GOVERNMENT PRINTING OFFICE: 1994 — 5 37 -9 7 3 wii C08LA&5091