Ethylene Dibromide (EDB) – A Guide for Decision Makers

ETHYLENE DIBROMIDE (EDB)

AGUIDEFOR DECISION MAKERS

Exposure, Heatrn Effects & Risk Assessment

Prepared by

ENvirolocic data

Portland, Maine

‘March 1984

‘About ENVIROLOGIC DATA, INC.

Envirologic Data performs health arc ery risk assessments related to toxic sursta affecting health. Located medical orientation that ‘management field. Human, animal, 2 are evaluated ty "ea7th cate ara” other toxic substames ic water, gir, at Se) addressed Dy the inmoweti we mettecs ogy Demet

ae a

ETHYLENE DIBROMIDE (EDB)

A GUIDE FOR DECISION MAKERS

Exposure, Health Effects And Risk Assessment

Frank H. Lawrence, M.D. &

Robert A. Michaels, Ph.D.

Envirologic Data PORTLAND, MAINE

Copyright 1984 by Envirologic Data, Inc.

All rights reserved. No part of this book may be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without permission in writing from Envirologic Data.

This document is based upon information obtained from numerous sources; research articles and references reviewed have been listed. Every reasonable effort has been made to provide reliable data and information; however, Envirologic Data cannot assume responsibility for the quality or validity of laboratory studies or other data reported in the literature or for the consequences of their use.

Published by: Envirologic Data

Printed in the United States of America by: Curry Copy Center, Portland, Maine

Envirologic Data P.O. Box 1438 Portland, ME 04104 207-773-3020

alae

Project Staff and Contributors

EDB Project Staff

Michael Kitch Freelance Writer

dane McCrodden, B.A.

Director, Nata Collection and Analysis

Mary Sauer, M.S.P.H. Research Analyst

Pam Boardman, M.P.H. Research Analyst

Envirologic Data Publishing Staff

Mark A. Maloney Director of Marketing and Sales

Lisa C. Wolf

Mary Lee Fowler

Other Contributors

Laurent J. Beauregard, Jr., Ph.D. Director of Genetics Program Eastern Maine Medical Center

James E, Haddow, M.D.

Associate Medical Director Foundation for Blood Research

-ifi-

Sandra Bisset, M.P.H. Research Analyst

Ellen Ebert, B.A. Research Analyst

David W. Crawford, M.P.H., P.E. Director, Program Development

Peter W. Rand, M.D. Director of Research Maine Medical Center

PREFACE

Quest for knowledge via scientific study may be analogous to that sought via geographic exploration. Unknowing and sometimes fearful, individuals and groups have repeatedly set out to explore, motivated by the desire to see just over the horizon, just around the bend. Incessantly, man has sought vantage points, hill and mountain tops from which topography is laid out in plain view, and sight of the next valley provides evidence where previously there was only conjecture.

Scientific study is entrenched in methodology; its underpinnings are likewise its objectives: discovery, proof and comprehension of the most subtle mechanisms. Hypotheses roll freely from the minds of astute men and women; experimental designs are well chosen and their methods now reflect not only age-old essences of analytical process but also new electronic and computer arrays which often defy description. Armed with thousands of data points, the researcher marches, with the craft of statistical analysis, to the arena of discussion where he climbs the final hill to written conclusion--that pinnacie from which he better sees the problem that he set out to study; that vantage point from which he learns of the next horizon.

Our world and its scientists are increasingly introspective. We seek to understand the natural facets of our planet as well as the complex instruments and chemicals that we add to it. ‘he need to learn has produced an explosion of fact, opinion, and conclusion. This morass of information engenders not only near instantaneous accountability, but an unusual and new burden of responsibility for the future. This burden and challenge rests squarely on the shoulders of our society's decision makers as they are charged with the management of risk. The increasingly defined discipline of human health risk assessment must follow the tradition of those who have carried out the basic

and applied research. Skillful analysts are required to assemble alli the information, draw relationships and use the results wisely to predict trends and hazards. If those who assess risk are to have vantage comprehension

rather than factual disarray, each bit of information, like a piece of a puzzle, requires examination, evaluation, and finally, insertion into the right place.

The database produced by the study of EDB is representative of the explosion of information surrounding pesticides and other toxic materials. Although many recognize the need to assemble the broadly dispersed conclusions of authors, it seems that few do this with purity of purpose or scientific method.

Envirologic Data's purpose is to bring together valid studies, extract then meld their essence to formulate best statements; then, importantly, the task is to communicate the information with clarity. Envirologic Data seeks to provide facts for the toxicologist as well as guidance for the individual or group which must determine use patterns and regulation.

EDB, A Guide for Decision Makers, is dedicated to those research scientists who have built the database as well as to those persons who have the tough task of exploring it.

F. Lawrence, M.D. March, 1984

-iy-

EXECUTIVE SUMMARY

EDB (1,2-dibromoethane, or ethylene dibromide) has become the object of public scrutiny because of its ubiquity in the human environment and its long-term toxicity. EDB is primarily used as an additive in leaded gasoline, resulting in mean ambient air levels of 0.026 parts per billion in large U.S. cities; and as a fumigant pesticide in citrus and grain products, resulting in residues in consumer products and some wells.

Envirologic Data has examined nearly 500 literature citations, including (predominantly) primary research articles as well as some secondary review articles and risk assessments. The focus of other EDB risk assessments examined by Envirologic Data was typically narrower than the focus adopted in the present document. In other documents, exposures were estimated for only one or two, but not all three of the major EDB sources. In some assessments, the focus was on occupational rather than general population exposure. In assessments of general population risk, a frequently encountered practice was to ignore actual exposure, and instead to calculate risks associated with a variety of regulatory options which would limit residues in grain and/or citrus products.

Envirologic Data has estimated annual body burdens of EDB in adult males and females, children, and infants in the general population, from all major sources: values were, respectively, 2175.3, 1957.4, 1326.4, and 317.7 to 377.2 micrograms per year. Urban air accounted for 74 to 88 percent, grain products for 11 to 25 percent, and citrus products for 0.4 to 0.8 percent of these total body burdens.

Absorption into the body from inhaled and ingested EDB were also examined. Ingested EDB is apparently absorbed with an efficiency of close to 80 percent. Data on inhalation efficiency were unavailable; consequently it was necessary to estimate inhalation absorption based upon the primary governing variable--solubility in the aqueous fluid in tne lung alveoli. Based upon comparison with the water solubility and adsorption efficiency of chloroform, Envirologic Data estimated an 30-percent absorption efficiency for EDB. The similarity of absorption efficiencies between inhaled and ingested sources of EDB suggests that air levels in cities, where most U.S. residents live, may constitute the primary source of EDB exposure. Regulatory agencies at the state and federal levels have focused on controlling the dietary route of exposure.

Once in the body, EDB is selectively distributed to the liver and <idneys, regardless of route of initial exposure. Metabolism is by two routes, each producing the same mercapturic acid derivative, which is =liminated primarily in the urine, with a half-life of less than one cay. During its passage through the body, metabolic activation appears to enhance the toxic effects of EDB.

Health effects in animals and in humans have been observed by Numerous investigators, and their results are presented and evaluated in this document. Envirologic Data has rated EDB on a scale of 0 to 3, as indicated, in each of the following categories:

--Acute toxicity: 2 --Reproductive effects: 3 --Teratogenicity: 1 --Mutagenicity: 2 --Carcinogenicity: 3

In the rating scale, 0 corresponds to the absence of the health effect, 1 to insufficient evidence, and 2 and 3 to increasing evidence or potency of the substance being rated.

Risks of EDB-related cancer mortality have been estimated by several investigators. Such estimates of risk are variable based upon exposure assumptions, models used to extrapolate from the animal to the human case, and models used to extrapolate from high (experimental) doses to low (U.S. population) doses. Exposure estimates varied 209-fold, whereas risk estimates varied by more than five orders of magnitude. No firm quantitative conclusion can be drawn regarding risks of cancer mortality from EDB, given the variability in the available data and the inadequacy of the state of the art in toxicology to discriminate among low dose extrapolation models, which account for much of the variability among risk estimates.

Although absolute cancer mortality risks from each major EDB exposure source are difficult to estimate, relative risks can be inferred from exposure, absorption, and health effects data. The exposure data indicate the order of relative importance of the three major EDB sources; air, grain, and citrus, respectively. The absorption data indicate that inhaled and ingested EDB are assimilated into the body with about equal efficiency. Finally, the health effects data indicate that carcinogenic risk is about the same for inhaled and ingested EDB, based upon the observed appearance of diverse malignancies remote from points of EDB exposure in test animals by both routes.

Thus, in the absence of firm quantitative conclusions regarding cancer mortality risks of EDB exposure, it 7s nevertheless possible to draw a qualitative conclusion. That is, the distribution of risks to urban U.S. populations appears to correspond to the distribution of EDB sources, with inhalation apparently predominating over ingestion from grain and citrus. It is important to emphasize that, whatever the

Magnitudes of such risks may be, Envirologic Data makes no judgement regarding their seriousness.

ea

TABLE OF CONTENTS

Section Page PREFACE iv EXECUTIVE SUMMARY v TABLE OF CONTENTS vii LIST OF FIGURES ix LIST OF TABLES x LIST OF ABBREVIATIONS x INTRODUCTION 1 Purpose and Scope 1 History of EDB 3 METHODS AND NATURE OF RISK ASSESSMENT 7 GENERAL INFORMATION AND PROPERTIES 10 Summary 10 Categorization 10 Synonyms 10 Manufacturers and Trade Names 10 Synthesis and Production Volume 13 Analytical Methods of Detection 15 Principal Uses and Use Breakdown 16 Formulations 19 PrP ication Rates and Dosage 20 Chemical and Physical Properties 21 SOURCES OF EXPOSURE 23 Summary 23 Introduction 23 Natural Background Levels of EDB 24 Man-Generated Sources of Exposure 24 Gecupa trons) ESpOSUTS 24 Non-Occupational Exposure 24 EDB Intake 30 Comparison with Other EDB Risk Assessments 33 ENVIRONMENTAL FATE, PERSISTENCE, AND EFFECTS 35 Summary 35 Air a0 Aquatic Environments 35 Terrestrial Environments 36 PHARMACOKINETICS 7 Summary 37 Absorption 37 Distribution and Retention 38 Metabolism 40 Excretion 4] HEALTH EFFECTS -- NONHUMAN 46 Summary 46 Acute Toxicity 47 Subchronic and Chronic Toxicity 52 Teratogenicity and Reproductive Effects 56 Pusogete ly 61 Carcinogenicity 76

-vii-

Section Page HEALTH EFFECTS -- HUMAN 89 cuvaaty 89 Envirologic Data Ratings 89 Acute Toxicity 90 Subchronic and Chronic Toxicity 90 Teratogenicity and Reproductive Effects 91 Puvagenictty 91 Carcinogenicity 93 Risk Assessment 93 MECHANISMS OF ACTION 96 Summary 96 Mechanisms of Acute and Subchronic Toxicity 96 Synergism 97 Mechanisms of Teratogenicity and Reproductive Effects 97 Mechanisms of Mutagenicity 98 Mechanisms of Carcinogenicity 98 MEDICAL/OCCUPATIONAL SURVEILLANCE 105 Summary 105 Clinical Presentation 105 Poison Management 105 Medical Surveillance 106 FEDERAL AND STATE GOVERNMENT REGULATORY ACTIONS 107 Summary 107 Federal Regulatory Actions 107 State Regulatory Actions 110 LITERATURE CITED 112 ABOUT THE PRINCIPAL AUTHORS 122

-viii-

LIST OF FIGURES

Figure No. Title Page ls Probable Metabolic Pathways of EDB 42 2. Proposed Mechanism of EDB Binding to 99

Polynucieotides

LIST OF TABLES

Tabie No. Title Page le List of EDB Trade Names and Manufacturers/ a] Distributors ee Estimated EDB Production in the U.S. 4 3. Production Volume of Four Major Manufacturers 14 4, Pesticide Use for EDB and Amount Used 7 §. Chemical and Physical Properties 2l 6. Examples of Industries Presenting Potential 25 Risks of Becoming Occupationally Exposed to EDB a. Examples of EDB Concentrations in Ambient Air in 26 Occupational Settings 8. Ambient Air EDB Concentrations in Urban 28 Environments S, Respiratory Yolumes for Reference Individual 31 (Liters of Air Breathed) 10. Inhalation of EDB (ug) from Urban Air Assumin: 31 pean fankenthaslon oF 0.026 ppb of EDB {ug/kgd ll. Estimated Annual EDB Body Burdens from Major U.S. 33 Sources 12s EDB Risk Assessments: The Exposure Component 34 13. Distribution of EDB in Male Guinea Pigs 39 14. Distribution of EDB in Mice 40 18, Summary of Data on Pharmacokinetics of EDB 43 16. Acute Toxicity of EDB in Animals 48 1%, Acute Effects of EDB 50 18. Subchronic and Chronic Toxicity of EDB 54 19. Teratogenicity of EOB 57 20. Reproductive Effects of EDB 59 Zi, Summary of EDB Mutagenicity Studies 67 22. EDB Mutagenicity by Category of Organism 15. 23. Summary of EDB Carcinogenicity Studies 80 24. EDB Carcinogenicity by Study 88 25. EDB Risk Assessments--The Risk Component 95 26. Summary of Data on Mechanisms of EDB Action 100 27. Federal EDB Regulatory Actions 108 28. State EDB Regulatory Actions Ww

LIST OF ABBREVIATIONS

Abbreviation Full Word or Phrase

ACGIH American Conference of Governmental Industrial Hygienists

BTU British thermal units

ba Centigrade degrees

cal calorie(s)

cm centimeters

d day(s)

DBCP dibromoch] oropropane

opc diethyldithiocarbamate

DNA deoxyribonucleic acid

EDB 1,2-dibromoethane; ethylene dibromide

EPA Environmental Protection Agency

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

ft foot, feet

g gram(s)

hr hour(s)

kg kilogram(s)

1 liter(s)

1b(s} pound(s)

LD Lethal Dose, LDsg, dose which is lethal to 50 percent of the animals tested

m meter(s)

M molar

ug microgram(s)

ul micromole(s)

mg milligram(s)

nm) milliliter(s)

mm millimolar

mo(s) month(s)

NIOSH National Institute for Occupational Health and Safety

OSHA Occupational Health and Safety Administration

ppb parts per billion

ppm parts per million

ppt parts per trillion

RCRA Resource Conservation and Recovery Act

RNA ribonucleic acid

wk(s) week(s)

yr(s) year(s)

INTRODUCTION

Purpose and Scope

The purpose of this document is to provide a broad and thorough description of information for those involved in questioning fact and making decisions about the potential human health risks of EDB.

The document begins by providing background information, including a brief history of EDB's use and regulation and a discussion of the nature of risk and risk assessment procedures. It then describes Envirologic Data's methods of literature search, acquisition and analysis, including hazard identification rating methodology which has been used extensively in the public decision-making arena for pesticides in the State of Maine. Following this background section is a general information and properties section, which identifies EDB specifically based upon its chemical and physical properties, listing of manufacturers, synonyms and trade names; also included are formulation, application rates, principal uses, and methods of laboratory analysis.

Next is a full discussion of potential human exposure from such sources as diet, air and water. Highlighted in this section is the fact that elimination of dietary sources of EDB will not eliminate human exposure to EDB. The guide then continues with examination of the environmental fate of EDB and discusses physical and chemical changes which EDB may undergo in various media, including air, water, and soil. The persistence of EDB is examined in this section; persistence is an important determinant of environmental dynamics and exposure potential. Effects on species in the environment other than humans and laboratory

test animals are included here. In the section titled "Pharmacokinetics," EDB is examined from point of entry to point of exit in mammalian species. Processes of absorption, distribution,

concentration in particular tissues, metabolism, and excretion are dealt with.

Following this, the health effects in non-human species are considered, extracted from the extensive database which exists for EDB effects in laboratory animal studies. The acute toxicity, chronic toxicity, and potential for inducing cancer, birth defects and mutations in animal systems are described. Since there are few adequate studies of EDB effects in human beings, the insights provided in this section are of particular significance. Envirologic Data considers the animal health-effect database to be of special significance in the area of predicting human health effect.

In the portion of the study entitled "Health Effects--Human," effects of EDB on humans are considered. The section opens with a summary of Envirologic Data's hazard identification system as applied to EDB. A 0-3 rating scale, which indicates without strict attention to probability of exposure or dose, the inherent EDB risks in the areas of acute toxicity, reproductive effects, carcinogenicity, mutagenicity, and teratogenicity. A rating of O indicates a low or no hazard; a 3 indicates, based on

certain predictive assumptions, a strong risk that EDB will induce health disorders, and ratings of 1 and 2 are intermediate

in suggesting hazard. The rating system, its basis and components are discussed in the section of this document dealing with risk and risk assessment. Its value has been demonstrated in the public hearing and regulatory arena, and the value becomes most obvious when pesticides are rated and compared for possible human health effects. Envirologic Data has not yet done a comparative analysis of EDB substitutes.

EDB has been extensively studied to understand its biological actions. A summary of these studies appears in the section entitled "Mechanisms of Toxicity."

As the animal and human health effects of a toxic substance are discovered and when there is human exposure to that substance, one logically asks whether or not disease becomes discernible in exposed populations. Despite long use and with resultant human exposure to EDB, no definitive human epidemiologic studies have been performed to provide answers to all of the questions which scientists, government and individuals have about its health effects. In the section on medical and occupational surveillance, there is description of the symptoms and signs, which an acutely or chronically poisoned patient might present and description of appropriate clinical management of the patient. As pointed out in this section, there is no single "test" to provide an answer to such questions as “How much EDB have I been exposed to,” "What is the effect to date," and "What future health risks might I incur." However, brief attention is given in this section to diagnostic tests which may be appropriate in cases of EDB exposure.

In the final section of this document, the major federal and state regulatory actions relative to EDB are presented. Here, the results are reported of a fifty state survey conducted in February and March of 1984, including an outline of the actions of numerous state authorities who have moved to further limit exposure to EDB. Since, at the printing of this document, many state regulatory procedures were still in progress, it is anticipated that there will be significant modifications in the table.

Finally, in the bibliography, there is a list of the two hundred articles used in production of this document.

EDB--A Guide for Decision Makers is by design a document of limited length and scope; as such, the work of numerous authors is summarized. We invite comment and correction from readers.

History of EDB

Belonging to the earliest generation of commercial synthetic organic chemicals, EDB was first manufactured in the 1920s, at least 20 years before regulators of industrial and agricultural chemicals began to weigh risks to public health and natural environments. Not until the 1970s did agencies such as OSHA* and EPA** examine the human health effects of EOB. In 1980, the EPA, convinced of the dangers of ED8, proposed curbing its use. However, at the same time, agricultural uses of EDB began to grow and spread, winning it a prominent place in the production and distribution of foodstuffs at home and abroad. Although expanded use revealed the risks of E0B, it also spawned economic interests favoring EDB use. Regulators and users bargained against the background of federal efforts to relieve undue regulatory burdens until, during 1983,

steps taken by separate states prompted the EPA to restrict EDB early in 1984.

Since 1923, the bulk of EDB, some 90 percent of it, has been added to leaded gasoline where it scavenges and vaporizes lead which would otherwise collect on spark plugs and exhaust valves (Jacobs, 1980; U.S. EPA, 1984b). Since the EPA introduced measures to burn unleaded in place of leaded gasoline in 1973, EOB's use as a fuel additive has diminished. From a peak of 98 thousand metric tons in 1970, the amount of EDB in gasoline has decreased to an estimated 35 thousand metric tons in 1980, and will probably decline to some 22 thousand metric tons by 1985 (Jacobs, 1980).

As EDB use in fuel fell, its agricultural use expanded. Between 1976 and 1982, the use of EDB in agriculture underwent a near fivefold increase, rising from about 11 million to almost 55 million kilograms a year (calculated from: NIOSH, 1984a; U.S. EPA, 1983a; U.S. EPA, 1984b; and U.S. EPA, 1984f).

For forty years, EDB has been applied to grain stored on farms and in grain elevators to prevent infestation by insects. Estimates of the quantity of EDB applied to stored grain have varied from 374,800 to 1,386,000 kilograms a year, with the EPA citing the lesser figure (NIOSH, 1984a). Likewise, estimates of the share of grain stocks treated with EDB have varied considerably, ranging from 2 to 40 percent (U.S. EPA, 1983a). Of the EDB used to fumigate stored grain, 75 percent is applied to wheat. All but one of twenty-two samples of flour drawn from cities across the country in 1981 were found to contain EDB (U.S. EPA, 1983a; 1984c). In 1983, the EPA reported that residues of EDB appeared in 30

percent of the packaged grain products tested by federal and state agencies.

Flour milling machinery has been fumigated with EOB for the past two decades. The EPA has estimated that between 661,320 and 1,023,000

Occupational Safety and Health Administration (or Act)

*QSHA U.S. Environmental Protection Agency

EPA

“oo

kilograms of EDB were used each year for spot fumigation in flour mills processing 75 percent of the country's wheat (U.S. EPA, 1983a). Spot fumigation of milling machinery contributes to EDB residues in finished flour and packaged products.

In December 1980, after studying the risks and benefits of EDB for three years, the EPA proposed to forbid its use as a fumigant for both stored grain and milling machinery (U.S. EPA, 1981; 1983a). The EPA found that EDB posed unreasonable risks of inducing cancer, heritable genetic damage, and reproductive disorders. Longstanding use on grain and in mills, the Agency concluded, were the primary pathways EDB followed to “contaminate the human diet." However, the EPA stopped short of curtailing two other uses--the treatment of soil before seeding and the fumigation of citrus after harvesting--both of which grew and spread.

During the past five years, EDB has been increasingly injected into the soil to control nematodes, which are small worms which may attack the root systems of vegetables and fruit trees. In 1979, anticipating a ban on dibromochloropropane (DBCP), soybean growers in six states--Alabama, Florida, Georgia, Mississippi, South Carolina, and Tennessee--sought and received exemptions to use EDB beginning in 1980 (U.S. EPA, 1981; 1983a). Since 1980, some 50.6 million kilograms of EDB, about 90 percent of the total used in agriculture, has been applied to soil and over half

of it, 28.2 million kilograms, has been used to safeguard soybean crops (U.S. EPA, 1984f).

When the Mediterranean fruit fly threatened the California citrus crop in 1981, the role of EDB in the United States Department of Agriculture/Animal and Plant Health Inspection Service (USDA/APHIS) Quarantine Program aroused controversy. The USDA required California, which had forbidden application of EDB to harvested crops, to fumigate citrus fruit exported to Japan. California, in turn, required produce entering the state from Florida and Texas to be fumigated. Soon four other states--Florida, Texas, Arizona, and Hawaii--required all citrus fruit likely to host fruit flies to be treated before crossing their borders. EDB, between 110,000 and 183,700 kilograms of it a year, has been the primary agent used by citrus-growing states against the spread of different species of fruit fly. Likewise, the USDA/APHIS program requires EDB treatment of imported citrus and tropical fruit, much of it from the Caribbean Basin. Moreover, Japan, a valuable market for American citrus growers, has also required fumigation, though the Japanese have accepted produce treated by cold storage.

In its initial report of 1980, the EPA recommended ending the EDB fumigation of citrus and tropical fruit by July 1, 1983. During 1981, while the Administration considered this report, California grappled with its fumigation program. The California Division of Occupational Safety and Health, uneasy with the federal limit of workplace exposure of 20 parts per billion (ppb), adopted a more stringent level suggested by the National Institute of Occupational Safety and Health (NIOSH) as early as 1977 (Lash and Gillman, 1984). Meanwhile, pressure mounted on the EPA to act on its own proposals to restrict the use of EDB.

In the spring of 1982, the EPA proposed to postpone the end of fumigation of fruit until July 1, 1985 (Lash and Gillman, 1984; Sun,

470%). Ihe Giscovery oF EUG In groundwater of four states in the summer of 1983 hastened the regulatory process. In Georgia, Florida, California, and Hawaii, where EDB was used as a soil fumigant, 114 wells were contaminated with EDB, most at levels between 0.05 and 5.0 ppb, with some as high as 100.0 ppb (Lash and Gillman, 1984; U.S. EPA, 1983a). Since 1983, EDB has been detected in wells in Washington, Connecticut, South Carolina, and Oregon. Florida suspended the use of EDB as a soil fumigant and California followed suit in the five counties in which tainted wells were found.

Within days of the hearings, William Ruckelshaus, Administrator of the EPA, issued an emergency suspension, the stiffest measure the Agency may invoke, of EDB as a soil fumigant (U.S. EPA, 1984f). This was the only major use of EDB which the EPA had yet to propose curtailing. Ruckelshaus also ordered cancellation of EDB's other agricultural uses, though these orders may be challenged in court and the uses may continue while litigation proceeds. At this time, EPA declined to set residue tolerances for €DB in food products. EDB had been exempted from tolerances in 1956 on the grounds that any residue would dissipate from foodstuffs before they reached consumers. In 1980, however, the EPA found that EDB residue did not completely dissipate (U.S. EPA, 1980a; 1983a). In September 1983, the Agency increased its original estimates of re exposure to EDB in food products (see “Sources of Exposure” section).

In response to elevated exposure estimates nationwide, state health agencies assessed exposure and risk in their own states. Florida announced that EDB was found in a quarter of all products tested, and removed 77 items from grocers’ shelves.

Ruckelshaus suspended the use of EDB on stored grain and milling vachinery on February 3, 1984 (U.S. EPA, 1983a; 1984d). In addition, fuckelshaus established recommended guidelines, advising states against permitting the sale of flour containing more than 150.0 ppb and ready-to-eat products containing more than 30 ppb of EDB. The EPA stated chat only one or two percent of food in the marketplace would have to be withdrawn if states followed these guidelines.

Many states have adopted the federal guidelines, whereas others have chosen to set tighter standards. Massachusetts imposed the most stringent iimits: 10.0 ppb on all foods, to be reduced to 1.0 ppb on “arch 7, 1984. Other northeastern states--Vermont, New Hampshire, Maine, connecticut, New Jersey, and New York--spoke informally, agreeing with =PA that zero, the only acceptable level for EDB in foodstuffs, should be reached as quickly as possible. However, these states have set different tolerances or accepted the federal tolerances in pursuing this goal.

On March 2, 1984, the EPA moved to end the last major agricultural use of EDB, the fumigation of citrus fruit (U.S. EPA, 1983a; 1984a; 1984f). No users had challenged restriction more strenuously than those in the citrus industry. Their challenge had been upheld by the USDA as well as by the United States Agency for International Development, which represented the interests of Carribean growers seeking to keep their

place in the American market. Ruckelshaus announced tolerances for FDB residues of 250.0 ppb for the whole fruit and 30.0 ppb for the edible pulp on domestic and imported citrus fruit and papayas, These tolerances are slated to expire on September 1, 1984 when the EPA requires domestic use of EDB on fruit marketed within the country to cease.

Three years after it first sought to remove EDB from the U.S. diet, the EPA, in a flurry of decisions taken during the past seven months, neared its goal. However, not all states kept precisely in step.

The public has become bewildered by claims and counterclaims abcut the safety of staple products which fill shopping baskets week after week

Overshadowed by headlines about regulations and tolerances, the scientific data indicating the risks of EDB to human health has seemed obscure; wariness and confusion have reigned amidst a staccato of official statements.

In presenting this risk assessment of EDB, Envirologic Data seeks to offer a comprehensive, accurate, and timely guide to the risk EDB poses to public health.

METHODS AND NATURE OF RISK ASSESSMENT

Risk assessment is the scientific prerequisite for risk management, informing and guiding the management of hazardous materials by technical and regulatory measures. By exploring the biological, medical, and environmental effects of chemicals, risk assessment determines whether or not a particular substance poses risk to either human health or natural environments. If jit does, risk assessment specifies the nature and weighs the severity of the risks associated with each use. Characterized by gathering, studying, distilling, and interpreting data, risk assessment is essentially a scientific undertaking.

Some legislation requires that assessments of risks to health or environment be integrated with other considerations--the benefits derived from using the chemical, the cost of lessening risks by controlling the chemical, the risks of abandoning or replacing the chemical, and the cost and feasibility of regulation itself. These cost-benefit studies, like risk assessments, are performed according to established analytical procedures. Together risk assessment and cost-benefit analysis provide the information required to manage the risk, whether by technical or regulatory means,

The three elements of risk management--risk assessment, cost-benefit analysis, and regulatory decisions--may be performed by a_ single institution, like the EPA. On the other hand, some suggest that risk assessment should be divorced from regulatory action to enhance the credibility of the first and the legitimacy of the second. Certainly there is widespread agreement that successful risk management requires striking a fine balance between sound scientific knowledge, accurate

economic analysis, and informed regulatory choices.

Recognizing that neutrality and objectivity measure the worth of a risk assessment, Envirologic Data has prepared this document without a specific client. The risk assessment offers a comprehensive profile of a chemical, explains its hazards and assesses its risks to individual and public health. The profile is shaped by a complex and lengthy process. First, literature is searched for information about the chemical. The explosion of scientific data has been matched by the power of computer searches to sift, sort, and seize information. The risk assessment of EDB culled 876 citations from 11 databases, including Chemical Abstracts, Toxline, Medline, Enviroline, Pollution Abstracts, and Biosis. Altogether, nearly 500 citations have been examined, culminating in review and evaluation of the large number of literature citations supporting this document.

Once the information is in hand, it is read and evaluated. During the quality control phase, the information is reviewed by a panel of specialists. Preliminary evaluations are further reviewed by other

specialists to ensure accuracy of interpretation. Only after the information has been thoroughly and carefully considered is it utilized

in drafting the risk assessment. In the next phase, peer review occurs in two stages. There are: first, review within the project, and second, peer review by specialists outside the project staff. Each stage results in modifications, which respond to the critiques of the reviewing specialists. With this refined data, the risk assessment is constructed.

The risk assessment begins with a straightforward characterization of the chemical: the names it travels under; the companies which make it; the chemical class it belongs to; its structure; its properties; its uses; and the ways it is applied. This information indicates routes of exposure and population groups at risk; for instance, those whose occupation or diet increases their exposure to the substance.

The identification and assessment of risks, the centerpiece of risk assessment, presents a series of methodological problems. Reliable evidence of the effects of chemicals may be wanting. Clinical studies, providing data about individuals, and epidemiological studies, offering data about groups, may appear after initial exposure. This data is useful in tracing the effects of chemicals on those already exposed to them. However, the results of these studies may not appear until well after the time for risk management has passed. Consequently, risk assessment also draws on microbial and animal experiments. However, such studies may not have been performed for the full range of effects a chemical might possess.

Evidence from microbial and animal experiments must be applied or extrapolated to human beings in order to reach significant conclusions. Extrapolation itself poses two dilemmas. First, it must show that a biological effect observed in animals can be reasonably expected to occur in humans. Second, effects of low, actual doses or levels of exposure in the real world must be estimated from effects of high, experimental doses or levels of exposure under laboratory conditions. In short, two extrapolations are required, one from animals to humans and another from high to low doses.

Performed by modelling and statistical techniques, these extrapolations command confidence only in the context of stated assumptions. The results may not always possess the degree of certainty encountered in pure science. However, where risks to the public health of present and future generations are at stake, neither the regulators nor the public can afford to act only when the certainty of pure science is achieved. Rather, both must live with the best available scientific information even if imperfect or insufficient, from which sound, reasoned judgements can be drawn. It is important to remember that good science policy must be based upon good science.

Risk is a product of the hazard of the chemical as well as the probability of exposure at levels sufficient to generate adverse effects. Envirologic Data expresses hazard for a range of effects by means of a numerical rating procedure. Each effect is scored on a scale of D to 3, where the higher number designates either greater hazard or greater evidence of hazard. A score of 0 indicates no risk of the given effect. A score of 1 indicates that the data are insufficient. A a of Envirologic Data's ratings appears in "Health Effects--Human" section.

Perhaps the most obvious effect of a chemical is its acute toxicity. One measure of acute toxicity is the LDso, the concentration, expressed in milligrams per kilogram of body weight (mg/kg) or other units, sufficient to kill half the animals of a test sample. LDegs typically vary from species to species as well as to route of administration. The lower the L059, the more acutely toxic the material. For example, with an oral LOsq of 3 mg/kg, the pesticide parathion is a hundred times more acutely toxic to a test organism than another pesticide, phosmet, with an LDceq of 300 mg/kg for the same organism.

Of equal concern is a chemical's capacity to produce harmful effects long after initial exposure, that is, its chronic toxicity, always insidious and sometimes irreversible. Chronic toxicity may take many forms. A chemical may impair the work of organs, hinder the processes of reproduction, or enhance the spread of viruses. Chemicals may also possess the power to cause cancer (carcinogenicity), genetic damage (mutagenicity), and birth defects (teratogenicity). The hazard assessment considers each of these effects, measuring the hazard of each by the rating procedure.

Two other biologic properties of a chemical may enhance its harmful effects. The metabolic pathways of chemicals, the chemical changes they undergo as they are processed by the body, may produce by-products, called metabolites, sometimes as toxic as, or more toxic than, than the original material. The tendency of a chemical to accumulate in specific tissues, is known as bioconcentration. Bioaccumulation is defined as the increase in concentration of a substance as it is passed to successive levels in food chains. Both these aspects of E£DB are examined in the risk assessment.

How long a chemical persists in the environment may also influence the probability of exposure. Persistence may be affected by environmental circumstances such as the alkalinity or acidity of soil and

water or variations in temperature, humidity, and sunlight. Because of its influence on the probability of exposure, persistence, in soil and

water alike, is an important aspect of the risk assessment.

GENERAL INFORMATION AND PROPERTIES OF EDB Summary

EDB is an acronym which is usually applied to the 1,2- but not to the 1,1-isomer of ethylene dibromide. First synthesized in the U.S. in 1922, EDB is a halogenated hydrocarbon which exists as a highly volatile liquid at standard temperature and pressure. Its volatility, the fact that its vapors are 6.5 times as dense as air, and its insecticidal and nemotocidal action, suit it well for fumigation of fruits, grains, and soils. Moreover, in internal combustion engines, EDB scavenges tetraethy] lead, volatilizing it and preventing build-up of lead in engines burning leaded gasoline. Domestic production of EDB for these primary purposes exceeded 151 x 10© kilograms (332 million Ibs) in 1974. In recent years some 90 percent of this production has been allocated to leaded fuels, a use which is declining with the increasing use of unleaded fuels. The presence of EDB can be detected analytically in food at a concentration of about 1 ppb. In air EDB is detectable by a characteristic odor at levels of 10 to 25 ppm; these levels far exceed minimum biologically active doses (see “Health Effects--Nonhuman" and "Health Effects--Human" sections).

Categorization

Identification Numbers: Chemical Abstract Service Number (CAS): 106-93-4 National Cancer Institute: NCI-C00522 Pesticide Family: brominated hydrocarbon fumigant Chemical Family : haloalkane, halogenated hydrocarbon, brominated

hydrocarbon Chemical Name: 1,2-Dibromoethane Common Name: ethylene dibromide (EDB) Synonyms alpha-beta-dibromoethane; celmide; DBE; dibromoethane;

dibromoethane (DOT); dibromure; EDB; ethane, 1-2-dibromo; ethylene bromide; 1,2-ethylene dibromide; Fumo-gas; glycol bromide; glycol dibromide; sanhyuum; sym-dibromoethane; aethylenbromid (German); bromuro di etile (Italian); dibromure d'ethylene; DWU bromoetan (Polish) (NIOSH, 1983b; NLM, 1984)

Manufacturers and Trade Names

Four manufacturers have accounted for almost all EDB production in the U.S. They are: Dow Chemical Co., Magnolia, Arkansas and Midland, Michigan Ethyl Corporation, Magnolia, Arkansas Great Lakes Chemical Corporation, El Dorado, Arkansas PPG Industries (Houston Chemical), Beaumont, Texas* (NLM, 1984)

Trade names of EDB products are listed along with their manufacturer or distributor in Table 1.

*no longer manufactures EDB

-10-

Table 1. List of EDB Trade Names and Manufacturers/Distributors

REGISTERED TRADE NAME MANUF ACTURER/DISTRIBUTORS

Agway Sera-fume

Brayton EB-5 Grain Fumigant

Coast-0-Fume W-85

Coop New Activate Weevil Killer Fumigant

Cotton Producing Association EDB 40

Dawson (R) 37 Fumigant

Dawson 73 Fumigant

De-Pester Fumigant No. 2

De-Pester Grain Conditioner and Weevil Killer

De-Pester Weevil Killer

Douglas Suffokato No. 3 Grain and Mill Spot Fumigant

Douglas Tetrakil Weevil Killer and Grain Conditioner

Douglas Tetrafume Weevil Killer and Grain Conditioner

Dow Dowfume EB-7D Spot Fumigant for Milling Machinery

Dow Fume (R) EB-30

Dowfume C

Dowfume EB-5 Effective Grain Fumigant

Dowfume EB-15 Inhibited

Dowfume EB-59

Dowfume F

Dowfume MC-2

Dowfume 40

Dowfume W-40

Dowfume W-85

Dowfume W-90

Dowfume W-100

Dynafume

ED-40

EDB-86

EDB-85

EDB-93

EDB-100

E-D-BEE

Ethylene Dibromide

Ethylene Dibromide

Fascofume EDB-40

Fascofume EDB-85

FC-4 $x Grain Storage Fumigant FC-? Grain Fumigant

Formula 635 (FC-2) Grain Fumigant Formual 815 (FC-3) Grain Fumigant Frontier Chloro-Fume Grain Fumigant Grain Fumigant No. 1

Grain Fumigant No. 1

Grain Fumigant and Conditioner Grain Fumigant and Spot Treatment

Altix

Agway

Brayton Chemicals Coastal Agricultural Chemical Farmland Industries Gold Krist, Inc. Ferguson Fumigants Ferguson Fumigants Uniroyal Chemical Uniroyal Chemical Uniroyal Chemical Douglas Chemical

Douglas Chemical Douglas Chemical Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Dow Chemical USA

Parry Moore Co. and Griffin Co.

Dow Chemical

Dow Chemical

Dow Chemical

Atomic Chemical

Ethyl Corp.

Asgrow, FLA

Ethyl Corp.

Ethy] Corp.

Ethyl Corp.

Great Lakes

Dow Chemicals

Great Lakes Chemical

Kerr-McGee

Kerr-McGee

Vulcan Materials

Vulcan Materials

Cardinal Chemical

Vulcan Materials

Yulcan Materials

Mid American Chemical

Kaw Valley Warehousing

Kaw Valley Warehousing

Kaw Valley Warehousing

(continued on following page)

Table 1. (continued)

REGISTERED TRADE NAME

MANUFACTURER/DISTRIBUTORS

Grainfume MB

Hortus Larvae-Tox 431

Hortus Larvae-Tox 440

Infuco Debrome Spot Fumigant

Infuco 50-50 Spot Fumigant

Infuco Two-in-One Grain Fumigant

d-Fume C

d-Fume 20

Kill-fume 40

Leitte Spotfume 60

Macco Grain Fumigant and Conditioner

Macco Grain Fumigant and Spot Treatment

Maclean's S-T-D

Max Kill Spot- 59 Spot Fumigant for Mills and Milling Machinery

Max Ki11 10 Liquid Grain Fumigant

Max Spot Kill Machinery Fumigant

(FC-13) Mil) Machinery Fumigant

(FC-13) Mil] Machinery Fumigant

Patterson's Weevil Killer

Parsons Lethogas Fumigant

Pestmaster Fumigant EDB-85

Pioneer Brand Grain Fumigant

Professional ORS/MIS Soil Fumigant

Selig's Grain Storage Fumigant

Selig's Grain Fumigant No. 15

Serfume

Soilbrom-40 Soil Fumigant

Soilbrom-85

Soilbrom-90

Soilbrom-90 EB

Soi ]lbrom-100

Spot Fumigant

Staffel's Grain Fumigant Interstate

Terr-0-Cide 15

Terr-0-Cide 30

Terr-0-Cide 54-45

Terr-0-Cide 72-27

Terr-0-Cide 93.5

Tetrafume

Tetrakil]

Tetraspot

Trifum-A Grain Fumigant

Vulcan Formula 635 (FC-2) Grain Fumigant

Waco-50

914 Weevil Killer and Grain Conditioner

Selig Chemical Industries Hortus Chemical Hortus Chemical Industrial Fumigant Industrial Fumigant Industrial Fumigant d-Chem, Fumigators Jd-Chem, Fumigators Asgro Company

Leitte E. H. Company Mid America Chemical Mid America Chemical Yan Waters and Rogers Research Products

Research Products Researcn Products Cardinal Chemical

Vulcan Materials Patterson Chemical Prentiss Drug and Chemical Velsicci Chemical Corp. Bartel and Shores Chemical Dettel Bach Pesticide Corp. Selig Chemical Industries Selig Chemical Industries Dow Chemical USA

Great Lakes Chemical Great Lakes Chemical Great Lakes Chemical Great Lakes Chemical Great Lakes Chemical Uniroyal Chemical

The Staffel Company

Great Lakes Chemical Great Lakes Chemical Great Lakes Chemical Great Lakes Chemical Great Lakes Chemical Douglas Chemical

Douglas Chemical

Douglas Chemical

Stauffer Chemical

Vulcan Materials

Ferguson Fumigants PBI-Gordon Corporation

Note: many of these products have been discontinued (Ruckelshaus, 1984)

Sources: U.S. EPA, undated

-12-

Synthesis and Production Volume

The primary synthesis of EDB is by the addition of elemental bromine to ethylene (Fishbein, 1980). Natural bromide-containing brines are treated with chlorine to release elemental bromine through anionic replacement (NIOSH, 1977a). Gaseous ethylene and, liquid bromine (which may contain traces of chlorine) are reacted at 35 °- 85 °C at atmospheric pressure with EDB itself, as the solvent (Fishbein, 1980). After cooling the reaction mixture is neutralized to remove free acid and the product is purified by distillation. The reaction by-products can include vinyl Dromide, ethyl bromide, and ethyl chlorobromide. The purity of EDB can reach 99.5 percent, depending upon the purity of the inital EDB solvent used (Fishbein, 1980). EDB can also be produced by the hydrobromination of acetylene, although this process is of little economic value, and thus uncommonly used (NIOSH, 1977a).

EDB was first synthesized by Balard in 1826, by the reaction of ethylene with bromine (IARC, 1977). EDB production in ths U.S. was first reported in 1922 (IARC, 1977). Since 1923, EDB and ethylene dichloride nave been added to gasoline as lead scavengers to reduce the lead deposits created by Jead antiknock compounds. The use of EDB as a fumigant was first reported in 1925 by Neifert (IARC, 1977). It was later registered as a pesticide in 1948.

The U.S. production of EDB has increased in proportion to the demand of leaded gasoline. While estimates of domestic EDB production vary, the U.S. production of EDB has increased from 29 x 10© kg (64 million pounds) in 194D to highs of 151 x 106 kg to 159 x 106 (332 to 350 million pounds) in the 1970s. EDB production levels are shown in Table 2 and the production volume of four major manufacturers in Table 3.

The EDB production volume for 1979, as shown in Table 3, was estimated to be 159 x 106 kg/yr (350 million pounds) and was produced orimarily by four major manufacturers. The bulk of this production, 104 x 10© kg/yr (230 million pounds) was used as an additive for leaded gasoline (U.S. EPA, 1980a). Approximately 5.9-6.8 x 10° kg/yr (13-15 million pounds) of the total EDB produced annually was for pesticidal usage (U.S. EPA, 1980a). The EPA, in 1983, estimated that over 127 x 106 kg/yr (280 million lbs/yr) of EDB were produced annually, and of that 118 x 106 kg/yr (260 million lbs/yr) are used as an additive to leaded gasoline, and the remaining 9 x 106 kg/yr (20 million 1bs/yr) for agricultural pesticidal uses (U.S. EPA, 1984b).

The production volumes for EDB's use as a solvent in resins and waxes and as a synthetic chemical intermediate for pharmaceuticals and dyes were only available for 1976 (Johns, 1976). Of the total U.S. EDB zroduction of 150 x 106 kg/yr (331 million lbs/yr) during 1976, 13.3 x 10® kg/yr (29.3 million ibs/yr) were used in solvents and 7.4 x 100 «g/yr (16.3 million lbs/yr) were used in synthetic intermediates. The zercent of the total produced for these purposes was 0.09 percent and -.05 percent respectively.

= =

Table 2. Estimated EDB Production in the U.S.@

Production Production Year (kg/yr x 108) (1bs/yr x 106) Reference T1974 TSI 332 U.S. 1100, 1976 1975 124 275 USs TGs 19e7 1976 91 201 U.S. ITC, 1977 1977 11 244 U.S. ITC, 1978 127 280 U.S. Bureau of Mines, 1981 1978 104 230 U.S. ITC, 1979 TF 259 U.S. Bureau of Mines, 1980 1979 130 286 U.S. ITC, 1980 98 215 Lyday, 1980 116 256 Anonymous major producer, 1980 1980 80 176 Ibid. 1982 68 150 Ibid.

(projected)

aAdapted from Winzler and Kelly, 1982 bu.S. International Trade Commission

Table 3. Production Yolume of Four Major Manufacturers (June 1979)

Production Volume Percent Manufacturers Kg/yr x 10° Too Tb/yr of Total Dow Chemical Co., Magnolia, Ark. 52 115 33 Ethyl Corp., Magnolia, Ark. 638 150 43 Great Lakes Chemical Corp., El Dorado, Arkansas 23 50 14 PPG Industries, Beaumont, TX 16 _35 a. Totals: 159 350 91

Adapted from Winzler and Kelly, 1982

-14-

Analytical Methods of Detection

Analytical methods to determine food residues have been recommended by the EPA*. Separate methods are recommended for detecting EDB in whole grain, in intermediate (prepared) grain products, in ready-to-eat grain products, and in citrus. These are set forth below.

Detection in Whole Grain. The Association of Official Analytical Chemists (AOAC) method for determination of fumigant residues in grains has been modified by Clower (1980b). The modified method uses the basic procedural steps of the AOAC as_well as gas liquid chromatography equipped with a constant current ®3Ni electron capture detector. EDB residues are extracted by soaking the sample in a mixture of acetone and water. Water is partially removed by salting out with NaCl. After final drying with CaCl2, the acetone solution is analyzed by gas liquid chromatography and an electron capture detector. The detection level is 2 ppb.

Detection in Intermediate Products. EDB residues in biscuits and commercial flour are analyzed by a method developed by Rains and Holder (1981). Flour samples are extracted with hexane at room temperature with maximum extraction of EDB in 4 days. Biscuits are extracted by steam distillation with hexane. Three extractions are considered optimal. Each of the extracts are analyzed separa i Samples are injected into a gas chromatograph equipped with a electron capture detector. The limit of detection by this method is 0. 5 ppb.

Ready-to-Eat (cooked foods)- Recommended test procedure is Rains and Holder method as mentioned in intermeditate products.

Detection in Citrus. For imported citrus, papayas, and mangoes, the EPA recommends the King method (King, 1980). The method involves steam distillation from a benzene-water mixture for pr tab and cleanup. By using gas chromatography coupled with a electron capture detector, the limit of detection is 0.38 ppb.

Another method (Clower 1980b) is used for citrus fruit. This method uses ethyl] acetate as the solvent and distilled water. The mixture is distilled, and then cooled. The cloudy ethyl acetate is drained and sodium sulfate is added to clear the ethyl acetate. Then using gas

liquid chromatography coupled with a §3Ni electron capture detector, the limit of detection is 1 ppb of EDB.

Air Sampling Analytical Methods. The primary method of analyzing EDB in air is by taking a known volume of air through a charcoal tube to trap the EDB vapor (NIOSH, 1978b). The charcoal in the tube is transferred to a flask and the analyte is desorbed with 10.0 ml of 99:1 benzene-methane. A sample is then put into a gas chromatographic analyzer equipped with an ejectron capture detector. The range of detection is 0.002 to 8.0 mg/m? for a 25-liter air sample.

*Telelphone communication with Sami Malak, Residue Chemist, EPA, Office of Pesticide Programs, March 12, 1984

a6:

Water Sampling. In the sample preparation 37 ml of the sample are shaken with i Tm) of hexane for 1 minute in the original field sample vial, then injected into a gas chromatograph. (The separation of EDB is

done in two capillary columns). The detection limit for these analyses was determined to be approximately 0.01 ug/L (Jovanovich, 1983).

Principal Uses and Use Breakdown

Based upon 1976 data, 90 percent of the EDB produced annually was used as an additive in leaded gasoline (Jacobs, 1980). In 1923, Midgely discovered that an organic compound of bromine and chlorine added to tetraethy] lead would reduce the lead oxides and sulfates which otherwise deposit on the combustion chamber walls, piston tops, and exhaust valves of internal combustion engines (Jacobs, 1980) Since 1923, all alkyl lead compounds used in gasoline to suppress engine knock have contained EDB and ethylene dichloride as lead scavengers. The overall use of EDB in leaded gasoline is expected to be reduced by 80 percent by 1986 from its peak use in 1970, due to the phase out of leaded gasoline (Jacobs, 1980).

The remaining 8 to 10 percent of EDB produced is used as a fumigant insecticide and nematocide, i.e., root worm nematode killer (U.S. EPA, 1984b). In 1978, EDB used as a soil fumigant accounted for over 90 percent of the total pesticide use (U.S. EPA 1983c). In 1983, the use of EDB had increased to approximately 10 x 106 kg/yr (23 million pounds) annually for nematocide control (U.S. EPA, 1983a).

Pesticide uses of EDB are divided into four main categories: pre-plant soil fumigation; post-harvest fumigation; spot fumigation; and minor uses, as shown in Table 4. EDB is used before the planting

(pre-plant) of tobacco and soybeans to kill the rootknot nematodes. Grain and citrus are fumigated after they are harvested (post-harvest) in large fumigation chambers in preparation for storage. EDB also has been used for more than twenty years as a spot fumigant to control insect pests that infest flour milling machinery. Additional minor uses of EDB are in the fumigation of: beehive supers (top structure of the beehive) and honeycombs; structures for termite control; felled log fumigation to control the mountain beetle; storage vault for clothing and furniture; and for the Japanese Beetle domestic quarantine program for plants, soil and grass sod.

The EPA announced on September 28, 1983, an emergency order suspending the sale or use of registered products for soil fumigation as well as the Notice of Intent to Cancel pesticide products containing EDB for stored grain fumigation, spot fumigation of grain milling machinery, and felled log fumigation (U.S. EPA, 1983b). The other Notice of Intent to Cancel was for products registered for quarantine fumigation of citrus, tropical fruits, and vegetables, and was not made effective until September 1, 1984, allowing time to develop alternatives. Administrative hearings are scheduled for registration of EDB products for felled log fumigation. On February 3, 1984, an emergency order was announced suspending the use of EDB products for fumigation of stored grains and spot fumigation of milling machinery.

-16-

Table 4,

Crop

Pesticide Use for EDB and Amount Used

EOB Used Annually

Target Pest thousands kg

thousands 1b

PRE~PLANT SOIL FUMIGATION:

Soybean Tomatoes4 Beans@ Carrots?

Other Produce@ Cotton

Peanuts

Tobacco

Citrus

Pineapple

Peaches

Sweet Potatoes

White Potatoes

5800.

Nematodes 80. 78, 30. Sls

wWonP Oo

Root-knot nematode 2041.1 975.6

Root-knot, Root Lesion 414.4 Stunt nematode

Citrus and burrowing Sat nematode

Root-knot, Reinform 367.4 (meloidogyne javanica) nematode

Ring, Root-knot, Tal, dogger nematode

72.5-107.0 435 .4-489.9

POST-HARVEST FUMIGATION:

Stored grain Wheat Corn

Insects 226.8

Oats, rye, rice, barley

Quarantine Fumigation:

Citrus, mangoes, papayas

Mexican fruit fly 22.6 (Anastrephia Ludens)

12,787.05 177.3 173.65

68.2D 70.5b 4,500.0 2,151.05

913.65 20.0¢

g10.0°

170.0¢

160-236.1 960-1080.0b

500.04

50.0€

(continued on following page)

ci.

75 10€ 10&

NA

Table 4. (continued)

Crop

EDB Used Annually Target Pest thousands kg thousands 1b

Vegetables and other fruit NA

SPOT FUMIGATION:

Grain milling

Machinery

Flour mills (elevators)

MINOR USES:

Beehive supers/ honeycombs Felled logs

Structures

Storage vaults for clothing/ furniture

Quarantine plants, soil, grass, sod

South Amer. fruit fly (Anastrephia fraterculus) Caribbean fruit fly

(Anastrephia suspensa Mediterranean fruit fly (Ceratitis Capitata) Oriental fruit fly (Dacus dorealis)

MeTon fruit fly (Dacus cucurbitae)

0.39 0.850¢ Insects 226.7 500.04 45.3¢ 100.04

Greater Way moths Mountain pine beetle (Dendroctonus deoderone)

Termites

Insects

Japanese beetle

Adapted from:

stimates may be 1 . EPA, 1983a . EPA, 1980a . EPA, 1983b - EPA, 1984f

a Est : U.S

U.S d us eus

ower due to California suspending use in 4 counties.

=1§=

The use of registered EDB products remains restricted for the fumigation of beehive supers and honeycombs, storage vaults, termite control and quarantine use for Japanese Beetles. The restricted use status allows the continued registration of these products under the condition that special changes be made on the product labels to reduce the exposure risks associated with their use.

Other uses of EDB are as a chemical intermediate, in the manufacturing of dyes, pharmaceuticals, and gauge fluids, and as a solvent in resins, gums and waxes (NLM, 1984). EDB has also been used in fire extinguishers (Sittig, 1981).

Formulations

Motor-mix formulations. EDB has been used in the manufacture of antiknock additives which are blended to gasoline in varying amounts. More than 100 different blends are produced (OSHA, 1983). The composition of a typical gasoline blend produced by Dupont consists of ethylene dibromide (17.86 percent weight), ethylene dichloride (18.81 percent weight) and tetramethyl and tetraethy] leads (Jacobs, 1980; OSHA, 1983). The current conventional motor mix formulations contain 1 mole ethylene dichloride and 0.5 mole EDB per mole of tetraalkyl lead (Jacobs, 1980). The antiknock mixtures are formulated such that each contains 39.39 percent by weight of lead calculated as lead metal (Jacobs, 1980) The remaining constituents of the antiknock mixtures are small quantities of dye (to aid in leak detection), antioxidant, (for stability) and kerosene (an inert diluent) (Jacobs, 1980). Aviation antiknock mixer contains 35.68 percent weight EDB and no ethylene dichloride (OSHA, 1983).

Pre-plant Soil Fumigation. EDB has been injected directly into the soil to ki nematodes. The formulation for products registered for soil fumigation range from 40-100 percent EDB active ingredient.

Post-harvest Fumigation. Stored grains: EDB is mixed with carbon tetrachloride, ethylene dichloride, and/or carbon disulfide. EDB constitutes 1 percent to 9 percent of the weight of the liquid fumigant mixture. A frequently used product is Dowfume EB-5, which contains carbon tetrachloride, ethyl dichloride and EDB in 63:30:7 ratio (U.S. EPA, 1983c). An example af a maximum dosage formulation of EDB is the Douglas Suffokato No. 3 (R grain fumigant; its constituents are carbon disulfide (14,54 percent), EDB (9.99 percent), SO2 (1.54 percent) and pentane (0.37 percent)”.

Quarantine fumigations. Quarantine specifications call for use of

technical grade EDB of at least 97 percent purity. Fruit fumigation formulations contain 100 percent EDB.

Spot Fumigation. Product formulations range from 7 to 70 percent EDB. Examples of maximum dose spot-fumigants are:

1) Dowfume 70 (R): 70 percent EDB and 30 percent methyl bromide (U.S. EPA, 1980a);

2) Vulcan §R): 59.9 percent carbon tetrachloride 19.6 percent EDB n 20.4 percent methyl bromide”;

x. Telephone communication, William Miller, EPA, Office of Pesticide Registration Division, April 4, 1984.

-19-

3) Macco {R) Spot Treatment: 63.6 percent carbon tetrachloride 29.2 percent ethylene dichloride

7.2 percent EDB

Minor Uses. The five minor uses of EDB are as follows:

Beehive supers and honeycombs: an 84.5 percent EDB formulation is currently registered. EDB (1-2 tablespoons) is placed on absorbent Material which jis set on stacked supers, and then the beehive jis covered.

Felled logs: the formulations used jin Colorado are in a 5:1 volume/volume dilution of a 23 percent EDB formulation. This corresponds to a 4.76 percent weight/weight concentration (U.S. EPA, 1983a).

Structures for termite control: formulations range from 15-83 percent

Vaults for stored clothing/furniture: two products containing 5 percent EDB are registered for use in vault fumigation (U.S. EPA, 1980a)

Fumigation of quarantined plants, soil and grass sod: lid registered formulation is EDB No. 434, containing 2.5 percent EDB*

Application Rates and Dosage

The major use categories for EDB and particular application rates follow:

Pre-plant soil fumigation. E0B is applied by a chisel-injector into the soil to a depth of 3-4 inches, where it interacts with the soil environment. The application rate is approximately 160 lbs/acre, depending upon particular crop and formulation. EDB has been applied in citrus groves in barrier zones from 16 to 32 feet wide (U.S. EPA, 1984f).

Post-harvest Fumi gation. Stored grain: EDB liquid fumigant formulations are Tabele or use on the farm and in grain storage elevators. Typically, the fumigant is applied to the upper layer of the grain. An example of application rates for bulk fumigation of st a8 kt

using the maximum dose formulation, Douglas Suffokato No. is follows :

Sorghum: 11 Ibs. active ingredient/1,000 bushels Corn: 9 lbs. active ingredient/1,000 bushels Barley, oats: 7.2 lbs. active ingredient/1,000 bushels

*Telephone communication, Sami Malak, EPA, Office of Pesticide Programs, Residue Chemist, March 12, 1984

**Telephone communication, William Miller, Office of Pesticides, Registration Division, April 4, 1984.

-20-

Post harvest quarantine fumigation of citrus. EDB is applied at a rate of T6 ounces per 1,000 cubic feet of storage area. The EDB is volatized in a chamber for two hours. This application is for grapefruit, bananas, mangoes, papayas, pineapples, string beans, melons, cantaloupe, cucumbers, guavas, nuts, peppers, and zucchini squash. Oranges are treated with up to 24 ounces active ingredient/1,000 cu. feet of storage space. The amount of EDB applied is dependent upon the air temperature within the chamber and percentage capacity of the chamber occupied by the fruit and type of fruit fly (U.S. EPA, 1984f).

Spot fumigants. The spot fumigants containing EDB are applied at various locations in grain mills, including elevator heads, elevator boot, rollstand, endspouts, conveyors, sifters, conveyor dust trunk, and dusters. The formulation of 70/30 mixture of ethylene dibromide and

methyl bromide is commonly used. The drift application is 2-12 ounces. Fumigation of the average mill requires approximately 10.5 gallons of the

mixture, and 10 to 11 treatments annually (U.S. EPA, 1984d).

Chemical and Physical Properties

The chemical and physical properties of EDB are listed in Table 5.

Table 5.

Chemical and Physical Properties

Reference

Physical properties:

Molecular structure:

Molecular formula Molecular weight Boiling point

Melting point

Vapor pressure

Specific gravity Density

heavy, colorless liquid with a chloroform-

like odor

CoH4Bro

187.88 131-132°C 94°C, 9.97°6 11 mm at 25°C, 17.4 mm at 30°C

2.1707 at 25°C/4°C 2.18 g/ml at 20°C

Merck, 1983 NLM, 1984

IARC, 1977

IARC, 1977 Merck, 1983 Merck, 1983

Sax, 1975 IARC, 1977

Merck, 1983 Sax, 1975

Sax, 1975 NIOSH, 1977a

(continued on following page)

Table 5. (continued)

Reference Refractive Index nd29 1.53789 IARC, 1977 Viscosity 38.5 centistokes 20°C NLM, 1984

Vapor density

Percent in saturated air

Heat of vaporization: Liquid Surface tension:

Solubility

Stability

Reactivity

Flash point

Flammability

Incompatabilities

Detectable by odor :

Conversion factors :

6.487 (Air=1)

1.5 at 25°C

82.1 BTU/Ib = 45.6 cal/G = 1.91 x 10° Joules/kg NLM, 1984

38.75 Dynes/cm = 0.03875 Newtons/m at 20°C slightly soluble in water (0.43g/100g of water at 30 C); soluble in acetone, alcohol, benzene, ethanol, ether, and most organic solvents

slowly decomposes in presence of light and turns brown. Hydrolyzes to ethylene glycol and bromoethanol when heated with water. Resistant to atmospheric oxidation by peroxides and ozone. EDB may also react with hydroxyl radicals.

bifunctional alkylating agent, liberates bromides

none

nonflammable by standard tests in air; has been used in fire extinguishers

incompatible with chemically active metals: sodium, potassium, calcium powdered aluminum, zinc, magnesium, liquid ammonia, strong oxidizers

10 ppm (77 mg/m3) to 25 ppm (192.5 mg/m)

1 U.S. gallon = 18.11 1bs = 8.23 kg 1 1b liquid = 208 mi at 25°C

1 02/1000 ft3 = 1.mg/liter = 1 gm/m3 1 ppm = 7.69 mg/m?

1 mg/m? = 130 ppb

U.S. EPA,1980a

NLM, 1984

NLM, 1984

IARC, 1977 NLM, 1984

NLM, 1984 IARC, 1977 Leinster, et

al., 1978

NLM, 1984 U.S. EPA, 1980a

Sittig, 1981

Sittig, 198)

NIOSH, 1977 U.S. EPA, 1980a

=22-

SOURCES OF EXPOSURE

Summary

Natural background levels of EDB are, effectively, zero; all detectable residues may be regarded as man-generated. Occupational exposure to EQB is encountered primarily in EDB manufacture; EOB fumigation of soits, citrus, grains and grain milling machinery; formulation of automotive fuel additives; and the pumping of automotive fuels at gas stations. Levels encountered are highly variable, depending upon industry and occupational setting. Non-occupational exposure may originate from non-point sources, such as automobiles and gasoline stations, or from dietary components, principally citrus and grain products.

Envirologic Data has calculated mean EDB levels in these major sources. Ambient air in urban environments exhibits mean residues of 0.026 ppb. EDB levels in some two percent of the domestic citrus crop which is treated with EDB exhibits a mean of about 101 ppb in edible portions. Imported fruits are all fumigated, and exhibit a mean of 766 ppb. Grain products exhibit a mean residue level of 50.50 ppb for raw products, 22.66 ppb for prepared (intermediate) products, and 2.32 ppb for ready-to-eat products.

Envirologic Data has used these calculated residue levels, along with total air and dietary intakes of each EDB source, to estimate total annual intake of EDB by adult men, adult women, children, and infants. Total annual intakes are, respectively, 2,175, 1,957, 1,326, and 318-377 micrograms per year. By far, the largest input into this total is urban air, accounting for 74 to 88 percent. It is, however, important to remember that effective EDB dose depends not only upon intake, but

absorption into the body from the lungs and gastrointestinal tract. This process is evaluated in the "Pharmacokinetics" section.

Introduction

The risk that any individual will experience adverse health effects--such as cancer--caused by EDB depends upon two factors: first, the likelihood of exposure to EDB, and second, potency in causing the adverse effect. This general relationship can be expressed as follows:

Risk = exposure + effect.

Accordingly, this section of the Risk Assessment document considers the sources of human exposure, and the next section describes the behavior of EDB in the environment, with emphasis on EDB dynamics which may lead to human exposure. Later sections consider routes of EDB entry into the body, EDB distribution, and EDB health effects.

=

Natural Background Levels of EDB

It is likely that, over several million years of evolution, the human organism has effectively adapted to those substances which have been ubiquitously present in the environment. Consequently, in assessing the risk associated with exposure to any particular substance, we focus on incremental exposure levels, i.e. amounts above the natural background levels which are assumed to be harmless or, in any case, tolerable to humans by any standards of environmental concern. Unlike such naturally occurring substances as lead, chromium, and the other heavy metals, EDB is a man-made substance which was unlikely to have been encountered by organisms prior to recent years when it was first synthesized. Any level detected must, therefore, be regarded as above natural background levels, and be subjected to health risk scrutiny.

Man-Generated Sources of Exposure

Man-generated sources of EDB exposure may be occupational or non-occupational and, if non-occupational, may be from non-point sources (automobiles, gas stations) or from dietary components. Principal dietary sources of EDB are grains and citrus fruits. The following paragraphs analyze levels of EDB exposure from these principal sources.

Occupational Exposure

Examples of industries in which occupational exposure to EDB may occur are listed in Table 6. (NIOSH, 1977b). Occupational exposure to EDB is encountered primarily in such industries as EDB manufacture; EDB fumigation of soils, vegetables, fruits, grains, and grain milling machinery; formulation of automotive fuel additives; and the pumping of automotive fuels at gas stations. The primary route of occupational

exposure appears to be inhalation, although skin contact may also occur. Several investigations of occupational exposure have revealed highly

variable levels among sites and job descriptions (Table 7.). For example, as the table indicates, EDB in ambient air at a fruit packing plant exhibited levels ranging from below detection to as much as 2960 ppb (Markel, 1981).

Non-Occupational Exposure

Non-point Source Exposure. EDB in leaded automotive fuels results in emissions from moving vehicles as well as from such widely dispersed sources as gasoline stations. Leinster et al. (1978) showed that exposure from such emissions could be divided into three components: exposure from evaporative loss from gasoline, from EDB in exhaust from leaded fuels, and from crankcase blow-by gases. Only the evaporative component would, in temperate climates, vary seasonally, that is, be higher in warmer weather. As indicated by direct measurements, the evaporative component predominates in the summer, creating seasonally variable EDB concentrations in ambient air. Such factors as wind

-24..

Table 6,

Examples of Industries Presenting Potential

Occupational Exposure to EDB

Industry

Occupation

Chemical

Agricultural

Exterminating Pharmaceutical Manufacturing

Textile

antiknock compound makers fat processor

gum processors

lead scavenger makers resin makers

tetraethy] lead makers

cabbage growers

farmers

fumigators of citrus and grain

termite controllers

drug makers

fire extinguisher makers

wool reclaimers

Adapted from:

NIOSH, 1977b

-25-

Table 7. Examples of EDB Concentrations in Ambient Air in Occupational Settings

EDB Concentration in Ambient Air

Occupational Setting (ppb) Reference EDB production plant 0.75 - 42.74 (500 m Johns, 1976 downwind, wind at 6 m/sec) Houston Chemical Company nbb - 1068 White and Lybarger, 1978 Fuel additive formulation 0.2 - 54 Rumsey and Tanita, 1978 and use Oil refinery 0.03 - 0.208 Going and Long, 1975 0i7 refinery 0.4 - 40 American Petroleum Inst., 1982; Centaur Assoc., 1983 Grain fumigating plant NDD- 21 NIOSH, 1984a Fruit packing plant NDD_ 29608 Markel and Peter, 1981 operators 1604 fork lift operators 1804 loader 2004 EDB introduction point 7904 fumigation chamber doors 9504

4 authors' units transformed by Envirologic Data, Inc. b ND = not detected

<0

velocity, density of both automobiles and gasoline stations, and EDB

content of local fuel mixtures (e.g., 0.91 g EDB/gallon in one mixture; Jacobs, 1980) constitute additional “variables. Chcentraston of EDB in

tailpipe emissions varied from undetectable levels to as much as 1300 ug/mile (Sigsby et al. 1982), but it was not possible to relate this unit to others used 7m this report.

Several investigators have reported EDB concentrations in ambient air in urban environments (Going and Long 1975; Leinster et al., 1978; Singh et al., 1981; 1982). These are summarized in Table 8, Which indicates a mean level of approximately 0.026 ppb in the U.S. cities investigated.

Dietary exposure. Dietary exposure to EDB occurs primarily through ingestion of grains and fruits (mostly citrus) fumigated with EDB, although pollution of surface and well waters cannot be ruled out as potentially significant dietary exposure sources (Going and Long, 1975; Page, 1981; Sun, 1984).

Going and Long (1975) reported that a stream near an oi] refinery was contaminated with 1.13 ppb of EDB, and that a stream near an EDB manufacturing site contained 1.05 ppb. Sun (1984) summarized evidence of EDB contamination of wells: some wells in California, Florida, Hawaii, and Georgia were found to be contaminated with EDB following soil fumigation. Page (1981) reported detecting EDB in 6 percent of 175 surface water samples and 8 percent of 421 ground water samples in New Jersey. The mean, modal, and 90th percentile concentrations of EDB were all zero. More extensive data are required from more states to define the extent of EDB groundwater contamination.

Two approaches can be adopted for determining dietary exposure from fruits and grains: empirical or experimental. Using the experimental approach, Dumas (1973) and Dumas and Bond (1975) fumigated apples with EDB. In the latter study, the investigators applied 8 to 24 mg/l (ppm) of EDB for 4 hours at 13°C and found that within 13 days the EDB levels had dropped below 0.1 ppm; however, apples stored at 4°C required 4 weeks to reach 0.1 ppm. Other experiments have been designed to measure the effect of varying such conditions as the initial EDB concentration, the temperature, the type of container, the moisture levels, and so forth. Such investigations indicate that the gradual loss of EDB over time is a complex variable; it is, therefore, difficult to quantify potential human exposure to EDB from dietary sources without specifying the conditions and duration of storage of the foodstuff.

These difficulties have given rise to the empirical approach to determining EDB expesure, that is, the approach of simply measuring EDB levels directly in random or selected samples of fruits and grains. Although this method generates a great quantity of data, it does not easily control for the many variables, and therefore requires large sample sizes to ensure that the data reflect actual consumer exposure.

The EPA reported residue levels in citrus fruits from numerous

sources, and statistically analyzed only those samples exhibiting residues above the detection limit of about 1 ppb (U.S. EPA, 1984f).

-27-

Table 8. Ambient Air EDB Concentrations in Urban Environments

EDB Concentration

Location (ppb) Reference London, UK ND@ - 0,22b Leinster et al., 1978 Houston, TX 0.059 Singh et al., 1982 St. Louis, MO 0.016 " Denver, CO 0.031 : Riverside, CA 0.022 " Staten Island, NY 0.020 - Pittsburgh, PA 0.016 S Chicago, IL 0.026 Los Angeles, CA 0.033 0.026 (SD)@> Singh et al., 1981 Oakland, CA 0.016 0.013 (sp)ab " Phoenix, AZ 0.040 0.038 (sp)ab *

0.0083 Going and Long, 1975

a b

ND = not detected; SD = standard deviation authors' units transformed by Envirologic Data, Inc.

=28=

Residue levels jin whole fruit” (from domestic and imported sources) exhibited a median EDB concentration of 250 ppb, a mean concentration of 1633 ppb, and a maximum concentration of 16,000 ppb. However, levels in the pulp {edible portion) of domestic and imported fruit were lower: median 265 ppb; mean 373 ppb, and maximum 6,250 ppb. Levels in imported citrus fruit were higher than in domestic: in pulp, concentration in imported citrus was 766 ppb (maximum 6,250 ppb), compared with a mean of 101 ppb (maximum of 520 ppb) in the pulp of domestic citrus.

Quantifying exposure to EDB from grain is more complex than for citrus fruit because grain may be consumed raw or cooked. Cooking reduces EDB levels in the food {see below). Moreover, many grain products are sold as ready-to-eat products in grocery stores. The U.S. EPA (1984d) reported residue levels for raw grains. EDB levels are primarily determined by duration of storage and degree of dilution with non-fumigated grain; other factors include EDB application rate, amount of aeration, and temperature (U.S. EPA 1984d). Although only 5 percent of U.S. grain is fumigated with EDB, about 60 percent of all raw grain tested for EDB contains detectable levels.

Envirologic Data calculated mean levels of EDB in raw wheat, corn, and all raw grain combined from data submitted to the EPA from six sources (U.S. EPA, 1984d). For wheat, the mean EDB level in 862 samples was 40.56 ppb; for corn, the mean level in 290 samples was 56.31 ppb; and in all raw grains combined, the mean level in 1264 samples was 50.50 ppb.

Milling of grains was found to reduce EDB levels in the flour by 68 to 95 percent. In one study, wheat flour exhibited a 68 percent

reduction (Wit et al., 1969); in another, Berk (1974), (as cited in EPA 1984a), the reduction was 74 percent. However, levels in the bran, that is, the external, fumigated part of the grain, were higher, in one study by a factor of about 1.5 (Wit et al., 1969).

Uncooked, intermediate grain products were monitored in eight studies reported to the EPA since 1983 (U.S. EPA, 1984d). Envirologic Data calculated mean levels of EDB in uncooked, intermediate grain products, as follows: for uncooked wheat products, the mean EDB level in 638 samples was 14.54 ppb; for uncooked corn products, the mean in 303 samples was 44.07 ppb; and for all grains combined, the mean level in 1007 samples was 22.66 ppb.

Cooking of commercial grain products reduces EDB levels through volatilization, decomposition, and dilution with uncontaminated ingredients (U.S. EPA, 1984d). Results of cooking experiments might depend upon such factors as cooking temperature, cooking duration, rate of stirring, and exposed surface area (open pans greater than covered pots). Data submitted to the EPA from four studies in which uncooked products containing more than 3 ppb of EDB were subjected to experimental cooking were reported by U.S. EPA (1984d). The weighted average reduction of EDB residues was 82.0 percent.

*including such non-edible parts as skin and pits.

-29-

In ready-to-eat (cooked) grain products, data were submitted te EPA from seven studies. Wheat products exhibited a mean level calculated by Envirologic Data’ of 2.31 ppb (sample size=272); in corn products the mean EDB level was 3.85 (sample size=92); and for all grains combined, the mean EDB level was 2.32 ppb (sample size=453).

EDB Intake

It is possible to calculate total intake of EDB by using levels of exposure reported earlier for non-point (ambient air) and dietary sources of EDB. In the following analysis, occupational exposure levels are not included; hence, individuals occupationally exposed to EDB will have the body burdens derived for the general population plus additional burdens associated with their specific occupations. However, it is outside the intended scope of this document to analyze health risks of populations at high occupational risk; existing analyses of EDB health risks have, understandably, already emphasized these populations.

Inhalation. To analyze body burdens of EDB due to inhalation exposure, it is necessary to consider the individual's respiratory rate averaged over meaningfully long periods of time, such as a day or year Table 9 gives respiratory volumes for adults, children and infants. Based upon these estimates, Table 10 was constructed to present EDB inhalation intake for populations.

Dietary. To analyze body burdens of EDB due to dietary exposure, it jis necessary to consider the individual's dietary intake of citrus fruits and grains. Overall, grain consumption by the U.S. population js 74 percent wheat, 10 percent corn, 8 percent rice and 8 percent other grains (U.S. EPA, 1984d). Consumption by infants who are not nursing is quite different from the national average: 37 percent milled rice, 29 percent wheat, 21 percent oats, 8 percent corn, and 4 percent barley.

The U.S. population as a whole was reported to consume 1.4 grams of wheat per kilogram of body weight per day (511 grams/kg/year), 0.19 g/kg of corn (69.35 g/kg/year), 0.16 g/kg of rice (58.4 g/kg/year), and 0.15 g/kg of other grains (54.75 g/kg/year) by U.S. EPA (1984d). Non-nursing infants consumed 1.32 g/kg per day of rice (481.8 g/kg/yr), 1.03 g/kg of wheat (375.95 g/kg/yr), 0.29 g/kg of corn (105.85 g/kg/yr), and 0.90 g/kg of other grains (328.5 g/kg/yr). Total grain consumption was reported to be 1.9 g/kg per day (693.5 g/kg/yr) for adults, and 2.5 g/kg per day (1277.5 g/kg/year) for non-nursing infants.

Using these values, and assuming that 10 ppb of EDB is an average concentration for all grains, U.S. EPA (1984d) estimated the dietary EDB burden contributed by grains. EPA also considered the dietary burden of infants separately by assuming only a 1 ppb concentration in rice, an

important component of infants’ grain diets. The procedure used by EPA appears to be reasonable assuming that raw grain, which was found to have

*Envirologic Data assumed, as did EPA, that samples containing EDB at levels below the detection limit of 1 ppb actually contained 0.75 ppb.

ae

Table 9. Respiratory Volumes for Reference Individual (Liters of air breathed)4

AduTt Adult Child Infant man woman 10 yr lyr

8 hours working, 9,600 9,100 6,240 2,500 (10 hr) light activity 8 hours 9,600 9,100 6,240 nonoccupational 8 hours resting 3,600 2,900 2,300 1,300 (14 hr) Total (liters/day) 2.3 x104 2.1 x104 1.5 x 104 0.38 x 104 Total (liters/year) 8.4x106 7.7x 106 5.5 x 106 1.4 x 106

a Adapted from ICRP 1975, International Commission for Radiological Protection. Physiological data for reference man. In: Report of the task group on reference man, Nov. 23. New York, NY, Pergamon Press, pp. 335-65, 1975.

Table 10. Inhalation of EDB (ug) from Urban Air Assuming Mean Concentration of 0.026 ppb of EDB (yg/kg) in Air

Adult Adult Child Infant EDB Intake Man Woman 10 years 1 year per hour 0.2 0.2 0.1 0.03 per day 4.6 4.2 3.0 0.8 per year 1680 1540 1100 280

-31-

EDB levels greatly in excess of 10 ppb, do not constitute a major percent of the grain diet. However, this assumption may be in error for some individuals, particularly those consuming either a strictly or a largely vegetarian diet. With this caveat in mind, the EPA figures were: 6.9 micrograms per kilogram of body weight per year for adults, and 12.8 micrograms per kilogram of body weight per year for non-nursing infants. If rice has atypically low EDB levels of 1 ppb, the infant burden becomes 4.75 micrograms per kilogram of body weight per year.

In addition to grains, fresh citrus (excluding juice) is the only remaining major dietary input of EDB, primarily from _ oranges, grapefruits, lemons, limes, tangelos, and tangerines (U.S. EPA 1984f). Fresh citrus comprises only some ten percent of total citrus intake; 90 percent of total citrus intake is in the form of juice, which is prepared from citrus that is not treated with EDB. Average U.S. intake of fresh citrus is estimated at 0.24 grams per kilogram of body weight per day.

To determine total body burden, U.S. EPA (1984f) assumed a mean residue in edible portions of 100 ppb and a 2 percent fraction of the domestic citrus crop treated with EDB. Consumption of imported, tropical fruits is low on the average, but subpopulations of consumers may be exposed to higher levels from imported fruit. Under the U.S. EPA (1984f) assumptions, EPA estimated a mean daily body burden of 4.8 x 10-7 mg of EDB per kg of body weight. This estimate varied depending upon seasons, region, ethnic group, and age category, but the range of estimated body burdens was narrow, 6.8 x 107% to 9.4 x 107° mg EDB/kg/day. The value of 9.4 x 10-/ mg/kg/day, that is, nearly twice the mean value of 4.8 x 10°’, corresponded to the body burden of infants.

Envirologic Data calculated a mean residue level of 101 ppb of EDB in the edible portions of domestic citrus fruits in which EDB was detected, based upon data presented by U.S. EPA (1984f) from diverse sources. With new regulations (see “Federal and State Government Regulatory Actions"),

these levels will be reduced, as will the fraction of EOB-treated citrus fruit.

Consequently, in estimating EDB body burdens, it is important to distinguish between past and future exposure Jevels; however, for purposes of risk estimation, future risks may depend upon past as well as future exposures. Envirologic data assumes that the historical body burden to date has been 101 ppb in two percent of fresh citrus (one percent higher than EPA's assumption). Based upon the foregoing

discussion, the U.S. total EDB body burden from all sources is given in Table 11.

U.S. EPA (1983a) reported that in California, the only state for which information was available, 17 percent of fresh oranges, 35 percent of fresh grapefruits, and 4.4 percent of other fresh citrus were fumigated with EDB. However, California is atypical of most other states in two respects: first, in receiving a disproportionate share of Mexican fruits, which are all fumigated with EDB, and second, in requiring fumigation of incoming fresh citrus from Florida (U.S. EPA 1983a). Envirologic Data, therefore, has no basis to reject the EPA's 2-percent estimate of fresh citrus treated with EDB.

~32-

Table 11. Estimated Annual EDB Body Burdens from Major U.S. Sources

Source Adult Adult Child Infant man woman of II yrs of T year (70 kg) (59 kg) (32 kg) (7.4 kg) ug/yr percent ug/yr percent ug/yr percent _ug/yr percent Urban Air? = 1680 Pie 1540 78.6 1100 82.9 280 88.1 to 74.2 Grains 483 22.2 407.1 20.8 220.8 16.6 Sz TTT fo 26.1 to 94.7

Citrus Y2e3" O28 10.3 0.5 5.6 0.4 2.5 0.7 to 0.8 TOTAL 2175.3 1957.4 1326.4 317.7 to 377.2

4 assuming mean ambient air concentration of 0.026 ppb

Comparison with Other EDB Risk Assessments

Envirologic Data has examined EDB risk assessments by a variety of sponsors (Table 12), including federal and state agencies, as well as industry. As the table indicates, the focus of these assessments was typically narrower than that adopted by Envirologic Data, that is, exposures were estimated for only one or two, but not all three of the major EDB sources*. In some assessments, the focus was on occupational rather than general population exposure. The most frequently neglected source of general population exposure was ambient air ae comprising the predominant source of exposure according to Envirologic Data's calculations (Table 11).

Based upon consideration of all three major sources of EDB, each quantified by Envirologic Data, it was possible to calculate total EOB body burden from inhalation plus ingestion. From this total burden, it was also possible for Envirologic Data to determine the relative importance (percent of total body burden) of each major source contribution (see Table 6). This information may be valuable to the regulatory community, which may be required to assess incremental EDB risks as a fraction of total existing risk from all sources. This information may be valuable in selecting priorities for optimizing risk management; that is, the information can be used in formulating policies that will identify and reduce major sources of risk at a limited regulatory cost.

*In addition, a frequently encountered practice was to focus risk assessment on exposure levels corresponding to proposed regulatory options, rather than on observed residue levels of EDB.

**In some risk assessments, ambient air levels reported, but exposure was not quantified via a consideration of respiratory rates and volumes of exposed populations.

a

Table 12, EDB Risk Assessments: The Exposure Component

Risk Exposure (micrograms per year)

Assessment

Source Air Citrus Grain ___ Total Exposed Population

US EPA, 19844 -- -- 431 -- Adult -- - 69 -- Child

US EPA, 1984f -- 12.3* -- -- Adult male (70 kg) -- 10.3* -~ -- Adult female (59 kg) -- 5.6* = es Child (32 kg) = 2.5* = Infant (7.4 kg)

US EPA, 1980a -- 278,5* 88.7* = Adult male (70 kg) -- 234.7* 74,1* = Adult female (59 kg) -- 127,3* 40,.5* Child (32 kg)

US EPA, 1983a -- 10.5* 2192.2* -- Adult male (70 kg) -- 8.8* 1847.7* -- Adult female (59 kg) _ 4.8* 1002.1* = Child - (32 kg)

NYS DOH, 1984 1511 -- -- -- Adult (70 kg)

(Kim, 1984) 606 = -- == Child

Envirologic Data 1680 12.3 483 2175.3 Adult male (70 kg)

1984 1540 «10.3 407.1 1957.4 Adult female (59 kg) 1100 5.6 220.8 1326.4 Child (32 kg) 280 2.5 35.2-94.7 317.7- = Infant ( 7.4 kg)

377.2

Grocery Mfrs. of -- -- Adult

America, 1984 -- -- Child

Dynamac Corp., 1983 -- 84.3* -- -- Adult (70 kg) -- 38.5* -- -- Child (32 kg)

Brown, 1983 ms -- -- -- [Occupational]

Winzler and Kelly -- -- -- --

Ramsey et al., 1979 -- -- -- -- [Occupational]

SRI, 1982 -- -- -- -- [Occupational]

California DHS, 1984 -- -- -- -

CAG, 1980 -- =176,3* 86.4* = Adult male {70 kg) -- 148.6* 72.8* -- Adult female (59 kg)

80.6* 35.5* os Child (32 kg)

-- —-:18.6* 9.1% = Infant ( 7.4 kg)

CDC, 1984 ne -- -- =

“Asterisk indicates that, although author(s) did not report the value that

appears in this table, Envirologic Data was able to convert authors' units to standardized exposure units.

“gq

ENVIRONMENTAL FATE, PERSISTENCE AND EFFECTS Summary

EDB has appeared in surface and/or ground waters in several states and, therefore, may pose the threat of human exposure in drinking water.

The agricultural and urban use patterns cf EDB are likely to restrict its potential for drinking water contamination, and limit its introduction into natural ecosystems. Moreover, Envirologic Data has found no reports of EDB properties which suggest that it would bioaccumulate in food chains of natural ecosystems, if introduced. Based upon the above facts, EDB's human health effects, rather than its environmental effects exerted through food chain exposure, are of most concern. Although atmospheric transport of ED8 into natural ecosystems may occur, concentrations would be much lower than in urban environments. EDB, though it may decompose into free bromine in the stratosphere, is not regarded as a significant threat to ozone levels because other ozone-decomposing substances, such as chlorinated compounds, are released in vastly greater quantities.

Air

Very little information is available on the environmental fate of EDB in air. Its possible degradation products include vinyl bromide and bromoacetaldehyde (Brown et al., 1975), the former of which is a hazardous substance. According to one source (Winzler and Kelly, 1982), EDB is moderately resistant to degradation in the troposphere, and is more likely to enter the stratosphere, where it may be subject to intense ultraviolet irradiation and oxidative conditions, leading to the formation of free bromine atoms. These atoms may then react with ozone and decompose it. EDB is released into the atmosphere in smal] amounts compared to chlorinated compounds, so its effects on stratospheric ozone are probably comparatively minor.

Aquatic Environments

The environmental fate of EDB in water is largely unknown and the information available is conflicting. One review reported that the rate of hydrolysis was very slow, the half-life ranging from 274 to 3 x 105 years, the breakdown products being glycol and bromoethanol (Brown et al., 1975). Another review of the same data, however, reported’ @ hydrolysis half-life of 5 to 10 days, the reaction being favored by acidic conditions (Johns, 1976). EPA recently began a hydrolysis study (Jungclauss et al., in progress), and preliminary indications are that EDB's half-life at 20 degrees Celsius is greater than one year under neutral conditions and may be as long as 24 years. Because of the large discrepancy of results obtained on EDB's half-life, it is not possible to draw firm conclusions regarding the extent of EDB's persistence in water. Although data are Jacking regarding the propensity for EDB to bioaccumulate or biodegrade in aquatic environments, there is evidence that it is stable in water long enough to contaminate water supplies.

As of September, 1983, EDB had been found in water from wells in at

least five states. EDB's use as a soil fumigant was considered a likely cause of the contamination.

-35-

In 1980, the EPA helped design and sponsor a ground water contamination study by the California Department of Food and Agriculture. Two wells out of over two hundred sampled were contaminated with 0.1 ppb and 0.2 ppb EDB (Zalkin, et al., in progress). By the spring of 1983 about 18 wells sampled in five California counties contained amounts of EDB ranging from 0.1 to 2.0 ppb (U.S. EPA, 1983a).

Two areas of Florida have been sampled since mid-1983, and both contain ground water contaminated with EDB. Those areas are an eight county citrus growing region in the central part of the state, and a soybeans/peanuts area in the panhandle. Eighty-six out of 334 wells sampled contain detectable levels (>20 parts per trillion); 75 of these 86 positives contain EDB concentrations greater than the level 0.1 ppb (Bigler, 1983).

In June, 1982, EPA scientists were notified by EDB registrants in Georgia that three wells in one county were contaminated with EDB at levels as high as 100 ppb. Since further monitoring was not conducted by the registrants, the EPA and the United States Geological Survey (USGS) initiated and completed a reconnaisance study of the area in late summer, 1983, in cooperation with the county extension agent (U.S. EPA, 1983a). Nineteen wells and one surface water sample were analyzed, and six wells were found to be contaminated with between D.03 and 11.8 ppb EDB.

One hundred wells in Hawaii were analyzed for EDB in the summer of 1983 (U.S. EPA, 1983a). Four wells near pineapple fields sprayed with EDB contained EDB levels ranging from 0.02 to 0.10 ppb.

One study compared the frequency of ground water contamination by EDB to surface water contamination by EDB in New Jersey (Page, 1981). Samples of ground water were collected from 421 wells throughout the state, and samples of surface water were collected from more than 175 sites. Thirty-four ground water samples and 11 surface water samples had detectable levels of EDB, the highest concentration being 48.8 ppb.

Terrestrial Environments

Two studies have indicated that EDB can degrade in soil. Castro and Belser (1968) reported that EDB was degraded to ethylene and bromide ions in a California agricultural soil. Even in the same soil, however, the half-lives in different experiments varied from two weeks to eighteen weeks. Mink (1981) detected high levels of bromide jons in the perch water aquifer under the site of a several hundred-gallon EDB spill in

1977 in Oahu, indicating breakdown of EDB to an unknown extent in a three-year period.

Diffusion of EDB in soil depends upon several variables, including temperature, bulk density, and moisture level. One study looked at the effects of these three factors on diffusion of EDB in three soil types (Townshend, et al., 1980). In the two coarser-textured soils (fox loamy sand and vineland silt loam) diffusion was favored by high moisture, high temperature, and soil compaction, whereas almost no diffusion occurred in Lincoln clay, indicating no association with these factors.

-36-

PHARMACOKINETICS

Summary

A critical variable for quantifying EDB exposure risk is the efficiency with which EDB enters the human body through its primary routes: inhalation and ingestion. At present, inhalation exposure accounts for between 74 and 88+ percent of total exposure. Absorption of inhaled EDB appears to be about as efficient as absorption through the dietary route which approaches 80 percent. Consequently, the effective dose of EDB over any given time period, say one year, depends not only upon the applied dose, but upon the dose that actually is absorbed.

Inhalation absorption efficiency has apparently not been measured, and can only be estimated. Envirologic Data estimated that the absorption efficiency of most inhaled EDB is about 80 percent of that for chloroform, based upon their relative water solubilities. This is about the same as the ingestion absorption efficiency given above. However, it is likely that some inhaled EDB is absorbed with lower efficiency than 80 percent because it may remain in the tracheal and bronchial tubes rather than proceed to the alveoli, where it is in more intimate contact with absorbing membranes.

Once in the body, EDB is selectively distributed to the liver and kidneys, where it exerts much of its effect (see "Health Effects--Non-Human" and “Health Effects--Human" sections), and where it jis primarily metabolized. Metabolism is by two routes: oxidation by cytochrome enzymes, and conjugation with glutathione. The ratio of oxidative to conjugative metabolism is about 4:1. Although the metabolic intermediates of these routes differ, both routes ultimately produce the same mercapturic acid derivative, which is eliminated in the urine. EDB has a probable half-life in the body of less than a day.

Absorption

EDB may enter the body via inhalation, ingestion or dermal absorption. When introduced by the oral route, absorption is rapid. Kirby et al. (1980) found that in minipigs there was a serum level of 1 ppm (0.1 percent of dose) 15 minutes after oral administration of 56 mg/kg of body weight. As much as 8&0 percent of the orally administered dose is absorbed. Jones and Edwards, in a 1968 study (cited in CAG, 1977) found that only 20 percent of the orally administered dose was eliminated unchanged in expired air. In another study, Plotnick et al. (1979) found levels of approximately 73 percent of the administered dose present in the urine of rats given a single oral dose of EDB.

When absorption was due to continuous dermal exposure, the maximum EDB concentration in the blood occurred at approximately 1 hour in guinea pigs (Jakobson, 1982). After that period, the concentration of EOB in the blood remained essentially constant, indicating that the rate of influx into the blood was essentially at equilibrium with the rate of

efflux. There is no information available regarding the percent of dose that is absorbed into the body after dermal exposure.

ate

Envirologic Data attempted to find studies which indicate the percentage of inhaled EDB which is actually absorbed by the lungs. There were no studies found in either primary or secondary sources. According to Dr. Walter Weigel of NIOSH there have been no studies undertaken to address this question. Envirologic Data felt that this was an important aspect of the toxicology of EDB, and consequently decided to attempt to estimate the absorption of EDB by the lungs based upon a comparison with the known inhalation absorption efficiency of another substance.

The absorption of a soluble gas in the lungs across the alveolar capillary membranes into the blood depends primarily upon the gas solubility in blood (Doul] et al., 1980), which may be approximated by its solubility in water (Waldbott, 1978). The solubility of EDB is approximately 1 part per 250 parts of water (Merck, 1983). The solubility of chloroform is 1 part per 200 parts of water (Merck, 1983). Chloroform is a somewhat more polar alkyl halide and is absorbed readily into the lungs. Essentially 100 percent of the chloroform entering the lungs is dissolved in the alveolar fluid and moves into the bloodstream (Doull et al., 1980). Since the solubility of EDB is 80 percent of the solubility of chloroform, Envirologic Data made the assumption that about

80 percent of the EDB in the alveoli of the lungs is dissolved in the alveolar fluid. Because the alveolar capillary blood flow would remove the EDB absorbed into the alveoli and carry it away, the EDB concentration in the blood would always be lower than that in the alveolar membranes. Alveolar capillary blood flow would, therefore, maintain a concentration gradient across the alveolar membranes which would draw essentially 100 percent of the dissolved EDB into the bloodstream. Therefore, we have estimated that 80 percent of the dose received jin the alveoli crosses the alveolar membranes and enters the body. It is likely, however, that a lower absorption efficiency would apply to inhaled air which remains in the tracheal or bronchial tubes.

It is difficult to estimate the absorption efficiency of EDB in tracheal or bronchial tubes, which would be diffusion-limited rather than inhalation-rate-limited because of the less intimate contact of the EDB with these tubes than with the alveoli (Doull et al., 1980). According to Doull et al. (1980), the nasopharyngeal cavity can readily remove at least 50 percent of inhaled toxicants, including organic vapors. However, not all inhaled air enters the nose; mouth-breathing would by-pass nasopharyngeal absorption. About 70 percent of total ventilation in the Jungs is represented by alveolar ventilation. Thus, the uncertainties associated with the absorption efficiency of EDB in the upper airways does not appear to represent a major uncertainty in estimating overall inhalation efficiency.

Distribution and Retention

Once in the body, EDB jis rapidly distributed and selectively concentrated in certain tissues, especially the liver and kidney. Table 13 presents the distribution of EDB at 4 hours and 24 hours after the administration of 30 mg/kg of body weight via intraperitoneal injection into male guinea pigs (Plotnick and Conner, 1976).

*Personal communication, April 4, 1984.

-38-

Table 13. Distribution of EDB in Male Guinea Pigs

4 hours: 24 hours

distribution percent of distribution percent of

in tissues mean dose* jin tissues mean dose

Organ (mg/kg SEM**) (mean SEM) (mg/kg SEM) {mean SEM) kidney 236.6448.74 6.00#0.42 73.454#14.06 1.64#0,45 liver 129.03#14.94 16.2942.42 38.0743.47 4.7240.21 adrenals 60.75#10.74 0.0940.02 28.6343 .68 0.03#0.01 pancreas 35.0445 84 0,314#0.10 18.68+1.09 0.20#0.03 spleen 15.79#2.70 0.0740.01 14.89402.5 0.08+0.02 heart 14.01#2.53 0.1340 .02 9,51#0.40 0.104#0.01 Tung 20.9342.71 0.3540.06 15.43#1.58 0.2440.01 testes 10,.68#1.98 0,16#0.04 8.3440.05 0.12#0.01 brain 6.19#1.28 0.12#0.02 6.47#0.39 0.13+0.01 fat 21.394#4.09 not given 3.19#0.55 not given muscle 5.53#1.09 not given 4,59#0.20 not given stomach not given 1.14#0.44 not given 0.38#0.07

*Intraperi toneal injection of 30 mg/kg b.w. SEM = Standard Error of the Mean

Of the administered dose, 14.9#1.0 percent was excreted in the urine within 4 hours and 46.044.8 percent of the administered dose was excreted in the urine within 24 hours. Thus, the half-life of EDB in guinea pigs is approximately 24 hours (Brown, 1983). In another study, mice given intraperitoneal injections of 40 mg/kg retained EDB in selected tissues as shown in Table 14 (Edwards, et al., 1970).

Although distribution of EDB in the body is widespread, EDB is bioconcentrated in those tissues where it is primarily metabolized and

excreted, particularly in the liver and kidneys. A summary of the data is given in Table 15.

=39=

Table 14. Distribution of EDB in Mice

Percent of Dose*

Organ 1 hour 3 hours 24 hours small intestine 34.0 5.8 0.39 kidney 13.0 12.0 1.0 liver 12.0 6.6 0.42 Tung 0.9 1.0 0.14 spleen 4.1 4.7 0.61 plasma 12.0 12.0 2.6

*Intraperitoneal injection of 40 mg/kg b.w.

Metabolism

There are several pathways of EDB metabolism in animals. The primary route involves microsomal, enzyme-mediated oxidation by the P-450 cytochrome system; a secondary route is direct conjugation with glutathione, The ratio of oxidative to conjugative metabolism is approximately 4:1 (van Bladeren et al., 1981a). Both transformations take place principally in the liver. Both pathways yield the mercapturic acid derivative N-acetyl-S-2-hydroxyethyl-L-cysteine as the final metabolic product (Nachtomi et al., 1966; Nachtomi et al., 1968; van Bladeren et al., 1981b). However, the intermediate metabolites differ for the two pathways. A summary of data is given in Table 15. The probable metabolic pathways of EOB are given in Figure 1 (CAC, 1977; Edwards et al., 1970; van Bladeren et al., 1980; van Bladeren et al., 1981a; van Bladeren et al., 1981b).

The enzymatic conjugation of EDB with glutathione requires metabolic activation (that is, enhancement by metabolites rather than direct action by EDB) (Kluwe and Smithson, 1981). The activated metabolite produced by conjugation is S-2-bromoethy] glutathione. This intermediate rearranges to a thiiran ion by intramolecular attack of the sulphur ion on the carbon bearing bromine (Edwards et al., 1970). The thiiran ion then reacts with water, producing $-(B-hydroxyethy])glutathione. This undergoes enzymatic breakdown by peptidase and glutathione, forming S-(B-hydroxyethyl)cysteine. This undergoes acetylation, yielding the major urinary product, S-(B-hydroxyethy!) mercapturic acid sulfoxide Tagikt elimination in the urine (Nachtomi, 1970; van Bladeren et al., 1981b). an

-40-

Microsomal oxidation of EDB in the liver by the P-450 cytochrome system produces a second activated metabolite, bromoacetaldehyde. This

metabolite covalently binds to cell macromolecules. In addition, conjugation of bromoacetaldehyde with glutathione is followed by enzymatic reduction of the aldehyde moiety of the conjugate, leading to S-(B-hydroxyethy]) glutathione, that is, to the same metabolic intermediate as that produced by the conjugative pathway. Again, this undergoes further metabolism to the urinary products, as described above and depicted in Figure 1 (Nachtomi, 1970; van Bladeren et al., 1981b). Both activated metabolites, S-2-bromoethy1 glutathione and bromoacetaldehyde, bind to microsomal proteins and polynucleotides. However, the conjugative metabolite (S-2-bromoethy]l glutathione) binds primarily to polynucleotides, and only weakly to microsomal proteins (Shih and Hill, 1981) (see "Mechanism of Action" section, under "Carcinogenicity"). In contrast, the oxidative metabolite (bromoacetaldehyde) binds primarily to microsomal proteins (Banjeree, 1979b), and only weakly to polynucleotides.

EDB is also transformed to ethylene by a minor metabolic pathway in rats. This transformation is highly dependent upon the presence of reduced glutathione (GSH), and probably involves the enzyme GSH S-transferase, although the exact mechanism is not known (Anders and Livesey, 1980).

Excretion

The kidney is the major excretory organ for EDB with 80 percent of the dose in rats (Watanabe et al., 1978) and 80 percent of the dose in mice (Brown, 1983) being recovered within 24 hours. All EDB metabolites excreted in the urine are apparently derived from glutathione conjugates which are formed by both the conjugative and oxidative metabolic pathways (Watanabe, 1978). The half-life of EDB at doses ranging from 7 to 75 mg/kg in rats was 5.1 to 5.6 hours.

Other routes of excretion include 20 percent of dose in expired air (CAG, 1977), less than 3 percent in feces (CAG, 1977; Plotnick and Conner, 1976; Plotnick et al., 1980) and a detectable biliary excretion (CAG, 1977). No unchanged EDB jis eliminated via any route (Brown, 1983). Table 15 provides a summary of the data on excretion of EDB.

Rig

BrCHoCHoBr

(EDB) OXIDATION CONJUGATION by P450 (glutathione cytochromes (in liver) (in liver) S-transferase) s+ &- BrCHoCHO GSCH2CHpBr + ~—s HBr (bromoacetal dehyde) (S-2-bromoethylglutathi one) CONJUGATION (glutathione H9 with §-transferase) glutathione GS —— CH

a 5 + | Bre GSH | GSCH2CHO | HBr + ——> 6556 + Coz

CHp GSH (transient (thiiran ion) Rag intermediate) GSCHgCH2SG H20 (S-S'-ethylene- , bis-glutathione) GSCHgCH20H + HBr [S-(B-hydroxyethy] )glutathi one]

(glutathionase peptidase)

CHgCH OH

|

S~CHpCHCOOH

NHo ([S-(B-hydroxyethy] )cysteine] | (acetylase) CHpCH20H CHCHy0H (S-oxidase) | | > S-CHgcHcooH S-CH2CHCOOH HNCOCH HNCOCH3 [S-(B-hydroxyethy1) [S-(B-hydroxyethy] )mercapturic acid) mercapturic acid sulfoxide]

URINE ELIMINATION

Figure 1. Probable Metabolic Pathways of EDB

-42-

Table 15. Summary of Data on Pharmacokinetics of EDB

Dose Test Animal Route Reference or Species Duration Results ABSORPTION Jakobson et.al., 1982 guinea pigs 1 ml/skin depot Maxiumum level obtained in blood within 1 = conc. not given hour 4-12 hours Kirby et al., 1980 minipigs 55.6 mg/kg Serum level reached 1 ppm (0.1 percent of — intravenous dose) 15 minutes after administration of 15 minutes dose. DISTRIBUTION & RETENTION Brown, 1983, not given not given half-life approximately 24 hours Edwards et al., 1970 mice 40 mg/kg IP. Values for retention in selected tissues 1 - 3 hours given between 1 and 24 hours. Plotnick, 1976 guinea pigs 30 mg/kg IP. Highest concentrations found in the liver, 4 - 72 hours kidney and adrenals METABOLISM CAG, 1977 not given not given readily metabolized, pathway diagram Anders and Livesey, 1980 rats 14.1 mg/ml Biotransformation of EDB to ethylene. Banerjee et al, 1979b mouse liver 0.047 mg, total EDB undergoes metabolic activation =~ microsomes vol. not given Bromoacetaldehyde an activated intermediate NADPH & microsomes Edwards et al., 1970 rats § @ 10 mg/ kg EDB is rapidly metabolized. It removes ‘ subcutaneous sulphur from cysteine and produces a 2.5 hours a monofunctional mustard. mice 40 mg/kg IP. 1 - 3 hours Hill et al., 1978 rats 2.63 - 3.16 mg/kg EDB a substrate for glutathione natal IP. 24 hours s-transferase. Binding to macromolecules occurs, particu*arly in the liver and kidney. Kirby et al., 1980 minipigs 0.056-55.6 mg/kg EDB rapidly metabolized. Direct injection

et al. 190

orally or IV. 1, 4, 14 or 63 days

bypasses the liver and normal metabolic transformation is avoided.

{continued on following page)

Table 15. (continued)

Reference

Kluwe and Smithson, 1981

Nachtomi et al., 1968

Nachtomi E.,

et al. 1968

Nachtomi, 1970

Shih and Hill, 1981

van Bladeren et al., 1980

van Bladeren et al., 198la

van Bladeren et al., 1981b

CAG, 1977

Brown, 1983

Dose Test Animal Route or Species Duration Results mice dose not given EDB conjugation with GSH requires metabolic rats IP, 2 hours activation. A microsomal pathway also rats 100 mg/kg products in urine were S-B-hydroxyethy] stomach tube mercapturic acid (major metabolite) and 24 hours S-B-hydroxyethyl-L-cysteine. Ratio not

rats & chicks

rats

rat hepatocytes

rats

rats

rats

not given

not given

110 mg/kg stomach tube up to 20 hours

12.0 mg/kg stomach tube 4 hours

pretreatment with phenobarbital.

967.6 mg (total vol. not given), 30 min.

9.4-329.0 mg/kg orally, 24 hours

18.79 mg/ animal wel ane not given orally and IP.

duration omitted

47-282 mg/kg

stomach, with or

without disulfiram EXCRETION

not given

not given

given

liver weight, adrenal weight, alkaline phophatase and free sulfhydryl] groups ali increased. Liver NADP and ascorbic acid decreased.

In vivo, EDB goes to S-B-hydroxyethy1- ‘glutathione mostly in the liver and then degrades later to S-B-hydroxyethy] mercapturic acid and its sulphoxide in the kidney

Glutathione conjugate binds to polynucleotides. 2-bromoacetaldehyde binds to microsomal proteins.

Oxidative and conjugative pathways lead to same final product N-acety!-S-2-hydroxyethyl-L-cysteine.

EDB is oxidized to bromoacetal dehyde and conjugated to

S-2-bromoethyl glutathione in the ratio of 4:1.

oxidation and conjugation give rise to same

final product, N-acetyl -S-2-hydroxyethylL-cysteine.

half life in animals, 1 day. Primarily in urine, also in air and feces

84 percent excreted in 24 hours. None unchanged.

(continued on following page)

Table 15. (continued)

Dose Test Animal Route Reference or Species Duration Results

dakobson et al., 1982

Plotnick, 1976

Plotnick et al., 1980

Watanabe et al., 1978

guinea pigs

guinea pigs

rats

rats

1 ml/skin depot conc. not given

4 - 12 hours 30 mg/kg IP. 4 - 72 hours

15 mg/kg, oral w/ or w/o disulfiram. 24 hours

7 - 75 ppm for

6 hours inhalation evaluated for

48 hours

influx of EDB into blood after 1 hour is largely at equilibrium with the elimination.

excretion principally via the kidneys.

Fecal excretion approximately 1.7 percent 73.5 percent of dose excreted in urine within 48 hours.

Major route of excretion is urinary with metabolites derived from GSH conjugation. Half-life at all doses, 5.1-5.6 hours.

HEALTH EFFECTS -- NON-HUMAN

Summary

The effects of EDB in test organisms are significant as indicators of potential health effects on humans. Envirologic Data has examined health effects of EDB arising from the three major exposure regimens: acute (short-term), subchronic (intermediate-term), and chronic (long-term). Any of these exposure regimens might, for a particular substance, result in mutations, cancer, or abnormalities of fetal development. These three effect categories are discussed separately under the titles of mutagenicity, carcinogenicity, and teratogenicity/reproductive effects, respectively.

Acute Toxicity. Single exposures over a period of about a day are termed “acute. tbe has been shown to be lethal to a variety of test

organisms. LOsg's (doses lethal to 50 percent of the individuals in a sample of the exposed species) varied from 22 mg of EDB per kilogram of body weight (in mice) to 300 mg/kg (in rats and rabbits), with intermediate doses for guinea pigs and chickens. Other reported acute effects include loss of body weight, elevations of liver and kidney weights, alterations of enzyme levels, dermal effects, impaired renal function, stiffness, anorexia, prostration, karyopyknosis (cell nucleus shrinkage), junctional (skin cell) separation, dyspnea, diarrhea, lethargy, and asthenia. The doses causing these acute effects were much greater than doses to which the general human population is exposed by all routes combined. Acute effects of EDB are, therefore, of minor significance for the general human population in the U.S.

Subchronic and chronic toxicity. Repeated exposure over a period of up to three months 1s termed subchronic,’ whereas exposure over an 18- to 24-month period is termed 'chronic.' Few studies of subchronic and chronic effects have been performed; the studies have involved mice, rats, chickens, and quail. The effects observed include the following: liver cell necrosis and degeneration, reduced liver weights, elevated plasma enzyme levels, loss of body weight, hyperkeratosis, acanthosis, spleen atrophy, hemosiderosis, retinal degeneration, and epithelial hyperplasia of the respiratory system. As with acute effects, subchronic and chronic effects have been reported at levels far exceeding those to which the general U.S. population is exposed from all sources combined.

Teratogenicity and reproductive effects. There are apparently only four reports in the Titerature of teratogenicity studies using EDB. The results of three were negative. The last revealed increased incidence of hydrocephaly and rib abnormalities in fetuses of rats exposed to EDB. However, without further study, no firm conclusion can be made regarding EDB teratogenicity. Several studies have been performed regarding the effect of EDB on reproduction in bulls, rams, chickens, and rats. In chickens, EDB reduced egg size and fertility. In rats, EDB prevented males from impregnating females, caused atrophy of the testes, epididymis, prostate, and seminal vesicles; females exposed to EDB became sexually inactive and exhibited abnormal menstrual cycles. Bulls exposed to EDB exhibited deformed sperm, reduced numbers of sperm, inhibition of spermatogenesis, and inhibition of sperm maturation.

Lag...

Mutagenicity. Extensive research regarding EDB mutagenicity has been reported. Although scattered studies produced negative results, the

preponderance of evidence clearly indicates that EDB is mutagenic to test species among the bacteria, fungi, vascular plants, insects, and mammals. Experiments on laboratory cultured mammalian cells have produced positive results in all nine in vitro investigations. However, several (five) investigations of whole mammals were negative for mutagenicity, although three were positive for DNA damage. The negative results in mammalian test organisms do not cast doubt on the conclusion that EDB is a mutagen in non-human systems; moreover, the positive data on non-human systems are relevant to the possibility of EDB mutagenicity in humans (see "Health Effects--Human") .

Carcinogenicity. Carcinogenicity of EDB was reported in several investigations on mice and rats. Results were positive for both males and females, for all routes of EDB exposure (gavage, dermal, oral, inhalation), and for several strains of both test organisms. Numerous malignancies were observed, some at a distance from the site of EDB application. Reported tumor types include the following: papillomas of lungs and skin; squamous cell carcinoma of skin and forestomach; alveolar and bronchiolar adenoma; hemangiosarcoma of the spleen, liver, and mesentery; mammary gland adenocarcinoma; nasal cavity tumors, subcutaneous fibrosarcoma of the skin; hepatocellular tumors; kidney adenoma; adrenal tumors; and thyroid tumors. Some studies attempted to determine the shape of the dose-response curve, but were compromised by high-dose mortality due to acute toxicity of EDB. Extrapolation of carcinogenicity results to low doses is, therefore, difficult based upon the available data.

Acute Toxicity

Acute toxicity may be defined as il] effects resulting from either a single exposure or multiple exposures occurring within 24 hours (Doull et al., 1980). On an escalating toxicity scale of 1 to 6 presented in Clinical Toxicology of Commercial Products (CTCP; Gosselin et al., 1976), EDB is rated a 4, where 4 is “very toxic,” with the probable oral lethal dose in humans ranging from 50 to 500 mg/kg of body weight. A rating of 1 is "practically nontoxic," (probable oral lethal dose in humans being above 15 g/kg) and 6 is "super toxic," (probable oral lethal dose jin humans being less than 5 mg/kg). The rating is based upon mortality rather than morbidity. In addition, the rating is based on the acute toxicity of a single dose when taken by mouth or gavage (forced feeding through a tube passed into the stomach), and does not address toxicity due to other routes of administration, such as inhalation and dermal. Inasmuch as adequate data to establish a toxicity rating for humans exists for relatively few substances, most lethal dose values are based upon laboratory determinations made in laboratory animals.

In addition to the CTCP rating method, another measure of acute toxicity is the LDgg, the lethal dose of a compound that kills half of the animals in the test sample. LDS values for EDB have been established for several species, and are listed in Table 16.

“47x

Table 16.

Acute Toxicity of EDB in Animals

Route of Administration Species LD, (mg/kg) Oral Rat 108° Dermal Rat 3007 Dermal Rabbit 300° IP Mouse 208 Not reported Mouse 146° Oral Rat (male) 146P>¢ Oral Rat (female) 117>2¢ Oral Mouse (female) 420P2¢ Oral Rabbit (female) gaPac Oral Chicken 79° 9¢ Oral Guinea Pig 110P2¢

asource: NIOSH, 1983b

b

Source: Rowe et al., 1952

converted from g/kg to mg/kg

-48-

Laboratory studies of the acute toxicity of EDB have been conducted jin numerous species, including rats, guinea pigs, rabbits, sheep, calves, chickens, mice, and quail. Details of the studies are provided in Table 17. EDB has been shown to be toxic by most routes of administration (see Table 16); its acute toxicity in animals is manifested by loss of body weight, elevations in liver and kidney weights, depression of the central nervous system, alterations in tissue enzyme levels, and injury to skin cells. Kluwe et al. (1981) observed impairment of renal function in male but not female rats, and concluded that potential sex differences in EDB biotransformation may influence EDB nephrotoxicity.

Schlinke (1969) investigated the acute toxicity of EDB in livestock, and administered single injections of EDB to sheep and calves. At the higher doses, 25 and 50 mg/kg, one out of four and two out of three animals, respectively, died within 3 days, after exhibiting stiffness, anorexia and prostration.

Storer and Conolly (1983) investigated the acute hepatotoxicity and nephrotoxicity of EDB in male mice, and found statistically significant (p<0.05) elevations in liver and kidney weights, serum L-iditol dehydrogenase (IDH) and alanine aminotransferase (AAT) levels, and blood urea nitrogen (BUN) levels at a dose of 1.0 mmol/kg (188 ppm).

Kronevi et al. (1981) investigated the morphological skin changes induced by cutaneous exposure of guinea pigs to EDB, and found that karyopyknosis (cell nucleus shrinkage) and junctional (skin cell) separation progressed with exposure time, indicating that possibly irreversible cellular injury had occurred.

Two studies examined the acute effects of orally administered EDB on birds. Schlinke (1970) treated chickens with different levels of EDB, and observed depression and anorexia in EDB-treated birds. Chickens that died exhibited inflamed crop, excess pericardial fluid, and liver.

Westlake et al. (1981) examined EDB's acute and subacute effects on Japanese quail. Dyspnea, diarrhea, fluffed feathers, lethargy, asthenia, and loss of body weight were symptoms that appeared after 6 hours, and death occurred between 7 and 72 hours later. Quail which survived lost the physiological symptoms of EDB poisoning and returned to normal body weight 3 days after the cessation of treatments.

Rowe et al. (1952) investigated the acute and subacute toxicity of EDB in a series of experiments in rats, guinea pigs, rabbits, mice and chickens. Animals were exposed by several routes of administration - oral, eye contact, dermal absorption, and single and _ repeated inhalation. The results of the oral experiments , expressed as LDs5q's, are summarized in Table 16. Results of the other experiments are discussed below.

When undiluted EDB was introduced into the eyes of rabbits, and flushed out 3D seconds later, it caused obvious pain and conjunctival irritation, which cleared completely within 48 hours (Rowe et al., 1952). When a 10 percent solution of ED8 in propylene glycol was tested, it produced a similar but more severe response than did the undiluted material. Corneal injury was moderate to severe. Healing appeared to be complete 12 days after exposure, without corneal scarring.

-49-

Table 17. Acute Effects of EDB

Concentration Route of Number of and Observed Administration Species Animals Duration of Dose Effects Reference Intraperitoneal (IP) Rat not given single injection PAH” accumulation was reduced Kluwe, et al., 40 mg/kg at 96 hours post treatment in ns" males not given 4 injections (twice PAH accumulation was reduced a day for 2 days) in males (p 0.05) Dermal Guinea not given 1 m1 (absolute dose) Karyopyknosis and junctional Kronevi, et pig for 0.25, 1, 4, and separation progressed with al., 1981 16 hours exposure time =e Inhalation Rat 10 females 100 ppm, 7 hr/d; weight loss, increased weight of Rowe, et al., 7 exposures in 9 kidneys, lungs, and liver, cloudy 1952 days swelling of liver and congestion

of spleen, lung irritation, blood in stomach, 3 deaths

Inhalation Rat 23 females 25 ppm, 7 hr/d; no adverse effects Ibid. 13 exposures in 17 d Inhalation Guinea 7 females 25 ppm, 7 hr/d; decreased weight gain Ibid. pig 13 exposures in 17 days Inhalation Rabbit 4 females 100 ppm, 7 hr/d, fatty degeneration of liver, 3 Ibid. 2-4 days deaths Dermal Rabbit 30 210-1106 mg/kg CNS depression, erythema, edema, Ibid. for 24 hours and necrosis of skin, 12 deaths IP Sheep 4 sheep single injections of stiffness, prostration, and Schlinke, 1964 Cattle 3 calves 10, 25 and 50 mg/kg anorexia in one calf dosed at

50 mg/kg, and in two sheep at 50 and 25 mg/kg. Death within

3 days. Oral (capsule) Chicken 15 treated 50, 100 and 200 at 200 mg/kg, death occurred, Schlinke, 1970 18-24 mg/kg daily for 10 Crop inflammation, excess controls days pericardial fluid and liver

congestion were evident

(continued on following page)

Table 17. (continued)

Concentration Route of Number of and Observed

Administration Species Animals Duration of Dose Effects Reference

1p Mouse not given 0.5, 0.75, 1.0 and at 1.0 mmol/kg, elevations in Storer and 1.4 mmol/kg (94, liver and kidney weights, serum Conolly, 1983 141, 188 and IDH and AAT, and BUN. 282 mg/kg)

Oral (capsule), IP Quail 25-30 single dose ranging dyspnea, diarrhea, fluffed Westlake, et

from 57 to 272 mg/kg feathers, lethargy, asthenia, al., 1981 loss of body weight

* -ami nohippurate +P Mitol dehydrogenase, alanine aminotransferase, blood urea nitrogen

Rabbits were dermally exposed to four dose levels of EDB for 24 hours and were observed for two weeks (Rowe et al., 1952). At all dose levels erythema, edema, and necrosis of the skin were evident, as well as marked

depression of the central nervous system (CNS). Twelve animals died within four days.

The single inhalation exposures were measured in groups of 4 to 30 rats exposed for varying periods using 8 EDB concentrations ranging from 100 to 10,000 ppm and in guinea pigs exposed to 200 or 400 ppm for durations of 0.02 to 16 hours (Rowe, et al., 1952). Rats were exposed for 9 hours to 200 ppm before 50 percent mortality occurred, whereas exposure to 5,000 ppm resulted in 50 percent mortality at 0.07 hours. Guinea pigs were less susceptible to fatal effects than rats exposed to the same concentrations.

Repeated inhalation exposures were measured in rats, guinea pigs, and rabbits (Rowe, et al., 1952). Weight loss, increased organ weights, lung irritation, and spleen congestion were some of the health effects observed in rats exposed. No adverse effects were noted in guinea pigs, and fatty degeneration was observed in rabbits.

Subchronic and Chronic Toxicity

Several studies have examined the subchronic and chronic toxicity of EOB. A '‘subchronic' study is generally concerned with health effects which develop in the test species during an exposure period of three months, whereas a ‘chronic’ study is concerned with health effects which develop during an 18-month to two-year exposure period. The studies to be discussed are summarized in Table 18, which presents details of sample size, dose, duration of exposure and response. Two studies have investigated the subchronic effects of EOB when administered by gavage to rats and two species of birds. Rats and chickens were administered a single dose of EDB, subgroups were killed at regular intervals following the dosage, and all animals examined for morphological changes in the liver (Broda et al., 1976). Both species exhibited lesions in the liver, but the damage was more pronounced in rats.

Westlake et al. (1981) administered EDB to Japanese quail for either 7 or 21 days, and then sacrificed surviving birds 16 hours after the last exposure. In survivors, the observed health effects included dyspnea, diarrhea, fluffed feathers, lethargy, asthenia, and loss of body weight. These effects occurred after one day and death occured within one to seven days. Quail (as noted in the previous section) which survived the dosing regimen lost the physiological symptoms of EOB poisoning and returned to normal body weights after three days following the cessation of EDB treatments, Liver weights were significantly (p<0.005) elevated after the seven-day treatment, compared to controls. Hepatic total lipid showed a time related decrease, which became statistically significant (p<0.005) at 21 days, and several plasma enzyme activities were also significantly (p<0.05) increased. The authors concluded that chronic doses of EDB exert physiologically significant effects on several test organisms, but that the changes are dose dependent and may be reversible up to the level where hepatic necrosis occurs.

=67-

Several other studies were conducted on rats and mice. A National Cancer Institute (NCI, 1978) study of both rats and mice, in which EDB was adminstered by gavage, revealed that weight loss and skin diseases, such as hyperkeratosis (enlargement of the corneous layer of the skin) and acanthosis (thickening of the prickle-cell layer of the skin) were evident at high dose levels in the two species. Inhalation studies using both rats and mice, revealed a wide range of health effects. Wong et al. (1980) noted spleen atrophy, hemosiderosis (increase in tissue iron stores), and increased liver weight in rats exposed to EDB for 18 months. Nitschke, et al. (1981) observed an increase in liver and kidney weights, a decrease in body weight gain, and hyperplasia of the nasal turbinates in rats exposed to EDB for 13 weeks. The National Toxicology Program (NTP, 1982a) found hepatic necrosis and toxic nephropathy in rats, retinal degeneration in female rats, and epithelial hyperplasia of the respiratory system in mice in a two-year study.

Rowe et al. (1952) investigated the chronic toxicity of EDB in rats, guinea pigs, rabbits, and monkeys, and found that all groups tolerated repeated 7-hour exposures by inhalation to 25 ppm EDB without adverse effects. These same species, however, did not tolerate well repeated exposures to 50 ppm. Health effects included increased weights of kidneys, lungs, and liver, and weight loss. The authors suggested that EDB does not have a high degree of chronicity since the dose which

produced severe acute effects and the dose which produce chronic toxicity were very close for all periods of exposure.

Kluwe et al., (1982) investigated the toxic effects of EDB in rats on renal tubular cells. An intraperitoneal injection of 50 mg/kg caused renal proximal tubular degeneration in both the cortex and the outer medulla of the kidney. In addition, the renal concentration of reduced nonprotein sulfhydryl groups has decreased within 2 hours, but was increased at 24 hours after treatment.

-53-

Table 18.

Subchronic and Chronic Toxicity of EDB

Concentration Route of Number of and Observed Administration Species Animals Duration of Dose Effects Author Gavage Rat 50 single dose of 110 in rats, degeneration of Broda et al., Chicken 15 mg/kg hepatocytes, liver necrosis in 1976 unknown number chickens, small lesions in liver of controls Intraperitoneal Rat not given single dose of 50 renal proximal tubular Kluwe et al., mg/kg degeneration 1982 Gavage Rat 200 treated rats-40 and 80 mg/kg weight loss, hyperkeratosis and NCI, 1978 80 controls up to 49 weeks acanthosis occurred at high Mouse 200 treated mice-60, 100, 120 doses in both species 80 controls and 200 mg/kg Inhalation Rat 180 treated 3, 10 and 40 ppm at 10 and 40 ppm hyperplasia of Nitschke, et 60 controls 8 hr/d 5 d/wk, nasal turbinates; at 40 ppm al., 1981 for 1, 6 and 13 wks increase in liver and kidney weights, body weight loss Inhalation Rat 30 treated 3, 15, and 75 ppm NTP, 1982 10 controls 6 hr/d, 5 d/wk Mouse 60 treated for 13 weeks hepatic necrosis and toxic 20 controls nephropathy in rats, retinal degeneration in female rats, Rat 200 treated 10 and 40 ppm for up epithelial hyperplasia of the 100 controls to 103 weeks respiratory system in mice Mouse 200 treated 100 controls Inhalation Rat 40 50 ppm, 7 hr/d increased weight of kidneys, Rowe et al., 63 exposures in 91 lungs, and liver; decreased 1952 days weight of testes and spleen, 24 deaths (10 male, 4 female) 40 25 ppm, 7 hr/d, 151 exposures in 213 days 13 deaths Inhalation Guinea 16 50 ppm, 7 hr/d, 57 weight loss, decreased growth Ibid pig exposures in ab days rate, congeston and parenchymatous degeneration of kidneys, fatty degeneration of liver 16 25 ppm, 7 hr/d, 145 6 deaths due to pulmonary

exposures in 2D5 days

infection

(continued on following page)

Table 18. (continued)

Concentration Route of Number of and Observed Administration Species Animals Duration of Dose Effects Author Inhalation Rabbit 4 50 ppm, 7 hr/d, small increase of liver and Ibid. 59 exposures in kidney weight 84 days 4 25 ppm, 7 hr/d, no adverse effects 152 exposures in 214 days Inhalation Monkey 2 50 ppm, 7 hr/d increased weight and slight Ibid. a exposures in 70 fatty degeneration of liver jays 2 25 ppm, 7 hr/d, 156 no adverse effects exposures in 220 days Gavage Quail 16 treated uy for 7 or 21 liver weights and plagma enzyme Westlake et 8 controls days (absolute dose) levels were elevated;, hepatic al., 19817 total lipid decreased Inhalation Rat 96 treated 20 ppm, 7 hr/d, spleen atrophy, hemosiderosis,” Wong et al., 96 controls 5 d/wk for 18 trend toward increased liver 1980

months

weight

*statistically different from controls

Teratogenicity and Reproductive Effects

Teratogenicity and Reproductive Effects. Teratogenic effects of EDB, that is, effects on fetal development and offspring behavior, have been investigated in four studies, which are summarized in Table 19,

Short et al. (1976) investigated teratogenic effects in rats and mice. Pregnant animals were exposed by inhalation to 32 ppm of EDB for 23 hours per day from day 6 through 15 of gestation. Primary effects attributed to EDB exposure were an increase in hydrocephaly from fourth-ventricular obstruction, reduction in the occurrence of fourteenth rib, and an increase in the frequency of wavy ribs. Maternal toxicity in rats and mice was also observed, as evidenced by decreased food consumption and decreased weight gain.

A second study was conducted by Short et al. (1978) on the teratogenic effects of EDB in rats and mice. Pregnant animals were exposed by inhalation to 0, 20, 38, or 80 ppm of EDB, 23 hours a day from day 6 to 16 of gestation. Rats and mice were sacrificed on gestational day 20 and 18, respectively, and the uterine contents were examined. Fetal mortality was increased in rats exposed to 80 ppm of EDB and in mice exposed to 38 ppm of EDB, but the increase was not statistically significant. Body weights were reduced in fetuses from rats exposed to 38 ppm of EDB, and were significantly (p<0.05) reduced in mice exposed to 20 and 38 ppm of EDB. Some skeletal anomalies were significantly {p<0.05) increased in mouse fetuses from dams (mothers) exposed to 20 and 38 ppm EDB. EDB also produced adverse effects on maternal welfare, as measured by either weight change or feed consumption, in both species at all of the doses tested. A significant (p<0.05) increase in adult mortality occurred in rats exposed to 80 ppm of EDB and in mice exposed to 38 and 80 ppm of EDB. Fetal toxicity occurred at EDB concentrations that adversely affected the dam (mother), indicating that a causal relationship between the maternal and fetal effects may exist. Alterations in maternal homeostasis, which were associated with EDB exposure, may have produced fetal toxicity independently of a direct action of EDB on the fetus. The authors concluded that the inability to dissociate maternal and fetal effects and the failure to produce anomalies in the absence of developmental toxicity suggests that EDB has little primary effect on development, and is not a teratogen in rats or mice.

Hardin et al. (1981) investigated the teratogenic potential of EDB in rats. Ten to fifteen pregnant rats received an intraperitoneal injection with 55 mg/kg EDB on days 1 through 15 of gestation, and a control group of equal size received only corn oi] injections. Rats were killed on day 21 of gestation, and the uterine contents were examined. There was no evidence of reduced pre- or post-implantation survival of the fetus, reduced fetal body weight or length, or grossly visible external or internal (visceral or skeletal) malformations. Treatment-related effects of EDB were limited to evidence of maternal toxicity, defined as a significant (p<0.05) change in at least two maternal organ weights. The authors therefore concluded that fetal development was not. adversely affected by EDB.

LBG

Table 19. Teratogenicity of EDB Concentration Route of Number of and Observed Administration Species Animals Duration of Dose Effects Author IP Rat 10-15 55 mg/kg on days 1 fetal development was not Hardin et al., through 15 of adversely affected; maternal 1981 gestation toxicity was evident Inhalation Rat not given 32 ppm on days 6 increase in fourth-ventricular Short et al., Mouse through 15 of hydrocephaly, reduction in 1976 gestation occurrence of 14th rib, increase in wavy ribs; maternal toxicity Inhalation Rat 47 treated 20, 38, or 80 ppm, increase in fetal mortality, Short et al., Mouse 33 controls 23 hr/d on days 6 decrease in fetal body weight; 1978 through 16 of maternal toxicity was evident gestation Inhalation Rat 36 treated 0.43, 7, and 67 ppm, alterations in offspring Smith and 12 controls 4 hr/d, 3 d/wk, on behavior Goldman, 1983

day 3 through 20 of gestation

One study found that parental exposure to EDB caused alterations in the behavior of offspring. Smith and Goldman (1983) reported that pregnant rats exposed by inhalation to 7 and 67 ppm of EDB on days 3 through 20 of gestation produced significant (p<0.05) alterations in some offspring behavior parameters up to 90 days after exposure. Parameters which were measured included both rotorod and T-maze performance. Signs of stress, such as increased defecation and decreased gestational weights, were observed in mothers exposed to 7 and 67 ppm EDB. The results indicated that EDB produced long-term and possibly permanent alterations in the behavior of offspring, but the authors suggested that the changes were secondary to stress reactions in the mothers.

Reproductive Effects. Several studies have been performed on the reproductive effects of EDB in bulls, rams, chickens, and rats, which are summarized in Table 20.

Studies conducted on bulls demonstrated the effects of EDB on spermatogenesis. The earliest study (Amir and Volcani, 1967) reported abnormalities in spermatozoa and in the testes of bulls administered oral doses of EDB for a 17- to 22-month period. Later studies (Amir, 1973; Amir and Ben-David, 1973) also reported these abnormalities after both oral treatment and injection, noting that EDB adversely affected spermiogenesis and sperm maturation, resulting in spermatozoa with deformed heads and tails. After cessation of oral treatment, a period of 40 days was required for normal spermatozoa to reappear. After cessation of treatment by intratesticular injection, the recovery period for normal spermatozoa was 58 to 70 days. In examining the spermatogenic process more closely, Amir (1975) found that the early stages were adversely affected, and that the elimination of abnormal spermatozoa occurred more rapidly in young animals than in older ones. Courtens et al. (1980) reported that the late rather than the early spermatids were damaged.

In addition to the several studies documenting morphological defects in spermatozoa, one study revealed that the average sperm concentration in bulls receiving 4 mg/kg over a 20 day period was significantly (p<0.01) Jower than in the controls. When both rams and bulls were treated with EDB by injection, the rams detoxified the dose more rapidly (Amir and Tadmor, 1981).

Two studies examined the reproductive effects of EDB on chickens. Alumot et al. (1968) reported that feeding of EDB fumigated mash to chickens reduced egg size and egg fertility but exerted no effect on the onset of egg production or sexual development. Alumot (Olomucki) and Mandel (1969) investigated the potential of EDB to affect either the formation or the release of the follicle stimulating hormone (FSH) in egg~laying hens, and concluded that neither formation nor release of FSH was affected by EDB.

Another study by Short et al. (1979), examined the reproductive effects of EDB through inhalation exposure in male and female rats. None of the males exposed to 89 ppm of EDB impregnated any females, whereas at least 9D percent (9/10) of all males from the groups exposed to 0, 19, or 39 ppm EDB impregnated one or more females. In males, atrophy of the testes, epididymis, prostate and seminal vesicles were observed at 89 ppm

-58-

Table 20.

Reproductive Effects of EDB

Concentration Route of Number of and Observed Administration Species Animals Duration of Dose Effects Author Oral Chicken 30 treated exp. 1. 40 ppm for decrease in egg weight™ Alumot et al., 30 controls 6 mos 1968 12 treated exp. 2. 100 ppm for reduced fertilization rate 12 controls 6 wks 20 treated exp. 3. 80 and 180 slight growth depression low dose ppm for 3 mos up 6 weeks of age, not 20 treated statistically significant high dose 20 controls eke 25 treated exp. 4. 150 and 300 growth retardation low dose ppm for 3 mos 25 treated high dose 25 controls 11 treated exp. 5. 300 ppm no differences in semen 11 controls characteristics or fertilization capacity Oral Chicken 30 treated exp. 1. 10 mg/d gonadotropin levels were Atumot and 60 controls for 4 wks unaffected. Mandel, 1969 30 treated exp. 2. 10 mg/d pituitary hormone levels were 30 controis for 2 mos similar to controls Oral (capsule) Cattle 3 treated 2 mg ekg for 17.5 abnormalities were noted in Amir and to 22.5 mos spermatozoa and in the testes Volcani, 1967 Oral (capsule) Cattle 2 treated 4 mg/kg, 7 and 10 Amir, 1973 doses, every other day process of spermatogenesis and Oral (capsule) 1 treated 29, 10 doses, sperm maturation was adversely every other day affected; morphological IP 1 treated single dose 120 mg abnormalities were noted 1 treated single dose 220 mg 1 treated single dose 350 mg

{continued on following page)

Table 20. (continued)

Concentration Route of Number_of and Observed Administration Species Animals Duration of Dose Effects Author Oral ‘attie 3 treated 4 mg/kg every other morphological defects in Amir and day for 20 days spermatozoa were noted; recovery Ben-David, 1973 Injection 2 treated 110-120 mg single period for normal spermatozoa dose was 40 and 58-70 days after oral and injected treatments, respectively Oral (capsule) Cattle 13 treated 4 mg/kg every other early stages of the Amir, 1975 6 controls day for 20 days spermatogenic process were affected; elimination was more rapid in young animals than in older ones Oral (capsule) Cattle 3 treated 4 mg/kg every other average sperm concentrgtion was Amir et al., 3 controls day for 20 days lower than in controls 1977 Injection Cattle 10 treated singte dose of 200 rams were detoxified more Amir and mg rapidly than bulls; EDB Tadmor, 198] Ram 10 treated single dose of 135- concentrated in spermatids in 180 mg bulls IP Cattle 5 treated 270 mg single dose Courtens 5 controls Jate but not early spermatids et al., 1980 Oral 3 treated 4 mg/kg every other were damaged a 2 controls day for 20 days IP Rat males: not 1.25, 2.5, 5.0 and impairments of motor coordination Fanini et al., given 10 mg/kg for 5 days and motor actjvity devel opment 1984 in offspring Inhalation Rats males: 19, 39, and 89 ppm at 89 ppm atrophy of Short et al., 30 treated 7 hr/day, 5 days/wk reproductive organs was noted 1979 10 controls for 10 weeks and no females were impregnated; females: 20, 39, and 80 ppm, at 80 ppm fewer females 60 treated 7 hr/day, 7 days/wk mated during 10-day mating 20 controls for 3 weeks period * teP<0.01

ps0.05

exposure level. A decrease in body weight gain and food hs alg! and an increase in mortality were also noted in males. Females that mated with males exposed to 19 and 39 ppm produced litters that were normal in terms of total implants, viable implants, and fetal resorptions. Females exposed to 80 ppm of EDB for 3 weeks exhibited abnormal menstrual cycles and diestrus (sexual inactivity). As a result, fewer females mated in this group than in other groups during a 10-day mating period. All mated females were pregnant when killed at mid-gestation, and implants were normal. The authors concluded that adverse effects on reproduction were observed in both sexes exposed to EDB, but that the effects occurred only at concentrations that also produced morbidity and mortality.

In a study which investigated the effects of paternal exposure to EDB on the behavior of the offspring, male rats were treated with 5 daily intraperitoneal doses of EDB on 5 consecutive days, and bred with untreated females at 4 to 9 weeks after treatment (Fanini et al., 1984). One control group received corn oi] injections only, and an additional saline control group was used to control for the possible effects of corn oi? alone. Behavioral assessment of motor reflexes and motor coordination in the offspring were examined up to 21 days of age.

Significant (p<0.05) differences between treated animals and controls were observed in the development of motor coordination and motor activity

at dose levels as low as 1.25 mg/kg. The authors concluded that premeiotic stages of rat spermatogenesis are sensitive to the genotoxic effects of EDB, and demonstrate that behavioral assessments of developing

offspring from an EDB exposed male are capable of detecting such an effect induced during spermatogenesis.

Mutagenicity

Mutagenic effects include mutation induction and DNA damage. A mutation is a change in the genetic material of the cell that may be transmitted to descendant cells. When a mutation occurs in a germ cell, it may possibly affect future generations. In addition to measurements of mutation induction, measures of DNA damage also may be used to predict mutagenic activity. A number of short- and long-term tests have been developed to detect mutagenic action. Mutagenic effects of EDB have been studied in tests on bacteria, fungi, vascular plants, insects, mammalian cell cultures, and whole mammals. A summary of the results of EDB mutagenicity studies that were reviewed for this document is presented in Table 21. The table is arranged by category of organism, species or cell type, and mutagenesis test system. The conclusion of each study is given as positive if EDB caused DNA damage or induced mutations, and negative if these effects were not produced. The numbers of citations listed in Table 21 showing positive or negative responses in each category of organism are summarized in Table 22. The mutagenicity test results are discussed in the following sections.

Bacteria. The mutagenicity of EDB in bacteria was tested in four spectes: Salmonella typhimurium, Escherichia coli, Bacillus subtilis, and Streptomyces coelicolor. EDB was shown to be a bacterial mutagen tn all four species in a battery of tests (27 mutation induction tests and 1 DNA damage test). Positive mutagenic results for S. typhimurium, E. coli, and B. subtilis were corroborated in four or more tests each. EDB showed negative mutagenic activity in a few tests on strains of S. typhimurium and $. coelicolor. -

Bis

EDB has been tested for mutagenic activity in the Ames Salmonella test by several researchers. Strains typically used in this test contain point mutations that prevent the biosynthesis of the amino acid histidine. Therefore the bacteria cannot grow in the absence of histidine. Reversion to histidine independence can occur if a new mutation occurs at the site of the original mutation.

EDB induces point mutations in Salmonella typhimurium. Positive mutagenic results were obtained for S. typhimurium strains TA 98, TA 100, TA 1530, and TA 1535 (Brem et al., I974; ETT10t€ and Ashby, 1980; Barber et al., 1981; Principe et al., 1981, Moriya et al., 1983; Rannug et al., TS78; Rannug and Beije, 1979; Stolzenberg and Hine, 1980; and van Bladeren et al., 1980). EDB was not mutagenic to strains TA 1537 or TA 1538 in any of the studies reviewed (Brem et al., 1974; Principe et al., 1981; Moriya, et al., 1983). Strains TA 100, TA 1530, and TA 1535 are typically used to detect mutations caused by base-pair substitutions, whereas TA 98, TA 1537, and TA 1538 are typically used to detect frameshift mutations. The results shown in Table 21 indicate that EDB induces primarily base-pair substitution mutations. Several studies show dose-response relationships for EDB mutagenicity in S. typhi nate (Rannug et al., 1978; Stolzenberg and Hine, 1980; Barber et al., and Moriya é€ al., 1983).

Several Salmonella mutation tests were carried out in the presence of an S-9 metabolic activation mixture, which is added to in vitro mutagenicity tests to provide for the possibility that a substance may be transformed into a toxin in vivo. The S-9 mixture used in the Salmonella tests was prepared from rat liver homogenate. Shiau et al. (1980) showed positive EDB mutagenic activity in S. typhimurium strain TA 1535 only in the presence of S-9. Addition of S-9 metabolic activation enhanced the mutagenic effect of EDB in strain TA 1535 (Rannug et al., 1978) and in TA 100 (van Bladeren et al., 1980). Other studies showed that addition of metabolic activation either decreased or did not significantly affect mutagenic action of EDB in Salmonella assays (Stolzenberg and Hine, 1980; Barber et al., 1981; Principe et al., 1981; and Moriya et al., 1983). Rannug and Beije (1979) added EDB to a rat liver perfusion system and observed a mutagenic effect when the bile was tested in the Salmonella assay.

Four studies conducted on E. coli indicated that EDB induced mutations or caused DNA damage jn this bacterium. EDB induced point mutations in E. coli strain WP2 in the tryptophan reversion assay (Scott et al., 1978; Hemminki et al., 1980; Moriya et al., 1983). Mutation Frequency in E. coli was increased by the addition of plant extract prepared from Tradescantia (Spiderwort) in the study by Scott et al. (1978). In the same study, commercial EDB was distilled, and the residue and distillate were tested for mutagenic activity. The EDB residue showed greater mutagenic effect than the distillate, and the addition of plant extract increased the mutagenic activity of both samples. Brem et al. (1974) studied the DNA-modifying effect of EDB in a DNA polymerase deficiency assay. The enzyme DNA polymerase aids in the DNA repair process, Bacteria deficient in this enzyme are more sensitive

-62-

to DNA-modifying agents than the parent strain possessing the enzyme because they are unable to repair DNA efficiently. In this experiment, EDB preferentially inhibited growth of DNA polymerase-deficient E. coli compared to the parent strain, These results indicate that EDB may damage DNA and, therefore, may be a mutagen.

Mutagenicity tests conducted on Bacillus subtilis and Streptomyces coelicolor showed positive mutagenic responses in both species, although negative results also were obtained for both species. Shiau et al. (1980) studied mutation induction and DNA damage by EDB in B. subtilis. EDB induced mutagenic responses in two strains when tested in the presence of S-9 metabolic activation. DNA damage was measured as the decrease in survival of EDB-treated cells lacking certain DNA-repair enzymes compared to cells with intact repair systems. EDB did not show DNA-damaging activity in this study in the presence or absence of “metabolic activation. Principe et al. (1981) studied the mutagenicity of EDB in S. coelicolor. EDB caused forward mutations in S. coelicolor in a spot test but not in a plate test.

Fungi. Tests on two species showed that EDB was mutagenic in fungi. EDB induced mutations in Aspergillus nidulans in two assay systems (Principe et al., 1981, and Scott et al., 1978). Addition of plant extract enhanced the mutagenic activity of EDB in A. nidulans (Scott et al., 1978). In forward mutation assays on Neurospora crassa, EDB induced recessive lethal mutations (Malling, 1969, and de Serres and Malling, 1983).

Vascular Plants. Three tests conducted on the vascular plant Tradescantia (Spiderwort) showed that EDB was mutagenic in this plant system. Ma et al. (1978) investigated the mutagenicity of EDB in Tradescantia cells undergoing meiosis. The study measured damage to metotic chromosomes through micronuclei production in the early tetrad stage of microsporogenesis. Micronuclei were formed from chromosomes or chromosomal fragments that were not incorporated into daughter nuclei during meiosis. Tradescantia cuttings were exposed to 5 to 80 ppm of gaseous EDB for six hours. The investigators showed a positive correlation between the EDB dose and frequency of occurrence of micronuclei among cells of treated plants.

Tradescantia plants were also tested for mutagenicity in the stamen hair pigmentation system. In this test, mutations are detected from the change in color of cells in the stamen hairs. Sparrow et al. (1974) demonstrated induction of mutations in Tradescantia plants exposed to gaseous EDB for six hours. A dose-response relationship was reported over the range of EDB concentrations tested, which was 3.6 to 148.2 ppm. In a review article, Schairer et al. (1978) discussed experimental results for Tradescantia cuttings Exposed to a range of 0.14 to 100 ppm of gaseous EDB for a range of 2 to 144 hours. Statistically significant mutation frequencies were observed following EDB exposure to as little as 1 ppm for 6 hours and 0.14 ppm for 144 hours. At a given EDB concentration, the frequency of mutational events increased with duration of exposure. In this study, EDB was a potent mutagen and exhibited a dose-response relationship.

-63-

Insects. EDB was a mutagen in five of six mutagenicity studies conducted on the fruit fly, Drosophila melanogaster. Kale and Baum (1979a, 1979b, 1981, and 1983) studied induction of sex-linked recessive lethal mutations in Drosophila melanogaster exposed to gaseous EDB. Acute exposure of Drosophila males to ppm of EDB for 0.5 hours induced statistically significant frequencies of mutations in spermatids , spermatocytes, and spermatogonia (Kale and Baum, 1979a), In another experiment, germ-cell mutations were induced in Drosophila males exposed to 0.4 to 31.1 ppm for 0.58 to 2.6 hours (Kale and a 1981). Ina chronic exposure test, Kale and Baum (1979b) exposed Drosophila males to gaseous EDB concentrations of 0.21 to 2 ppm for 11 to 100 hours. EDB induced mutations at all concentrations tested. The dose-response effect was linear below the 60-ppm-hour exposure level for three germ-cell types: spermatozoa, spermatids, and spermatocytes. Pronounced germ-cell sensitivity differences were found: spermatids and spermatocytes were about 10 to 20 times more sensitive to mutation induction than were spermatozoa. Drosophila embryos were exposed to 0.24 to 145 ppm of gaseous EDB for 1.5 to 24 hours (Kale and Baum, 1983). DB induced mutations at exposures as low as 1 ppm for 3 hours. At exposure levels below 300 ppm-hour, the dose-response relationship was linear.

Two mutagenicity studies that exposed Drosophila to EDB in feeding solution showed conflicting results. In a study by Valencia (1981), EDB was administered in feeding solution at concentrations of 5 to 50 ppm for 72 hours. No mutagenic effect was observed. However, these results must be regarded as equivocal because the author noted that the exposure technique probably was inappropriate for EDB in that exposure may not have occurred if vapors escaped from food prior to ingestion. Vogel and Chandler (1974) also studied the effect of ingested EOB on sex-linked recessive lethal mutations in Drosophila. Males were fed 0.3 mM (26 ppm) of EDB in solution for 72 hours. Mutations were induced in the Drosophila male germ cells. Spermatids and spermatocytes were more afrected by EDB than were mature sperm. Positive results in this study support the hypothesis that in the study by Valencia (1981), EDB exposure had not occurred to the degree suggested by the applied dose.

Mammalian Cells. EDB has been tested in mutagenesis assays using four types of cultured mammalian cells: Chinese hamster ovary and Jung cells, mouse lymphoma cells, and human lymphocytes. Study results shown in Table 21 indicate positive mutagenic responses for all four cell types. The individual studies are discussed in this section.

Mutagenicity of EDB in Chinese hamster ovary cells in_ the hypoxanthine guanine phosphoribosy] transferase (HGPRT) enzyme system was investigated in two studies. Tan and Hsie (1981) added 0.05 to 5 mM (4 to 433 ppm) of EDB to Chinese hamster ovary cells, which were then incubated for 5 hours. Cells were subcultured on days 1, 3, and 6 following EDB treatment to obtain a stable phenotypic expression. Cells were plated, incubated for 7 days, and mutation frequency was determined. EDB exhibited statistically significant mutagenic activity and dose-response relationships in the presence and absence of S-9 metabolic activation. St es obtained by Brimer et al. (1982) (p<0.01) are shown in

able .

264-

Tezuka et al. (1980) investigated induction of sister-chromatid exchanges (SCEs), chromosomal aberrations, and polyploids in cultured Chinese hamster V79 lung cells treated with EDB. Sister-chromatid exchange is the exchange of DNA segments between the chromatids of a chromosome during cell division. Chromosomal aberrations consisted mainly of gaps and breaks in this study. Cell cultures were treated with EDB at concentrations of 5x10-4 to 5x10-3 M (43-433 ppm) for 26.5 hours. Statistically significant increases in frequencies of SCEs, chromosomal aberrations, and polyploid cells were reported. Frequencies of SCEs and chromosomal aberrations showed dose-response relationships. The frequency of polyploid cells was increased only at the highest dose level.

Clive et al. (1979) investigated mutagenicity of EDB in mouse lymphoma cells in the thymidine kinase enzyme system. In the absence of metabolic activation, cells were treated with 94 to 451 ug/ml (43 to 208 ppm) of EDB and incubated for 2 hours. Cells were treated with 30 to 60 ug/ml (14 to 28 ppm) in the presence of S-9 metabolic activation and incubated for 4 _ hours, Significant numbers of mutations and dose-response relationships were found in the presence and absence of S-9 metabolic activation. Clive (1973) also showed that EDB was mutagenic to mouse lymphoma cells in the absence of S-9 at similar dose levels.

Perocco and Prodi (1981) investigated DNA damage by EDB in human lymphocytes cultured in vitro. Lymphocytes were separated from human blood and cultured with a DNA precursor, tritiated thymidine, and EDB concentrations of 2.5 to 10 yl/ml (2500 to 10000 ppm) for 4 hours. Inhibition of scheduled (duplicative) and unscheduled (reparative) DNA synthesis were measured by the tritiated thymidine uptake. Reparative synthesis is an indicator of previous damage to DNA. EDB inhibited thymidine uptake, indicating that it inhibited scheduled DNA synthesis. When hydroxyurea was added to block the thymidine uptake due to scheduled DNA synthesis, thymidine uptake was decreased compared to the control in the absence of S-9 metabolic activation and increased in the presence of S-9, The magnitude of the difference between the uptakes in the presence and absence of S-9 indicates the extent of unscheduled DNA reparative synthesis when EDB is metabolized by S-9 mix. Results of this study indicate that EDB may cause DNA damage through its ability to elicit DNA reparative synthesis in human lymphocytes.

Sister chromatid exchange induction in human lymphocytes in vitro by EDB was investigated by Geddes (1982) and reported in an abstract. In one part of the study, human lymphocytes were cultured for 74 hours with EDB at concentrations from 1x10-8 to 1x10-3 M (0.0009 to 87 ppm) in the absence of S-9 metabolic activation. In the second part, cells were incubated with EDB with and without S-9 present for two hours, followed by a further incubation of 24 hours after washing. Doses of EDB were not reported for the latter experiments. The author concluded that EDB induced significant increases in numbers of SCEs in human lymphocytes compared to controls. This effect was enhanced by addition of S-9 metabolic activation. Exposure time and EDB doses are not reported in

Table 21 because the author did not specify whether one or both of the experiments elicited the positive response.

-65-

Mammals. EDB has been tested for dominant-lethal induction and DNA damage in vivo in two mammalian species, mice and rats. Dominant-lethal studies weré negative in both species. EDB caused DNA damage in hepatic cells in mice or rats in three studies. tudy findings are discussed in the following paragraphs.

A dominant-lethal mutation is one that kills an individual heterozygous for the mutation, and it is frequently due to a chromosome aberration. To test for dominant-lethal mutation in male germ cells, Males are exposed to a chemical, mated with females, and pregnant females examined for total implants and early and late fetal deaths. EDB has been tested for dominant-lethal induction in mice by gavage and intraperitoneal exposure and in rats by gavage and inhalation exposure.

Teramato et al. (1980) administered 100 or 150 mg/kg (ppm) of EDB to groups of 7 to 9 male mice, and 10 or 30 mg/kg (ppm) of EDB to groups of 15 male rats. Dosing was by gastric intubation (gavage) once a day for 5 successive days. Following treatment, male mice were allowed to mate with females for 6 weeks; male rats were allowed to mate with females for 10 weeks. Pregnant females from both mouse and rat experiments were killed 12 to 14 days after conception. Numbers of corpora lutea, implants, live embryos, and early and late embryonic deaths were recorded. EDB failed to induce dominant lethal mutations in either mice or rats in this study.

Dominant lethal tests in mice were conducted by Epstein et al. (1972) by both intraperitoneal injection and gavage. Results of EDB tests showed that early fetal deaths and total implant losses were within the control limits. (Actual data for EDB administration are not presented in the article.) Short et al. (1979) found no evidence of dominant lethal mutations in a study on rats exposed to gaseous ENB.

Hepatic cell DNA damage (DNA single-strand breaks) caused by EDB was investigated in mice in two studies and in rats in one study. Mkhite et al. (1981) treated male mice with 25 to 75 mg/kg (ppm) of EDB by ‘intraperitoneal injection three hours prior to sacrifice. Hepatic nuclei were isolated, lysed, and subjected to alkaline elution. Alkaline elution separates single-strand DNA on the basis of length. Elution rate constants calculated for the different treatment groups showed a dose-dependent increase in the amount of damage to hepatic DNA. Storer and Conolly (1983) also studied DNA single-strand breakage in mouse liver cells following in vivo intraperitoneal administration of DB. Statistically significant hepatic DNA damage was detected at a dose of 0.5 mmoles/kg (94 ppm). Both of these studies showed that EDB induces hepatic DNA damage in mice.

Nachtomi and Sarma (1977) studied DNA damage in rat liver cells as measured by sedimentation analysis in alkaline sucrose gradients. Rats were administered EDB by gavage. EDB induced DNA single-strand breaks at doses ranging from 75 to 220 mg/kg of EDR. Liver DNA damage induced by a dose of 7& mg/kg was almost completely repaired 96 hours after administration.

-66-

Table 21. Summary of EDB Mutagenicity Studies

EDB Doses b Category of Organism, Tested EDB Concentration Mutagenicity’ Organism and/or Exposure (as reported Range (Author's Cell Type Test System ime by authors) mg/pTate ‘ppm Conclusion) Reference BACTERIA Salmonelta Ames Salmonella/ typhimurtum microsome test TA 98 (plate test) 48 hr 14.10, 28.20, 2.6-21.0 nce +4 Barber et al., 56.41, 111.76 1981 umoles/plate TA 98 (type NR®) NR NR NC NC +d Moriya et al., 1983 TA 98 {spot test) NR 100 ul/plate 217 NC - Principe etal., 1981 TA 100 (plate test) 48 hr 14.10, 28.20 2.6-21.0 NC +d Barber et al., 56.41, 111.76 1981 umoles/plate TA 100 (type NR) NR 50 to 5000 ug/ 0.05 to 5.0 NC +4 Moriya et al., plate 1983 TA 100 (spot test) NR 100 ut/plate 217 NC Fla Principe et al., 198) TA 100 (plate test) NR 1, 2.5, 5.0 ul/ 2.2-10.9 NC +d Principe plate et al., 1981 TA 100 (type NR) NR O.14 Ty, 10 0.02-1.9 NC +d Stolzenberg umoles/plate and Hine, 1980 TA 100 (plate test) 48 hr 0.7 to 3.5 m™ NC 61-303 +4 van Bladeren et al., 1980 TA 1530 (spot test) 54 hr 10 umoles/plate 1.9 NC +4 en etal., TA 1535 (spot test) 54 hr 10 umoles/plate 1.9 NC +4 in etal., TA 1535 (plate test) 48 hr 14.10, 28.20 2.6-30.0 NC +4 Barber et 56.41, 111.76 al., 1987 umoles/plate TA 1535 (plate test) 72 hr = 4, 20, 100, 500, 0.004-2.5 NC +d Elliott and 2500 ug/plate Ashby, 1980

(continued on following page)

Table 21. (continued) EDB Doses ‘ Category of Organism, Tested EDB Concentration Mutagenicity Organism and/or Exposure (as reported Range® (Author's Cell Type Test System Time by authors) mg/plate ppm Conclusion) Reference TA 1535 (plate test) 72 hr 20, 100, 500, 0.02-2.5 NC +4f Elliott and 2500 ug EDB/plate Ashby, 1980 + 100 ug disulfiram/plate TA 1535 (type NR) NR NR NC NC +d Moriya et al., 1983 TA 1535 (spot test) NR 100 ul/plate 217 NC oe Principe et al., 1981 TA 1535 {plate test) NR 1, 2255 Siulsé 2.2-10.9 NC +d Principe plate et al., 1981 TA 1535 (plate test) NR 12 umoles EDB NC NC +4 (bile) Rannug and added to rat -d Beije, 1979 liver perfusion (perfusate) system. Perfusate and bile were tested. TA 1535 (plate test) 48 hr 0.5, 1.0, 2.0 0.09-0.38 NC <a Ranma umoles/plate oe TA 1535 (spot test) 48hr 5, 50, 100, 300 0.005-0.3 NC +4,F Shiau et al., ug/plate 1980 TA 1537 (type NR) NR NR NC NC a Moriya et al., 1983 TA 1537 (spot test) NR 100 ul/plate 217 NC - Principe et al., 1981 TA 1538 {spot test) 54 hr 10 umoles/plate 1.9 NC -4 bres etal., 1974 TA 1538 (type NR) NR NR NC NC -4 bide! et » 198F TA 1538 (spot test) NR 100 ul/plate 217 NC - ite et al., 1981

{continued on following page)

Table 21. Summary of EDB Mutagenicity Studies

EDB Doses Category of Organism, Tested EDB Concentration Mutagenicity? Organism and/or Exposure (as reported Range* (Author's Cell Type Test System Time by authors) mg/pTate ‘ppm Conclusion) Reference BACTERIA Salmonella Ames Salmonella/ typhimurTum microsome test TA 98 {plate test) 48 hr 14.10, 28.20, 2.6-21.0 nce +d Barber et al., 56.41, 111.76 1981 umoles/plate TA 98 (type NR®) NR NR NC NC +d Moriya et al., 1983 TA 98 (spot test) NR 100 ul/plate 217 NC - Principe et al., 19817 TA 100 (plate test) 48 hr 14.10, 28.20 2.6-21.0 NC +4 Barber et al., 56.41, 111.76 1981 umoles/plate TA 100 (type NR) NR 50 to 5000 ug/ 0.05 to 5.0 NC +d Moriya et al., plate 1983 TA 100 (spot test) NR 100 ul/plate 27 NC a Principe et al., 1981 TA 100 (plate test) NR 1, 2.5, 5.0 aif 262-1059 NC +d Principe plate et al., 198) TA 100 (type NR) NR 0.1, 1, 10 0.02-1.9 NC +4 Stolzenberg umoles/plate and Hine, 1980 TA 100 {plate test) 48 hr 0.7 to 3.5 mM NC 61-303 +d van Bladeren et al., 1980 TA 1530 (spot test) 54 hr 10 umoles/plate 1.9 NC +d oe etal., TA 1535 (spot test) 54 hr 10 umoles/plate 1.9 NC +4 Rr etal., TA 1535 (plate test) 48 hr 14.10, 28.20 2.6-30.0 NC +4 Barber et 56.41, 111.76 al., 1987 umoles/plate TA 1535 (plate test) 72 hr 4, 20, 100, 500, 0.004-2.5 NC +d Elliott and 2500 ug/plate Ashby, 1980

(continued on following page)

Table 21. (continued)

EDB Doses Category of Organism, Tested EDB Concentration Mutagenicity Organism and/or eapestrs (as reported Range? (Author's Cell Type Test System ime by authors) __mg/pTate “pp Conclusion) Reference Escherichia coli WP2 uvrA WP2/Microsome 18 hr 20-10,000 uM NC 1. 7-865 +4 Hemminki tryptophan ipeubagedll in incubation et al., 1980 reversion 8 hr flask) i assay (plated) (type NR) WP2 her (plate test) 48 hr NR NC NC +d Moriya et al., 1983 wP2 (type NR) NR NR NC NC +4 Scott et al., 1978 polat, polAg DNA damage 8 hr 10 ul/plate ARTs NC 4 Brem et al., (polymerase 1974 deficiency assay) (spot test) Bacillus subtilis TKI5211 Mutagenicity 72 hr 5, 50, 100, 300, 0.005-0.3 NC +4,F Shiau et al., assay ug/plate 1980 (spot test) TKI5211 (plate test) 48-96 5, 25, 50, 100, 0.005-0.3 NC +4sf Shiau et al., hr 300 ug/plate 1980 Tkd6321 Mutagenicity 72 hr 5, 50, 100, 300 0.005-0.3 NC +0,F Shiau et al., assay ug/plate 1980 (spot test) TKd 6321 (plate test) 48-96 5, 25, 50, 100, 0.005-0.3 NC af Shiau et al., hr 300 ug/plate Ta 15 strains DNA damage “overnight” 109 NC 4 Shiau et al., assay 50 ul/plate 1980 (spot test)

(continued on following page)

Table 21. (continued)

test

EDB Doses Category of Organism, Tested EDB Concentration Mutagenicity? Organism and/or Exposure (as reported Range® (Author's Cell Type Test System Time by authors) mg/plate __Ppm Conclusion) Reference Streptomyces coelicolor Forward NR 100 ul/plate 217 NC * Principe Mutation. assay et al., 198] (spot test) (plate test) NR 2, 10, 20, 100 4,3-217 NC - Principe ul/plate etal., 1981 FUNGI Aspergillus nidulans Strain 35 Forward NR 200 ul/plate 434 NC Ps Principe mutation assay et al., 1981 (8-azaguanine resistance) (spot test) (plate test) NR 10, 50, 100, 200 21.7-434 NC - Principe ul/plate etal., 1981 Glasgow Methionine 5-6 d NR NC NC +4 Scott et al., system 1978 (type NR) Neurospora crassa Heterokaryon 12 ad-3 forward 3 hr 1.25-1.63 ul/mi = NC 1250-1630 +4 de Serres and mutation assay Malling 1983 Two-component ad-3 forward 3 hr 1.6 ul /ml NC 1600 +d Malling heterokaryon mutation assay 1969 VASCULAR PLANTS Tradescantia sp. Tradescantia 6 hr <5, 10, 20, 40, NAY <5-80 +4 Ma et al., TTone 6850 micronucTeus 60, 80 ppm 1975

{continued on following page)

Table 21. (continued)

EDB Doses b Category of A Tested EDB Concentration Mutagenicity Organism and/or Exposure (as reported Range@ (Author's Cell Type Test System Time by authors) mg/plate ‘ppm Conclusion) Reference Clone 4430 Stamen hair 2-144-0.14-10D ppm NA 0.14-100 «+** Schairer pigmentation hr et al., 1978 system Clone 4430 Stamen hair 6 hr 3.6, 10.8, 19.3, NA 3.6-148.2 +4 Sparrow pigmentation aby 60.2;. 78.35 et al., 1974 system 148.2 ppm INSECTS Drosophila Sex-linked 0.5, 1 250 ppm NA 250 +* Kale and Baum melanogaster recessive hr 1979a lethal test (males exposed to gaseous EDB) (wales exposed 11-100 0.21, 0.212, NA 0.21-2 +4 Kale and Baum eagase0le hr 0.257, 1, 2 ppm 1979b 0B (males exposed 0.58- 0. ‘. 0.5, 0.7, NA 0.4-31.1 +4 Kale and Baum ‘oO geseaus 2.6 hr 31.1 ppm 1981 (eggs exposed 1.5-24 0.24, 1, 2, 7, NA 0.24-145 +4 Kale and Baum O gaseous hr 12, 21, 25, 50, 1983 5 125, 145 ppm (males exposed 72 hr 5, 10, 50 ppm NA 5-50 4 Valencia to EDB in 1981 food) (males exposed 72 hr 0.3 mM NA 26 +4 Vogel and to EDB in Chandler, food) 1974

(continued on following page)

Table 21. (continued)

EDB Doses Category of Organism, Tested EDB Concentration Mutagenicity? Organism and/or Exposure (as reported Range (Author's Cell_Type Test System Time by authors) _ mg/pTate ppm Conclusion) Reference MAMMALIAN CELLS Chinese hamster Hypoxanthine- 5 hr <0.02 - 5 mM NA 2-433 ail Brimer et ovary cells guanine Phosphoribosy1 al., 1982 ransferase (HGPRT) system HGPRT system Shr 0.05, 0:1, 0.2, NA 4-433 a Tan and Hsie, 5 4, 0. 2,3, 4,°5 mt Chinese hamster Sister- 26.5 5x 107%, NA 43-433 a Tezuka et V79 lung cells chromatid hr 1x 1073; al., 1980 exchange 2x 10-2, 5x 10-3'm Chromosomal 26.5 5 x 1074, NA 43-433 ial Tezuka et. aberrations hr 1x 10755 al., 1980 2x 1075, 5x 1073'm Polyploid 26.5 5x 1074, NA 43-433 <aal Tezuka et cells hr ix 103, al., 1980 x 5 x 107-3 'M Mouse lymphoma Thymidine 2 hr O55, 1.0, 165, NA 43-260 +d Clive et al., L5178Y ceils kinase system 2.0, 2.5, 3.0 mm 1973 Thymidine 2 hr 94, 188, 282, NA 43-208 + Clive et al., kinase system 376, 414, 451 1979 ug/ml Human DNA repair 4 hr 2.5, 5, 10 ul/ml NA 2500-10000 +%>f Perocco and lymphocytes (unscheduled Prodi, 1981 DNA synthesis) Sister NR NR NA NC +d Geddes, 1982 chromati exchange

(continued on following page)

Table 21. (continued)

EDB Doses Category of Organism, Tested EDB Concentration Mutagenicity? Organism and/or Exposure (as reported Range® (Author's Cell_ Type Test System ime by authors) mg/plate ppm Conclusion) Reference MAMMALS Mouse Strain ICR/ Dominant tethal Ha Swiss test ~intraperi- Single 18, 90 mg/kg NA 18-90 - Epstein toneal dose et al., 1972 -gavage Dosed 50, 100 mg/kg NA 50-100 - ‘Epstein once a et al., 1972 day for 5 successive days Strain BDF Dominant Oosed 100, 150 mg/kg NA 100-150 - Teramoto lethal test once a et al., 1980 -gavage day for 5 successive days Strain B6C3F1 Hepatic-cell Single 0.25, 0.5 NA 47-94 Storer and DNA, single- Dose mmo} /kg Conolly, strand breakage 1983 assay -intraperi toneal Swiss Webster Hepatic-cell Single 25, 50, 75 NA 25-75 White et al., DNA, single- dose mg/kg 1981 strand breakage assay -intraperitoneal Rat ~~ Strain SD Dominant Dosed 10, 30 mg/kg NA 10-30 - Teramoto lethal test once a et al., 1980 -gavage day for § successive days Strain CD Dominant Tnr/day 19, 39, 89 NA 19-89 - Short et al., lethal test 5 days/wk ppm 1979 ~inhalation for 10 wks Wistar Hepatic-cell Single 50, 75, 100, NA 50-220 +4 Nachtomi and

DNA single- dose strand breakage assay

-gavage

150, 220 mg/kg

Sarma, 1977

(continued on following page)

Doses as reported by authors were converted to mg/plate where appropriate for bacteria and fungi and to ppm elsewhere.

Author's conclusion as to whether EDB is a mutagen based on overall study: +, positive mutagenic response; -, negative mutagenic response. Unless otherwise noted, results were subjected to statistical analysis. Where statistical analysis was performed, the

significance level is reported as follows: = p 0.05, * = p 0.01, *** = p 0.001. (If different significance levels were given for different doses, the less significant value for

p 9s presented in table.) Where a mutagonye response was obtained only in the presence of §-9 metabolic activation, it is shown in the table by footnote f. NC = Not calculated due to insufficient information.

Results were not subjected to statistical analysis to determine if EDB dosed responses were significantly different from control response.

NR = Not reported Positive mutagenic response was induced in the presence of S-9 metabolic activation mix.

NA = Not applicable.

Table 22. EDB Mutagenicity by Category of Organism

Results Category Mutation Induct fon _ DMA Dariige Bacteria 27 8 1 1 Fungi 4 1 0 0 Vascular plants 3 0 0 0 Insects 5 1 0 0 Mammalian cells 8 0 1 0 Mammals 0 5 3 0

4Number of citations in Table 21 that gave positve or negative results for each type of test and category of organism are given in this table.

Carcinogenicity

A chemical is a carcinogen if it possesses the ability to induce cancer. EDB has been shown to be a carcinogen in studies on two animal species, mice and rats. Chronic or long-term bioassays were conducted using three routes of exposure: dermal, inhalation, and gastrointestinal via gavage (stomach intubation). EDB induced both local and remote tumors in animals when administered by all three exposure routes, including tumors of the skin, nasal cavity, stomach, lung, spleen, liver, adrenal glands, and reproductive organs. Tumors induced by the combined action of EDB and the anti-alcohol abuse drug, disulfiram, include tumors of the liver, kidney, thyroid, adrenals, mesentary, jung, nasal cavity, reproductive organs, and spleen. This section includes discussion of tumor incidence (animals showing a tumor/total number of animals in the experimental group) and dose-response effects (an increase in tumor incidence with increasing dose). In addition to the chronic carcinogenicity bioassays on EDB, one investigator examined an early histochemical marker for hepatocarcinogenesis in rats. A summary of the results of the chronic carcinogenicity studies on EDB is presented in Table 23, Results are arranged by tumor type, that is, data from all studies reporting incidences of nasal cavity tumors are given in the first section of the table. Table 24 presents the conclusions of each study regarding EDB induction of particular tumor types. Detailed explanation of these studies is provided below.

Skin Application Bioassays. The carcinogenicity of EDB administered by skin application was examined in a bioassay on Ha:ICR Swiss mice. In one experiment, van Duuren et al., 1979) tested EDB in a two-stage carcinogenesis assay. This assay tests the hypothesis that one substance (the initiator) initiates the cancer process and a second substance (the promoter) enhances the carcinogenic effect when given subsequently. EDB was tested as an initiator with phorbol myristate acetate (PMA) as the promoter. Van Duuren et al. administered EDB in a single dose of 75 mg/application to the skin followed 14 days later by administration of the promoter three times weekly for the duration of the experiment. (The exact duration of the test for EDB was not specified, but was reported to be between 428 and 576 days.) No significant carcinogenic effects were observed, therefore, EDB was not active as a skin tumor initiator in this study.

In a second experiment, EDB was tested by repeated skin application of 25 or 50 mg/application three times weekly (van Duuren et al., 1979). (The duration of the test for EDB was not specified, but was reported to be between 440 and 594 days). EDB induced a statistically significant increase in local tumors including skin papillomas and carcinomas at the higher dose level. Also, significant increases in tumors remote from the site of application, lung papillomas, were induced by EDB at both dose levels. (For the remainder of this section, the term "significant" refers to statistical significance.)

Gastrointestinal Exposure Bioassays. The carcinogenicity of EDB via gastrointestinal exposure in BOC3F1 mice and Osborne-Mendel rats was investigated by the National Cancer Institute (Olson et al., 1973; Ward and Haberman, 1974; Weisburger, 1977; NCI, 1978; NCI, 1979). Researchers administered EDB by gavage 5 times weekly at either of two dose levels to

~-76-

groups of 50 male and female mice and rats. Time-weighted average doses were 62 and 107 mg/kg for male and female mice, and 38 and 41 mg/kg for male rats and 37 and 39 mg/kg for female rats. The specific doses administered to the animals for different periods during the experiments are given in the appropriate footnotes to Table 23. The bioassays were terminated early due to poor survival and early onset of cancer. The authors noted that accelerated mortality in both species appeared to be associated with cancer of the forestomach.

Results of the mouse experiments shown in Table 23 indicate highly significant increased incidences of squamous cell carcinomas of the forestomach in low- and high-dose groups of both sexes. This malignant tumor was observed as early as 24 weeks following initial exposure to EDB. The tumors metastasized throughout the abdominal cavity; several metastasized to the lung. Significant positive associations between dosage and tumor indicence (that is, dose-response relationships) were demonstrated for both sexes of mice. A significantly increased incidence of alveolar/bronchiolar adenomas of the lung in mice was observed in high-dose male and low-dose female mice. A significant increase was also observed in high-dose females when the incidence was time-adjusted to include only those mice surviving until the first tumor was observed. (This time-adjusted analysis was performed for several tumor types. Results of this analysis are not included in Table 23, but are discussed in the text where appropriate. In the time-adjusted analysis, alveolar/bronchiolar adenomas were observed in 4/14 [29 percent] of high-dose females compared to 0/19 [0 percent] of controls [p<0.05]). Dose-response relationships were observed for lung tumor incidence in males, and in females when the data were time-adjusted.

Results of the rat bioassays showed significantly increased incidences and dose-response relationships for squamous-cell carcinomas of the forestomach in both sexes. This tumor type was observed as early as 12 weeks following onset of exposure to EDB and metastasized throughout the abdominal cavity as in the mouse bioassays. A significantly increased incidence of hepatic lesions (neoplastic nodules and hepatocellular carcinomas) was observed in high-dose female rats in the time-adjusted analysis. (In the time-adjusted analysis, hepatic lesions were observed in 6/25 [24 percent] of high-dose females compared to 0/20 [0 percent] of vehicle control rats [p<0.05]). Low-dose male rats showed a significant incidence of hemangiosarcomas of the spleen.

Based upon these bioassays, the authors concluded that EDB was carcinogenic to mice and rats. DB induced squamous-cell carcinomas of the forestomach in mice and rats of both sexes, alveolar/bronchiolar adenomas of the lung in mice of both sexes, hepatocellular carcinomas and neoplastic nodules in female rats, and hemangiosarcomas in male rats.

Inhalation Exposure Bioassays. Two studies examined carcinogenicity of — from inhalation exposure. In one study, the carcinogenicity of EDB by inhalation in B6C3F1 mice and Fischer 344 rats was investigated by the National Toxicology Program (NTP) (Stinson et al., 1981; NTP, 1982a; Huff, 1983). Mice and rats were exposed to gaseous concentrations of 10 or 40 ppm EDB for 78 to 103 weeks. £EDB was carcinogenic to mice in this study. Statistically significant increases in the following tumor types were observed: circulatory system hemangiosarcomas (mainly in the retroperitoneal area) in high-dose females, lung alveolar/bronchiolar carcinoma or adenoma in high-dose males and Ttow- and high-dose females, mammary gland adenocarcinomas in Tow- and high-dose females, nasal cavity

a7.

tumors in high-dose females, and subcutaneous fibrosarcomas in Tow- and high-dose females. Dose-response relationships were observed for the incidences of hemangiosarcomas in females, alveolar/bronchiolar adenomas or carcinomas in males and females, nasal cavity tumors in females, and subcutaneous fibrosarcomas in females. Tumors of the nasal cavity included adenomatous polyps, adenomas, and carcinomas arising from the nasal epithelium. While the tumor incidence in females generally exceeded that in males, this does not appear to be a true sex-related trend. Male mice of all groups exhibited high mortality, attributed to urinary tract infection, and the study was terminated at 79 weeks. Female mice in the control and low-dose groups were killed at 104-106 weeks, and high-dose females were killed at 91 weeks.

EDB also was carcinogenic to rats in the NTP inhalation study (NTP, 1982a). Incidences of the following tumors were significantly increased relative to control levels: hemangiosarcomas (mainly in the spleen) in high-dose males and females, Jung alveolar/bronchiolar carcinomas or adenomas in high-dose females, nasal cavity tumors in low- and high-dose groups of both sexes, and mesotheliomas of the tunica vaginalis in lowand high-dose males. Dose-response relationships were observed for the following tumor types: hemangiosarcomas in males and _ females, alveolar/bronchiolar adenomas or carcinomas in females, mammary gland fibroadenomas in females, nasal cavity tumors in males and females, and tunica vaginalis mesotheliomas in males. Included in the category of nasal cavity tumors were adenomas, adenocarcinomas, adenomatous polyps, Squamous cell carcinomas, and carcinomas.

In a second inhalation exposure study, the National Institute for Occupational Safety and Health (NIOSH) investigated the carcinogenic potential of EDB to Sprague-Dawley CD rats inhaling either EDB alone, or EDB in combination with disulfiram in the diet (Plotnick, 1978; Yodaiken, 1978; Wong et al., 1980; Wong et al., 1982). Rats were exposed to 20 ppm of EDR for 18 months under simulated occupational exposure conditions. Disulfiram was added to the food at a concentration of 0.05 percent by weight. Disulfiram was chosen for this study because of its known effect as an enzyme inhibitor and its use jin humans in alcoholism-control programs.

Male rats receiving disulfiram in their diet and no EDB exposure did not show any increased cancers. Female rats receiving disulfiram only had a significantly higher incidence of mammary tumors when compared to controls. Inhalation exposure to EDB with no disulfiram in the diet produced significantly higher tumor incidences in male rats in the spleen, adrenal glands, and subcutaneous mesenchymal tissue when compared to control] male rats. Female rats showed significantly higher tumor incidences in the spleen, adrenal glands, and mammary glands. Male and female rats exposed to EDB also showed a high incidence of nasal cavity tumors; however, statistical analyses were not performed.

The combination of EDB inhalation exposure and disulfiram in the diet produced significant increases in tumors of the liver, kidney, thyroid, and mesentary in male and female rats compared to rats receiving disulfiram or EDB alone. Increased incidences of hemangiosarcoma in the spleen and of adrenal tumors were also observed compared to rats

-78-

receiving disulfiram alone. Male rats treated with EDB and disulfiram exhibited a significantly higher incidence of lung tumors than male rats receiving EDB or disulfiram alone. Rats exposed to EDB in addition to disulfiram in the diet showed a higher incidence of nasal tumors than rats receiving no EDB, again, no statistical analysis was performed on these results. In summary, results of the NIOSH study show that EDB is an animal carcinogen under simulated occupational exposure conditions. Disulfiram in the diet enhances the carcinogenicity of inhaled EDB. Tumors that were produced by the combination of EDB and disulfiram that were not produced by EDB alone included tumors of the kidney, liver, lung, mesentary, and thyroid.

Indicator Test for Hepatocarcinogenesis. Milks et al. 1982 tested EDB in rats for early indication of liver cancer using the enzyme-altered foci test. The indicator used in these experiments was the incidence of gamma~glutamyl-transpeptidase (GGT)--positive foci in the liver tissue. Milks et al. conducted two experiments; one tested EDB as a_ tumor initiator and the other tested EDB as a tumor promoter. In the initiation experiment, four consecutive daily doses of 60 mg/kg of EDB were given orally to male Sprague-Dawley rats either before or after partial hepatectomy, or a single dose of 120 mg/kg EDB was given orally to partially hepatectomized rats. Hepatectomy is reported to enhance initiation of chemical carcinogenesis. Three days following EDB treatment, rats were given a promoting regimen of 500 ppm phenobarbital in their drinking water for the next 56 days. Animals were then sacrificed, and numbers of GGT-positive foci were measured. The authors observed no increase in incidence of GGT-postive foci in any of the treated groups. This suggests that EDB is without significant liver cancer initiating activity at doses up to 120 mg/kg. The authors noted that the reason for the lack of initiating activity is unclear, but it may be related to DNA repair in the liver or to the nature of the adducts formed when EDB binds to DNA.

In the second experiment, Milks et al. tested EDB as a promoter following initiation with N-nitrosodiethylamine. Following partial hepatectomy, rats received 30 mg/kg of the initiator. Three days later, animals were subjected to a promoting regimen of either 10 or 30 mg/kg of EDB by gavage five times weekly. Rats were sacrificed after 8 weeks of EDB treatment. Significant increases (p<0.02) in foci incidence were observed jin the treatment groups receiving partial hepatectomy, initiation, and EDB. Low foci incidences were found in initiator controls (rats given partial hepatectomy and EDB) sham controls (rats given initiator and EDB) and promoter controls (rats given partial hepatectomy and initiator). Results of this study suggest that EDB is a tumor promoter in rat liver. This promotor activity may contribute to EDB's carcinogenic activity.

-7$-

(continued on following page)

Table 23. Summary of EDB Carcinogenicity Studies Tumor Incidence? Route Duration Cresponders/sample size (percent) ] Tumor Type Species of of Dose tontro reate Reference Exposure Exposure Male Femate Mate Female

NASAL CAVITY All tumor types Mouse Inhalation 6hr/d, Sd/wk, 10 ppm 0/45 (0) 0/50 (0) 0/50 (0) 0/50 (0) NTP, 1982a; Reems cenG= | 78-103 wks 40 ppm | 0/50 (0) 12/50 (24) Huff, 1983 matous polyp carcinoma, Rat 10 ppm 0/50 (0) —-1/50(2) 39/50 (78)*"" 34/50 (68)\y, Ibid. hemangiosarcoma) 40 ppm | | 41/50 (82) 43/50 (86) Benign nasal Rat sabslation, Thr/d, 5d/wk, O ppm EDB 0/47 (0) 0/47 (0D) 0/45 (0) 0/45 (0) Wong et al., 1980; tumors DS” diet for 18 mos and D 1

(cnt1°/0S)

20 ppm EDB 32/42 (76) 27/42 (64) Ibid.

and no DS

(EDB/cnt1)

20 ppm EDB 22/27 (81) 17/18 (94) Ibid,

and DS

(EDB/DS) Malignant nasal O ppm EDB 0/47 (0) 0/47 (0) 0/45 (0) 0/45 (0) Ibid. tumors and DS

(cnt1/DS)

20 ppm EOB 27/42 (64) 19/42 (45) Ibid.

and no DS

(EDB/ent1)

20 ppm EDB 17/27 (63) 3/18 (17) Ibid.

and OS

(EDB/OS) LIVER Hepatocellular Rat Gavage 5x weekly 37-38 0/20 (o)k 0/20 (ok 3/50 (6) 1/41 (2) NCI, 1978 carcinoma or for 47-57 wks mg/kg® neoplastic nodule

5x weekly for 39-41 - 2/50 (4) 6/48 (13) Ibid, 34-44 wks mg/kg

Hepatocelluar Mouse Inhalation 6hr/d, 5d/wk, 10 ppm 3/41 {7) 2/50 (4) 1/48 (2) 6/50 (12) NTP, 1982a; Earet nome or | 78-103 wks 40 ppm | | 3/46 (7) 1/50 (2) Huff, 1983 adenoma

Table 23. (continued)

Tumor Incidence?

Route Duration Lresponders/sample size (percent)] Tumor Type Species of of Dose CTontroT Treated Reference Exposure Exposure Mate Female Wate Female Hepatocelluar Rat 10 ppm NR 2/50 (4) NR 1/49 (2) Ibid. carcinoma or 40 ppm NR NR 5/48 (10) Tbid. neoplastic nodule Hepatocel luar Rat Inhalation, 7hr/d, 5d/wk O ppm EDB 0/48 (0) 0/48 (0) 1/48 (2) 0/48 (0) Wong et al., 1980; tumor DS-diet for 18 mos and DS¢ 1982 ~~ r (cnt1/DS) 20 ppm EDB 2/46 (4) 3/48 (6) Ibid. and no DS (EDB/cnt1) 20 ppm EDB 36/48 (75)* 32/45 (71)* Ibid. and DS (vs. cntl/0S (vs. cnt1/DS (EDB/DS) and EDB/cnt1) and EDB/cnt1) Liver O ppm EDB 0/48 (0) 0/48 (0) 0/48 (0) 0/48 (0) Ibid. hemangiosarcoma and DS (cnt1/DS) 20 ppm EDB 1/46 (2) 0/48 (0) Ibid. and no 0S (EDB/cnt1) 20 ppm EDB 3/48 (6) 4/45 (9) Ibid. and DS (EDB/DS) STOMACH Forestomach Mouse Dermal 3x weekly 25.0 mg nai 5/30 (17) NA 3/30 (10) Van Duuren et al., papilloma and EDB/appli- 2/30 {7) (1/30 (3)]" 1979 squamous fell cation carcinoma 50.0 mg NA NA 3/30 (10) Ibid. EDB/appli- = cation Forestomach Mouse Gavage 5x weekly for 62 mg/kg! 0/20 (0)3 0/20 (0)5 45/50 (90)***»" 46/49 (94)***>" NCI, 1978 squamous cell 53 wks 0/20 (0)k 9/20 (a)k carcinoma | 107 mg/kg” | 29/49 (59)***5" 28/50 (56)***»" Ibid.

(continued on following page)

Table 23. (continued)

Tumor Incidence

Route Duration Lresponders/sample size (percent)] Tumor Type Species of of Dose Tontrot Treated Reference Exposure Exposure ale ‘emale e emate STOMACH Rat 5x weekly for 37-38 o/20 (0)K 0/20 (0)k 45/50 (90)*** 40/50 (80)*** Ibid. 47-57 wks mg/kg® ie a 5x weekly for 39-4 | 33/50 (66) 29/50 (58) Ibid, 34-44 wks mg/kg? <7 KIDNEY Hamartoma or Rat Gavage 5x weekly for 37-38 0/20 (0)k NR 2/49 (4) NR NCI, 1978 mixed tumor, 47-57 wks mg/kg® malignant 5x weekly for 39-4 NR 4/50 (8) NR Ibid. | 34-44 wks mg/kg? — Adenoma or Rat Inhalation, 7hr/d, 5d/wk, O ppm EDB 0/48 (0) 0/48 (0) 0/48 (0) 0/48 (0) Wong et al., 1980; adenocarcinoma DS-diet for 18 mos and DS 1982 (cnt1/DS) 20 ppm EDB 3/46 (7) 1/48 (2) Ibid. and no DS (EDB/cnt1) 20 ppm EDB 17/48 (35)* 7/45 (16)* Ibid. and DS (vs. EDB/cnt1 (vs. EDB/cnti (EDB/DS) and cnt1/DS) and cnt1/0S) Hemangiosarcoma Rat Inhalation, 7hr/d, 5d/wk, O ppm EDB 1/48 (2) 0/48 (0) 0/48 (0) 0/48 (0) Wong et al., 1980; DS-diet for 18 mos and DS 1982 (cnt1/DS) 20 ppm EDB 0/46 (0) 0/48 (0) Ibid. and no DS (£0B/cnt1) 20 ppm EDB 0/48 (0) 2/45 (4) Ibid. and DS (EDB/DS)

(continued on following page)

Table 23. (continued)

Route

Duration

Tumor Incidence?

nders/sample size (percent) ]

Cres, Dose TonEroT

Tumor Type Species of of Treated Reference Exposure Exposure fate emate fale emale THYROID Follicular cel) Rat Gavage 5x weekly for 37-38 0/20 (o)* NR 5/50 (10) NR NCI, 1978 adenoma or 47-57 wks mg/kg® carcinoma 5x weekly for 39-41 ¢ NR 8/49 (16) NR Ibid. 34-44 wks mg,’kg eee. Follicular cell Rat Inhalation 6hr/d, 5d/wk, 10 ppm 0/48 (0) NR 0/50 (0) NR NTP, 1982a; adenoma or 78-103 wks Huff, 1983 carcinoma 40 ppm NR 3/46 (7) NR Ibid. C-cell adenoma 10 ppm 3/48 (6) 1/49 (2) 5/50 (10) 3/48 (6) Ibid. or carcinoma 40 ppm 1/46 (2) 1/45 (2) Ibid. Follicular Rat Inhalation, 7hr/d, 5d/wk, O ppm EOB 4/48 (8) 5/48 (10) 1/4B (2) 1/48 (2) Wong et al., 1980; epithelial 0S-diet for 18 mos and DS. 198 adenama (cnt1/DS) 20 ppm EDB 3/46 (7) 1/48 (2) Ibid and no DS (EDB/cnt1) 20 ppm EDB 18/48 (38)* 18/45 (40)* Ibid. and DS (vs. EDB/cnt1 (vs. EDB/cnt1 . (EDB/DS) and cnt1/DS) and cntl/DS) ADRENAL Cortical adenoma Rat Gavage 5x weekly for 37-38 NR 0/20 (o)* NR 0/44 (0) NCI, 1978 or carcinoma 47-57 wks mg/kg® 5x weekly for 39-415 NR NR 4/45 (9) Ibid, 34-44 wks mg/kg Pheochromocytoma Rat Inhalation 5Shr/d, 5d/wk, 10 ppm 4/49 (8) 3/50 (6) 5/49 (10) 1/49 (2) NTP, 1982a; 78-103 wks Huff, 1983 | 40 ppm 0/48 (0) 0/47 (0) Ibid. Pheochromocytoma, Rat Inhalation, 7hr/d, Sd/wk, O ppm EDB 2/48 (4) 1/48 (2) 1/48 (2) 0/48 (0) Wong et al., 1980; cortical adenoma DS-diet for 18 mos and DS 82" or carcinoma (cnt1/DS) 20 ppm EDB 11/46 (24)* 6/48 (13)* Ibid. and no DS (vs. untreated (vs. untreated (EDB/cnt1) control) control) 20 ppm EDB 6/48 (13)* 8/45 (18)* Ibid. and 0S (vs. cnt1/DS) (vs. cnt1/DS)

(continued on following page)

Table 23. (continued)

Tumor Incidence?

(continued on following page)

Route Duration Lresponders/sample size (percent)] Tumor Type Species of of Dose Control Treated Reference Exposure Exposure ale emale SKIN Skin papillomas Mouse Dermal EDB-single dose 75.0 mg NA 6/90 (7) h NA 2/30 (7) Yan Duuren et al., and skin squamous PMA°-3x weekly EDB/appli- (2/90 (2)] 1979 cell carcinomas cation 40.005 mg PMA 3x weekly 25.0 mg/ NA 0/100 (0, NA 2/30 (7) h Ibid. application 0/30 (0) £2/30 {7)1 50.0 mg/ NA NA 8/30 (27)*** Ibid. application [3/30 (10)J" Skin subcutaneous Mouse Inhalation Ghr/d, 5d/wk, 10 ppm 0/45 (0) 0/50 (0) 0/50 (0) 5/50 (10)* NTP, 1982a; fibrosacrcoma 78-103 wks ea Huff, 1983 40 ppm 2/50 (4) 11/50 (22) Ibid. Skin subcutaneous Rat 10 ppm 3/50 (6) 0/50 (0) 6/50 (12) 0/50 (0) fibroma | 40 ppm 8/50 (16) 3/50 (6) Subcutaneous Rat Inhalation, 7hr/d, 5d/wk, O ppm EDB = 3/48 (6) 0/48 (0) 1/48 (2) 0/48 (0) Wong et al., 1980; mesenchymal tumor DS-diet for 18 mos and DS 198 “2 (cnt1/DS) 20 ppm EDB 11/46 (24)* 1/48 (2) Ibid. and no DS (vs. untreated (EDB/cnti) control) 20 ppm EDB 4/48 (8) 4/45 (9) Ibid. and DS (EDB/DS) MESENTARY (OMENTUM) Hemangiosarcoma Rat Inhalation, 7hr/d, 5d/wk, O ppm EDB 1/48 (2) 0/48 (0) 0/48 (0) 0/48 {0) Wong et al., 1980; DS-diet for 18 mos and DS 198 (cnt1/DS) 20 ppm EDB 0/46 (0) 0/48 (0) Ibid. and no DS (EDB/cnt?) 20 ppm EDB 11/48 (23)* 8/45 (18)* Ibid. and DS (vs. cnti/DS (vs. cnt1/DS (EDB/DS) and E0B/cnt1) and EDB/cntl)

Table 23. (continued)

Tumor Incidence?

Route Duration [responders/sample size (percent)] Tumor Type Species of of Dose ControT ‘Treated Reference Exposure Exposure fale emate fale emate LUNG Benign papilloma Mouse Dermal 3x weekly 25.0 mg/ NA 30/100 (30)3 NA 24/30 (80)** Van Duuren et al., application 11/30 (37) sid (1979) 50.0 mg/ NA | NA 26/30 (87) Ibid. application Alveotar/ Mouse Gavage 5x weekly 62 mg/kg! 0/18 (0)J 0/20 (03 4/45 (9) 10/43 (23)*»" nc, 1978 bronchiolar for 53 wks o/20 (0)K 0/20 (0) ie adenoma 107 mg/kg™ | 10/47 (21)"»" 6/46 (13) Ibid. Alveolar/ Mouse Inhalation 6hr/d, 5d/wk, 10 ppm 0/41 (0) 4/49 (8) 3/48 (6) 11/49 (22)* NTP, 1982a; bronchiolar adenoma 78-103 wks mae ek Huff, 1983 or carcinoma 40 ppm 23/46 (50) 41/50 (82) Ibid. Alveolar/ Rat 10 ppm 1/50 (2) 0/50 (0) 2/50 (4) 0/48 (0) Ibid. bronchiolar adenoma - or carcinoma 40 ppm 1/50 (2) 5/47 (11) Ibid. Lung tumor Rat Inhalation, 7hr/d, 5d/wk O ppm E0B 0/48 (0) 0/48 (0) 0/48 (0) 0/48 (0) Wong et al., 1980; (hemogiosarcoma, DS-diet for 18 mos and DS 1982 bronchogenic ({cnt1/DS) carcinoma) P 20 ppm EDB 3/46 (7) 0/48 (0) Ibid. and no OS (EDB/cnt1) 20 ppm EDB 9/48 (19)* 2/45 (4) Ibid. and 0S (vs. cntl/0S (EDB/DS) and EDB/cnt1) CIRCULATORY SYSTEM Hemangiosarcoma Rat Gavage 5x weekly for 37-38 0/20 (ayk 0/20 (o)k 11/50 (22)* 1/49 (2) NCI, 1978 of circulatory for 47-53 wks mg /k qd system (mainly 5x weekly for 39-4 | 4/50 (8) 3/48 (6) Ibid. spleen) 34-44 wks mg /kg® Hemangiosarcoma Mouse Inhalation 6hr/d, 5d/wk, 10 ppm 0/45 (0) 0/50 (0) 0/50 (0} 11/50 reir NTP, 1982; of circulatory 78-103 wks aii Huff, 1983 system (mainly in 40 ppm | 2/50 (4) 23/50 (46) Ibid. retroperitoneal area) Hemangiosarcoma Rat 10 ppm 0/50 (0) 0/50 (D) 1/50 (2) 0/50 (0) Ibid. of circulatory ‘ea s system (mainly 40 ppm | | 15/50 (30) 5/50 (10) Ibid,

spleen)

(continued on following page)

Table 23. (continued)

Tumor Incidence?

Route Duration Lresponders/sample size (percent)] Tumor Type Species of of Dose tontrol Treated Reference Exposure Exposure fale ‘emale @ emale SPLEEN Hemangiosarcoma Rat Inhalation, 7hr/d, 5d/wk, O ppm EDB 0/48 (0) 0/48 (0) 0/48 (0) 0/48 (0) Wong et al., 1980; Ds-diet for 18 mos and DS 1982 (cnt1/DS) 20 ppm EDB 10/46 (22)* 6/48 (13)* Ibid. and no DS (vs. untreated (vs. untreated (EDB/cnt1) control) control) 20 ppm EDB 15/48 (31)* 10/45 (22)* Ibid. and DS (vs. cntl/DS) (vs. cnt1/DS) (EDB/DS) REPRODUCTIVE Mammary gland Mouse Gavage 5x weekly 62 mg/kg! NA 0/20 (0)3 NA 3/48 (6) NCI, 1978 adenocarcinoma for 53 wks 0/20 (0)K 107 mg/kg™ NA NA 1/50 (2) Ibid. Mammary gland Rat Sx weekly for 37-38 NA 1/20 (5)k NA 0/50 (0) Ibid. adenoma or 47-57 wks mg /kg® adenocarcinoma 5x weekly for 39-4 NA NA 2/50 (4) Ibid. 34-44 wks mg/kg? ra Mammary gland 5x weekly for 37-38 NA 0/20 (o)k NA 0/50 (0) Ibid. adenoma or 47-57 wks mg/kg fibroadenoma 5x weekly for 39-4if NA NA 3/50 (6) Ibid. 34-44 wks mg/kg Mammary gland Mouse Inhalation 6hr/d, 6d/wk, 10 ppm NA 2/50 (4) NA 14/50 (28)*** —NTP, 1982; adenocarcinoma 78-103 wks ‘ Huff, 1983 40 ppm NA NA 8/50 (16) Ibid. Mammary gland Rat 10 ppm NA 4/50 (8) NA 29/50 (58) *** Ibid. fibroadenoma | 40 ppm NA NA 24/50 (48) Ibid. Mammary tumor Rat Inhalation, 7hr/d, 5d/wk, QO ppm EDB NA 2/48 (4) NA ayaa (17)* Wong et al., 1980; (adenoma, fibro- Ds-diet for 18 mos and DS (¥s. untreated 1982 adenoma, carcinoma, (cnt1/DS) control) adenocarcinoma) , 20 ppm EDB NA NA 25/48 (52) Ibid. and no DS {vs. untreated {EDB/cnt1) control) 20 ppm EDB NA NA 13/45 (29)* Ibid. and DS {¥s. EDB/ent1) (EDB/DS) Tunica vaginalis ‘at Inhalation 6hr/d, 5d/wk, 10 ppm 0/50 (D) NA 7/50 (14)** NA NTP, 1982; of testis- 78-103 wks og Huff, 1983 mesothelioma 40 ppm NA 25/50 (50) NA Ibid.

(continued on following page)

Significance levels if reported: =<p 0.05, * = p<0.01, *** = p<0.001

DS = disulfiram Disulfiram was incorporated in diet at 0.05 percent by weight. Cnti = control

Time-weighted average for low doses were 38 and 37 mg/kg EDB for males and females, respectively. Male and female rats were administered 40 mg/kg for 41 weeks followed by a cycle of--Idosage-free week followed by 4 weeks of 40 mg/kg--for a total treatment time of 47 weeks for males and 57 weeks for females.

Time-weighted average high doses were 41 and 39 mg/kg EDB for males and females, respectively. Male and female rats were administered 80 mg/kg for 16 weeks, no dose for 13 weeks, 40 mg/kg for 12 weeks followed by a cycle of--] gosa e-free week followed by 4 weeks of 40 mg/kg--for a total treatment time of 34 weeks for males and 44 weeks for emales.

NR = not reported

Incidence of squamous cell carcinomas, where greater than zero, is given in brackets. From information presented by authors, it is assumed that significance was determined by combining results for papillomas and carcinomas.

NA = not applicable Untreated control Vehicle control

Time-weighted average low dose was 62 mg/kg of body weight. Mice were administered 60 mg/kg for 10 weeks, 100 mg/kg for 2 weeks, 60 mg/kg for 51 weeks, and then observed for 25 to 37 weeks.

Time-weighted average high dose was 107 mg/kg of body weight. Mice were administered 120 mg/kg for 10 weeks, 200 mg/kg for weeks, 120 mg/kg for 27 weeks, 60 mg/kg for 14 weeks, and then observed for 24-25 weeks.

P-value computed for comparison of treated group to vehicle control.

PMA = phorbol myristate acetate

Table 24. EDB Carcinogenicity by Study

Route of Exposure Tumor Type ReproInhal- Nasal ductive Reference Species Dermal Gavage ation Adrenals Kidney Liver Lung Mesentary Cavity Organs Skin Spleen Stomach Thyroid van Duuren Mouse x x x et al., 1979 NCI, 1978; Mouse x x K Rat x x* x x NTP, 1982a; Mouse x x x x x Huff, 1983 Rat x x x x x Wong et al., Rat: EDB x x x x x x 1980; T98Z = alone Rat: EDB x x x x x x x x x x

and disulfiram

4significant in high-dose female rats in time-adjusted analysis only.

HEALTH EFFECTS--HUMAN

Summary

Envirologic Data has rated EDB on a scale of 0 to 3, as indicated, in each of the following categories:

Acute Toxicity -- 2 Reproductive Effects -- 3 Teratogenicity -- 1 Mutagenicity -- 2 Carcinogenicity -- 3

Zero corresponds to the absence of the health effect, 1 corresponds to insufficient evidence, and 2 and 3 refer to increasing evidence of or potency of the substance being rated (see "Methods" section). In the discussion which follows in this summary and in later sections regarding human health, the non-human data has been important. As it was discussed earlier in the "Health Effects--Non-Human" section, only the available data on humans is presented in detail here.

Acute toxicity was rated by Envirologic Data based upon experimental results on animals, aS well as several clinical experiences in which accidental exposure led to symptoms of acute toxicity, including death. In one case two workers died following exposure to a dilute (0.3-percent) solution of EDB.

Teratogenicity was rated 1 because insufficient data were available. Data on reproductive effects of EDB were more available, including abundant animal data. In addition, two epidemiological studies were available. In one, reproductive performance of workers in four chemical plants was compared with national averages; in one plant, fertility was reduced 50 percent. In another study, sperm counts of workers exposed to EDB were compared with the general population, and found to be normal.

Mutagenicity was rated 2 based upon animal studies, as well as studies of human lymphocytes cultured in vitro. There was unequivocal evidence for mutagenesis in microbial tests, but ambiguous results in whole mammals. All dominant lethal mutation assays conducted on whole mammals were negative, but DNA damage was noted in three studies of whole mammals. Such damage, measured in hepatic cells rather than germ cells, is usually repaired and, if not repaired, rarely passed on to future generations because of nonviability of daughter cells.

Carcinogenicity was rated 3 based upon animal studies presented earlier ("Health Effects--Non-Human"). Risks of cancer mortality due to EDB have been estimated by several authors. In all cases, it was Necessary to extrapolate from animals to humans, and from high experimental doses to actual human exposure levels, which are relatively low. Examination of risk assessments revealed great variability in exposure assumptions, variability in selection of low dose extrapolation models, and variation in risk estimates. No firm quantitative conclusion can be drawn regarding risks of cancer mortality from exposure of the general U.S. population to EDB, given the variability in available data and state of the art in toxicology at this time.

-89-

Acute Toxicity

Envirologic Data has rated EDB's acute toxicity to humans as a 2 0na scale of 0 to 3, where 0 is least toxicity and 3 is most. This rating was based upon a CTCP* rating of 4 on an ascending scale of 1 to 6 (Gosselin et al., 1976). Envirologic Data's rating is also based upon abundant evidence of acute toxicity in animals, including mammals (see "Health Effects--Non-Human" section), as well as evidence from clinical experiences with accidental and experimental human exposures to EDB. This evidence is presented below.

Kochmann (1978) reported that a worker who had been repeatedly exposed to EDB demonstrated conjunctival irritation, swelling of the lower eye lids, and glandular swelling under the chin and angle of the jaw. The Ttevel of exposure was not given, and a subsequent exposure caused the symptoms to recur with pharyngeal and bronchial irritation, loss of appetite, headaches and depression.

Pflessor (1938) found that when 0.5 ml of EDB was rubbed on the forearm, and washed with soap and water three minutes later, swelling, itching, and reddening developed in the exposed area within 24 hours. Symptoms subsided in two to three days. Longer exposure resulted in a 15- to 30-day healing period.

Olmstead (1960) reported a case of ingestion of capsules which contained a total of 4.5 ml of EDB (140 mg/kg). The victim complained of abdominal pain, nausea, vomiting, and diarrhea within 48 hours of ingestion. Death occurred within 54 hours. At autopsy, liver pathology, renal congestion, lung congestion, edema and reddening of the gastric mucosa without necrosis was found.

Two workers were killed while cleaning a tank containing 0.3-percent EDB in water and EDB in the air in excess of 28 ppm (Wade and Sakura 1982). The workers were exposed to 2 to 4 inches of liquid solution for several minutes before losing consciousness. Skin exposure occurred for up to 15 minutes. Death occurred at 12 and 72 hours after the victims lost consciousness and had vomiting and diarrhea. Dermal burns extensive kidney, liver, heart and lung damage were seen, and tissue levels of EDB ranged from 0.5 ppm in the brain to 3.6 ppm on skin and in mesothelial fat.

Subchronic and Chronic Effects

Although there are no experimental data on subchronic or chronic effects of EDB on humans, it is safe to infer the effects from animal studies. As indicated in the "Health Effects--Non-Human" section, data are available on exposure of mice, rats, chickens, and quail. Effects are likely to be qualitatively similar, although there may be quantitative differences in effective doses. Envirologic Data has elected not to examine this question because general U.S. population exposure is far lower than any levels of EDB likely to approach these experimental levels; moreover, actual exposure levels are far lower than those likely to cause short- or intermediate-term effects.

*CTCP is Clinical Toxicology of Commercial Products

-90-

Teratogenicity and Reproductive Effects

Because of the equivocal data in this area, Envirologic Data has assigned a rating of 1 to teratogenicity. No data has been published regarding the teratogenic potential of EDB in humans. Although four studies examined teratogenicity in mice and rats, only one produced positive results (Short et al., 1975).

Envirologic Data has assigned a rating of 3 to reproductive effects because EDB has been shown to affect reproduction adversely in two species of mammals: bulls and rats. A series of studies by Amir et al. (1967, 1973, 1975, 1977) reported morphological defects in spermatozoa in bulls, decreased average sperm concentrations, and testicular abnormalities. Both male and female reproduction was adversely affected in rats (Short, et al. 1979). Males exposed by inhalation to 89 ppm of EDB failed to impregnate any females and exhibited atrophy of the testis, epididymis, prostate, and seminal vesicles. Females exposed by inhalation to 80 ppm exhibited diestrus (sexual inactivity) and abnormal menstrual cycles until 3 to 4 days after exposure ended.

Two epidemiologic studies have been conducted on reproductive effects in workers exposed to EDB. In one study, initiated by chemical companies, Wong et al. (1979) examined the reproductive performance of male workers in Four chemical plants. All married men (297) working at four plants in Arkansas and Texas who had been exposed to 0.5 to 5 ppm of EDB (duration not given) were included in the study. Male fertility was measured by examining the reproductive performance of the wives. Using information obtained from a questionnaire administered to the men, the number of live births to wives of workers was compared to national age-adjusted fertility rates. In three of the four plants studied, no difference in fertility rates was noted. In the fourth plant, however, there was a significant (p<0.05) 50-percent deficit of live births relative to expected numbers, indicating a decrease in fertility of plant workers. Exposure data on individual workers in this plant was not reported, but the group average was reported to be less than 5 ppm. Moreover, no information was provided on exposures to other chemicals used at the plants. Although these shortcomings make it difficult to draw firm conclusions regarding the effect of EDB on human male fertility, the authors did not provide viable alternative explanations of the effect.

Ter Haar (1980) conducted a survey of reproductive effects of EDB as measured by sperm concentration. Fifty-nine sperm samples were collected in 1977, and 24 in a follow-up study in 1978. The sperm counts were not different from published levels for the general population. Forty workers had EDB exposures of less than 0.5 ppm and 19 workers had exposures of 0.5 to 5 ppm.

Mutagenicity

Envirologic Data has rated the mutagenicity of EDB 2 on a scale of 0 to 3, where 0 corresponds to non-mutagenicity, 1 to insufficient evidence, and 2 and 3 to increasing evidence of mutagenicity. Based upon abundant data regarding EDB effects on non-human genetic systems (see "Health Effects--Non-Human"), there is little doubt that EDB is mutagenic in genetic systems upon exposure to them. Indeed, EDB has been repeatedly demonstrated to be mutagenic in test organisms among the

-91-

bacteria, fungi, vascular plants, insects, and mammals. Positive

mutagenic results accounted for 52 of 68 mutagenesis assays assessed by Envirologic Data.

Despite unequivocal evidence of mutagenicity upon exposure to genetic systems, the degree to which the genes and chromosomes of mammals may become exposed to EDB ingested or inhaled by whole organisms is not known, (The distribution, metabolism, and excretion of EDB by test organisms, including mammals, were discussed earlier in the "Pharmacokinetics" section of this document.) Among the 68 mutagenesis assays evaluated by Envirologic Data, 17 were conducted on mammals. Of the 17, nine were assays of mutagenicity in mammalian cells cultured in vitro; all nine were positive.

A different mammalian mutagenesis picture emerges from an examination of mutagenesis assays using whole organisms. In contrast to the positive tests on cultured cells, five in vivo dominant lethal mutation assays were applied to caged mice and rats; in all five assays, exposure to EDB failed to induce dominant lethal mutations. Positive mutagenicity tests in cells cultured in vitro are not inconsistent with negative tests conducted in vivo using whole organisms, because the dominant lethal assay detects only a small subclass of possible mutations (those that are lethal to progeny of the test organism). Moreover, detoxification, excretion, compartmentalization and other pharmacokinetic mechanisms may isolate EDB from mammalian genetic systems; such potential isolating mechanisms may exist only as a result of the organ systems which operate jin vivo, but which are absent in test systems consisting of cells cultured in vitro. Lastly, it is generally assumed that most dominant lethal mutations may be described as lesions of chromosomes, and that the mutations induced by EDB occur at the gene level rather than at the chromosome level. Consequently, negative results in dominant lethal assays do not assure absence of mutagenesis, whereas positive results would clearly signify mutagenesis. Envirologic Data's rating of 2 rather than 3 reflects this uncertainty regarding mutagenesis to whole organisms.

Envirologic Data has encountered three studies of mutagenesis of EDB in intact mammals (Nachtomi and Sarma, 1977; White et al., 1981; Storer and Conolly, 1983). These assays were discussed earlier in the “Health Effects--Non-Human" section. Single-strand DNA damage was detected in the liver cells of caged mice and/or rats injected intraperitoneally or by gavage with EDB. Nevertheless, Envirologic Data regards these tests as equivocal regarding potential EDB mutagenicity to humans for three reasons: first, in two studies, exposure was via intraperitoneal injection, whereas the major routes of human exposure are inhalation and ingestion, These routes would enable some potential isolating mechanisms to act which would be bypassed by intraperitoneal injection; second, there is some question of whether DNA strand breaks are heritable, as cells in which such breaks occur are likely to either repair the DNA damage or fail to produce viable daughter cells upon division; and third, the validity of these methods in predicting mutagenicity may be questioned since the DNA damage may be secondary to EDB toxicity to the cell rather than a true mutation. In the latter event, cell death and necrosis will lead to artifactual DNA breakage. Hence, the paucity of unequivocal data on EDB mutagenicity to test organisms in vivo supports

Envirologic Data's rating of 2 rather than 3.

~92-

Carcinogenicity

Envirologic Data has rated the carcinogenicity of EDB 3 on a scale of 0 to 3, in which QO corresponds to non-carcinogenicity, 1 to insufficient evidence, and 2 and 3 to increasing evidence of carcinogenicity. The rating of 3 reflects unequivocal data on both mice and rats, which was discussed in detail earlier, in the "Health Effects--Non-Human" section. EDB was carcinogenic by all routes of exposure; response was very high (over 80 percent in some cases); malignancies were of many varieties; and carcinogenicity was induced at sites remote from the point of application.

Experimental results regarding carcinogenicity in non-human systems were discussed earlier (see "Health Effects--Non-Human"), and will not be discussed again here, That is not to say, however, that the non-human data are irrelevant to human carcinogenicity. To the contrary: two standard means of transforming cancer causation in animals to estimates of potential cancer causation in humans are commonly employed. First, a biotagical extrapolation from animal to human systems, taking into consideration such factors as life expectancy, is necessary. Second, a numerical extrapolation is necessary to relate high (experimental) doses in animals to low (actual) exposure in the general human population. Potential human carcinogenicity may be assessed also by = any epidemiological or clinical studies which appear in the scientific or medical literature. These are limited in the case of EDB, and are discussed below.

In a human epidemiology study, Ott et al. (1980) examined the cancer mortality experience of employees occupationally exposed to EDB in two manufacturing plants. Two deaths from malignant neoplasms were observed versus 3.6 expected in one plant. In the second plant, 5 deaths from malignant neoplasm were observed versus 2.2 expected (p<0.072). Workers at the second plant were exposed to various other chemical agents in addition to EDB. The authors pooled the results from the two plants and examined mortality in relation to duration of exposure and interval since first exposure to EDB. No statistically significant increases in deaths were observed relative to duration of exposure. The authors concluded that their findings neither rule out EDB carcinogenicity to humans, nor establish it.

Risk Assessment

Introduction. Having examined exposure to EOB (see "Sources of

Exposure" section) and health effects of EDB, it is important to examine the issue or risk, where:

Risk = exposure + effect.

A typical unit of risk is deaths from cancer over a lifetime of chemical exposure per million individuals. This would correspond to an incremental risk factor of 10-6 (that jis, 0.000001). About one individual in five, that is, 20 percent, typically dies of cancer in the U.S. population (Epstein, 1979). This corresponds to a risk factor of 0.2. Hence, an added (or incremental) risk factor of 10-° would elevate this to 0.200001. Likewise, a risk factor of 10-5 (that is 0.00001) would elevate a one-in-five cancer mortality risk to 0.20001, Ae . risk factor of 10-4 would elevate the cancer mortality risk to 0.2001.

-93-

In the U.S., where there are some 230 million individuals, levels of any chemical corresponding to an incremental risk factor of 10-® would increase the number of deaths from cancer by 230 added deaths in the nation, that is, from a base rate of 20 percent, or 46,000,000 deaths*. An incremental risk factor of 10-5 would add some 2,300 cancer deaths, and a 10-4 risk factor would add about 23,000 deaths.

The acceptability or non-acceptability of a given risk level is quite different when viewed from the perspective of the individual vs. the public policy maker. An individual might accept an incremental personal risk of one in 10,000, whereas the public policy maker must consider the 23,000 deaths that this same risk factor would imply nationally. Although Envirologic Data cannot advise the public or the public policy maker regarding what constitutes acceptable risk, it has historically been the case that EPA's regulations have implicitly ‘accepted’ risks of LO=?' te 10-9 -

Examination of EDB Risk. Envirologic Data has examined EDB risk assessments by a variety of sponsors (Table 25), including federal and state agencies, as well as industry. The risk they predict depends upon the exposure assumptions used as inputs, and the low dose extrapolation model employed. The exposure assumptions were discussed earlier (see "Sources of Exposure" section). Table 25 sets forth the low dose extrapolation models and risks.

Table 25 reveals that, among the risk assessments examined by Envirologic Data, only two estimated risks to children. These incremental risk estimates ranged from 0.25 to 100 deaths per million individuals, that is, a 400-fold variation. Five risk assessments estimated risk to adults, producing incremental risk estimates ranging from 0.08 to 3780 deaths per million individuals--a variation of more than five orders of magnitude.

An examination of the exposure assumptions (Table 12) reveals a much narrower range of variation, that is, for adults, a range of 10.5 to 2192.2 micrograms per year, which corresponds to a 209-fold variation. It would, in principle, be possible to come to a single risk estimate by adjusting the exposure assumptions to a most reasonable figure. However, jin the present case, the predominant source of variability in risk arises from the selection of the low dose extrapolation model. At present, there is no scientific basis for preferring any one model (OTA 1981), as the sciences of risk assessment and toxicology are insufficiently advanced to discriminate among most models.

Perhaps the most salient conclusion that can be drawn from the data at hand is that there is a need to address the philosophy of risk assessment models, and to rank them in order of risks estimated from a single exposure input. It would then be possible for regulators to address the question of how conservative a model should be to protect public health in an uncertain world.

*Of course, cancer-producing exposures to chemicals may result in elevated cancer incidence some 10 to 40 years from now, but at these future dates, treatment technologies may have improved, and the death rate may have decreased for some types of cancer.

-94-

Table 25,

Risk Assessment Source

EDB Risk Assessments:

Low Dose

Extrapolation

Model(s)

The Risk Component

Risk*

(deaths per

million individuals)

U.S. EPA, 1984d U.S. EPA, 1984f

U.S. EPA, 1980a

U.S. EPA, 1983a

NYS DOH (Kim, 1984)

Grocery Mfrs. of American 1984

Dynamac Corp. 1983

Brown, 1983

Winzler and Kelly, 1982

Ramsey et al., 1979 SRI, 1982

California DHS, 1984

CAG, 1980 coc, 1984

one-hit and one-hit with Weibull timing

one-hit with Weibull timing

one-hit

one-hit with Weibull timing

Crump 1980 EPA Weibull

one-hit Guess and Crump 1976 multistage

one-hit, multi-stage, probit, logit,

Adult Child 1 to 10 1 to 100 264 -- 3780 -- 0.08 0.25 1 wz occupational

Weibull, gamma multi-hit

one-hit

multistage (Global), gamma multi-hit

one-hit with Weibull timing

one-hit

occupational

occupational

165 to 820

*Units converted by Envirologic Data for purposes of standardization

and comparison.

Moreover,

risks were calculated based upon varying

sources and durations of exposure, as well as varying source contribution assumptions, which were presented earlier (see Table 12).

-95-

MECHANISMS OF ACTION

Summary

EDB exerts its toxic effects primarily in the liver and kidneys, where it concentrates. In the liver, EDB is cytotoxic, and interferes with such enzyme systems as the cytochromes, which are required for normal cell metabolism. Enzyme poisoning causes cell-death (necrosis) and numerous functional abnormalities in surviving cells. Acute, subchronic, and chronic effects appear to result from the accumulated effects of toxic action at the cellular level. Moreover, EDB may also

act through toxic metabolites, a phenomenon known as _ ‘metabolic activation. '

EDB is known to be synergistic with two substances to which humans are frequently exposed: the anti-alcohol abuse drug disulfiram and the fungicide diethyldithiocarbamate (DDC). Both substances enhance EDB toxicity by prolonging the life of EDB or its toxic metabolite, bromoacetaldehyde, in the body.

Reproductive effects on bulls and other male test organisms include inhibition of sperm formation, reduction in sperm counts, and induction of morphological abnormalities in sperm. These EDB effects appear to be exerted through removal of sulphur from cysteine, a major component of nuclear basic proteins of spermatozoa.

Mutagenic effects of EDB appear to be exerted through conjugation of EDB with glutathione, producing a metabolically activated intermediate which alkylates sites on DNA molecules. Such alkylation damages the sites and causes breaks in the DNA strands, which alter genes and chromosomes.

Carcinogenic effects of EDB appear to be primarily related to its ability to bind with RNA, and secondarily to its ability to bind with DNA. Binding with RNA polynucleotides would interfere with nomnal binding of protein to RNA, leading to abnormal (cancerous) growth of cells. The mechanism of EDB binding requires metabolic activation through conjugation of EDB with glutathione; it is the conjugate that binds to polynucleotides, damaging the structural organization of the chromatin, impairing nuclear function and cell growth.

Mechanisms of Acute and Subchronic Toxicity

EDB probably exerts its toxic effect by poisoning the cytochrome P-450 enzyme system in target tissues (Moody et al., 1982). This system is the terminal component of an electron-transport chain found in adrenal mitochondria and liver microsomes. P-450 is an important link in the detoxification of foreign substances, acting via hydroxylation. The introduction of hydroxyl groups provides sites for conjugation with polar substances. This increases the solubility of the compound, thereby facilitating its excretion. P-450 also activates the synthesis of bile salts and steroid hormones.

-96-

In rats, EDB altered the cytochrome P-450 and the fatty acid composition of microsomes, resulting in a significant increase in the relative content of palmitic, oleic and linoleic acids (p<0.01), a decrease jin arachidonic acid (p<0.01) and a reduction in P-450 to 64 percent of control (Moody et al., 1980). The mechanisms involved were not stated. Details of the Studies indicating the effect of EDB on cytochrome P-450 and fatty acid composition are presented in Table 26,

Treatment of rats with 110 mg of EOB per kilogram of body weight by gavage resulted in increased liver weight, increased DNA synthesis in the liver and increased levels of the enzymes glutamic pyruvic transaminase and sorbital dehydrogenase. All of these effects are typical of liver cytotoxicity (Nachtomi, 1980). In guinea pigs treated with 30 mg/kg, the tissues with the highest levels of EDB were the same tissues as those reported to exhibit granular degeneration of parenchymal tissue after EDB administration (Plotnick and Connor, 1976). These mechanisms are unknown.

Synergism

Disulfiram (Antabuse) and diethyldithiocarbamate (DDC, a fungicide) have each been shown to exhibit synergism with EDB (Table 26). Disulfiram inhibits microsomal oxidation of EDB by inhibiting the enzyme, aldehyde dehydrogenase (van Bladeren et al., 1981b). This causes longer retention of the active metabolite bromoacetaldehyde, resulting in enhanced toxicity (Plotnick et al., 1980; van Bladeren et al., 1981b).

DDC is known to reduce microsomal cytochrome P-450. It is assumed that active intermediates, s-bromoethy]1-glutathione and s-bromoethylcysteine, are formed via this reduction route. By its interference with metabolism, DDC prolongs the life of EDB and the availability of its active intermediates, thereby enhancing EDB's effect on covalent binding to macromolecules, protein, RNA and DNA.

Mechanism of Teratogenicity and Reproductive Effects

The deleterious reproductive effects of EDB on spermatozoa are given in Table 26. (Edwards et al., 1970; Amir, 1973; Amir and Lavon, 1976; Amir et al., 1977; Short et al., 1979; Courtens et al., 1980) are probably due to the fact that the nuclear basic proteins of spermatozoa contain relatively large numbers of cysteine residues. EDB readily removes sulphur from cysteine, leaving a reactive (positively charged) carbonium ion at the point of excision (Edwards, 1970). Reaction of EDB with cysteinal thiol (SH) units causes an alkylating effect (Edwards, 1970; Courtens et al., 1980) which disturbs the normal binding of basic nuclear protein moTecules to DNA (Amir et al., 1977), causing improper chromatin packing (Amir, 1973; Amir and Lavon, 1976). The resulting sperm abnormalities are responsible for the teratogenic and reproductive effects of EDB (see "Health--Non-Human" section).

-97-

Mechanism of Mutagenicity

The mutagenic effects of EDB (see "Health Effects--Non-Human" section) occur both with and without metabolic activation. EDB has a natural electrophilicity and therefore is attracted to nucleophilic sites on DNA. This may relate to the capability of EDB to alkylate (that js, covalently bind to) DNA (CAG, 1977; Banerjee and van Duuren, 1979, 1983; Hil] et al., 1978; Ozawa and Guenger:ch, 1983; Shih and Hill, 1981; Sundheimer et al., 1982; White et al., 1981). Alkylation may weaken sites on DNA Teading to DNA ‘strand breaks (White et al., 1981).

Alkylation of DNA by EDB also may lead to other types of mutations.

In addition to acting as a direct mutagen, EDB also undergoes activation that leads to activated metabolites possessing enhanced mutagenic activity. Of the two activated metabolites depicted in Figure 1, S-2-bromoethy] glutathione, produced by the conjugative route, is a stronger mutagen than bromoacetaldehyde, produced by the oxidative route (Nachtomi, 1980; van Bladeren et al., 1980; White et al., 1983).

Mechanism of Carcinogenicity

EDB binds to DNA and RNA (CAG, 1977; Hill et al., 1978; Banerjee and van Duuren, 1979; Shih and Hill, 1981; White et al., 1981; Sundheimer et al., 1982; Banerjee and van Duuren, 1983; Ozawa and Guengerich, 1983) Tsee Table 26). There is a strong preference for EDB to bind with RNA over DNA: 5 times greater in rat liver (Hill et al., 1978; Sundheimer et al., 1982) liver and 10 times greater’ in rat kidneys (Hill et al., T5978). A high percentage of hepatocellular carcinomas have been reported in studies after administration of EDB (see "Health Effects--Non-Human”™ section under "Carcinogenicity"), and EDB shows a_ preferential concentration in rat liver (Plotnick et al., 1980). It is thought that cancer causation is related to the tendency of EDB to bind with polynucleotides. This binding would interfere with normal binding of proteins to RNA, resulting in abnormal cell growth.

The mechanism of the binding of EDB to nucleic acids requires Metabolic activation, that is, the toxicity-enhancing action of (EDB) metabolites (Banerjee et al., 1979; Banerjee and van Duuren, 1979). Although there is some evidence indicating that bromoacetaldehyde (produced by the P-450 pathway) damages DNA by alkylation (Nachtomi and Sarma, 1977; Hill et al., 1978; White et al., 1981; Banerjee and van Duuren, 1983), there is general agreement among researchers that it is primarily the conjugative pathway that is responsible for the carcinogenic activity of EDB (CAG, 1977; Hill et al., 197B; Shih and Hill, 1981; van Bladeren et al., 1981b; Sundheimer et al., 1982; Ozawa and Guengerich, 1983; White et al., 1983). 2-Bromoacetaldehyde binds preferentially to microsomal proteins, but the activated conjugate with glutathione, S-2-bromoethyl-glutathione, binds to polynucleotides (Shih and Hill, 1981) (Figure 2).

-98-

It is believed that EDB is a substrate for glutathione S-transferase (CAG, 1977; Hill et al., 1978; Sundheimer et al., 1982; Ozawa and Guengerich, 1983), Attack on the first bromine by glutathione results in an unstable reactive alkylator which reacts with nucleophilic (positively charged) sites on nucleic acids (CAG, 1977; Sundheimer et al., 1982). The equimolar binding of glutathione and EDB to DNA suggests that glutathione and a metabolite of EDB are linked together in zn adduct with DNA which has been tentatively identified as S-[2-(N’-guanyl)ethy1] GSH. However, the intact molecule has not been isolated (Ozawa and Guengerich, 1983).

Figure 2. Proposed Mechanism of EDB Binding to Polynucleotides (Shih and Hill, 1981).

GSH Polynucleotide

>Bound to polynucleotide

BrCH,CH BR >SCHACH,Br

EDB GSH S- S-(2-bromoieae transferase ethy1)GSH

The binding of the activated EDB metabolite S-2-bromoethy1-GSH to nucleic acids most likely damages the structural organization of the chromatin. This may impair nuclear function which can lead to defective

growth.

-99-

Table 26.

Summary of Data on Mechanisms of EDB Action

Dose Test Animal Route Author and Date or Species Ouration Effect Proposed Mechanism ACUTE AND SUBCHRONIC TOXICITY Moody et al., rat dose not given, alteration of fatty acid NR 1980 microsomes astric and cytochrome P-450 a ntubation 18 hr composition of microsomes

Moody et al., rats 0.16 mg/kg decreased levels of toxicity via poisoning of 1982 gastric intubation cytochrome P-450 in the cytochrome P-450 in target

liver, kidney, lung and tissues

testis Nachtomi, 1980 rats 10-110 mg/kg for liver cytotoxicity mitosis and liver cell damage

Plotnick and Connor, 1976

Nachtomi, 1980

Plotnick, et al., 1980

van Bladeren et al., 1981b

guinea pigs

rats

rats

rats

48 hours-- 18 days gavage

30_mg/kg IP. 4-72 hours

indicated

tissues with highest concentrations of EDB

were kidney and liver

‘SYNERGISM

10-110 mg/kg food or gavage 48 hrs - 18 days

15 mg/kg oral w/ or w/o disulfiram pretreatment, 24 hrs

47-282 mg/kg stomach tube w/ or w/o disulfiram

ODC synergistic

Disulfiram synergistic

metabolite, 2-bromo-

Disulfiram synergistic

indicated by enhanced DNA labelling, elevated calcium

content, liver AP activity and GPT in serum

NR

intereferes with metabolism, thus slowing the rate at which the active intermediates are degraded

interferes with breakdown of the EDB

acetaldehyde

inhibits the microsomal

oxidation of EDB and induces transferases

(continued on following page)

Table 26. (continued)

misshapen sperm heads

malformed heads and

decreased sperm concen-

evolution of spermatids

damaged spermatogenic

Dose Test Animal Route Author and Date or Species Duration Effect TERATOGENICITY AND REPRODUCTIVE EFFECTS Amir, 1973 bulls 4 mg/kg orally alternate days 12-21 days Amir and Lavon, bulls 10 doses @ 4 mg/kg 1976 alternate days changed amino acid 20 days composition of the sperm proteins Amir et al., bulls 4 mg/kg in 10 doses 1977 orally on alternate tration days for 8 weeks Courtens et al., bulls 10 doses @ 4 mg/kg damaged late stage 1980 orally, alternate days for 20 days Edwards et al., rats 5 @ 10 mg/kg 1970 subcut, 2.5 hr cells mice 40 mg/kg IP 1-3 hr Short et al., rats 0 - 89 ppm, 7 brs/

Banerjee et al., 1979

mouse liver microsomes

day, 5 days/week for 10 weeks inhalation

reduced reproductive activity in rats at

concentrations that also

caused morbidity and mortality

MUTAGENICITY

0.047 mg. (conc. not given) + NADPH and microsomes

binding to macro-

molecules dependent on

metabolic activation

Bromoacetaldehyde is a

potential activated intermediate.

Proposed Mechanism

interferes with normal nuclear chromatin condensation

couples with cysteine residues in sperm histones, interrupting the normal

binding of protein to DNA

may disturb the normal binding of the basic nuclear protein molecule to DNA

coupled with the thiol groups of the cysteine residues causing an alkylating effect

removes sulphur from cysteine and produces a non-functional mustard

NR

Bromoacetaldehyde and bromoethanol bind to protein and DNA without activation. EDB requires activation

(continued on following page)

Table 26. (continued)

Dose Test Animal Route Author and Date or Species Duration Effect Proposed Mechanism Banerjee, and Mouse liver and 0.047 mg/mixture metabolically activated damages structural organizavan Duuren, 1983 forestomach total vol. omitted EDB is more reactive to tion of chromatin by binding chromatin 1 hour homologous than to histone and nonhistone exogenous DNA and to proteins which are involved nonhistone than to in the structural and funchistone proteins tional organization of nucleosomes and chromatin fibers Nachtomi, 1980 rats 10-110 mg/kg DDC enhances muta- interfere with metabolism, food or gavage genicity of E0B thereby increasing the time of 48 hrs. to 18 days retention of metabolites that bind to macromolecules. van Bladeren et al-, Salmonella modified Ames test mutagenic in Ames test caused by formation of 1980 typhimurd um w/ and w/o activation. reactive GSH conjugates 48 hours White et al., Hepatic nuclei 3 hours dose dependent increase DNA lesion characterized as 1981 from mouse 25-75 mg/kg IP. in the amount of damage strand breaks due to to hepatic DNA alkylation by E0B White et al., mice 50 mg/kg IP. genotoxicity of EDB GSH S-transferase catalyzed 1983 duration not given enhanced by deuterium substitution CARCINOGENICITY CAG, 1977 not given not given NR nucleophilic intermediates

Banerjee and van Duuren, 1979

Banerjee et al., 1979

mouse and rat microsomes

mouse liver microsomes

0.047 - 0.14 mg

total vol. omitted 1 hour

0.047 mg, total vol. unknown

EDB bound to macromolecules

Bound to protein and DNA

bind to nucleic acids

dependent upon microsomal activation. Indirect-acting carcinogen by way of an activated intermediate.

Binding dependent on metabolic activation

(continued on following page)

Table 26. (continued)

Dose Test Animal Route Author and Date or Species Duration Effect Proposed Mechanism Banerjee and van Mouse 0.047 mg/mixture metabolically activated damages structural organizaDuuren, 1983 chromatin total vol. omitted EDB is more reactive zation of chromatin by binding 1 hour to homologous than to histone and nonhistone exogenous DNA and to proteins which are involved nonhistone than to in the structural and histone proteins functional organization of nucleosomes and chromatin fibers Hill et al., rats 2,.63-3.16 mg/kg binding to macro- EDB is a substrate for ‘ae = IP. 24 hr molecules occurs glutathione S-transferase particularly in liver and kidney Nachtomi and rats 120 mg/kg damages DNA. Lesion probably causes strand breaks Sarma, 1977 stomach tube generally repaired via alkylation 4 hours within 96 hours. Ozawa and rat 939 mg (total EDB metabollically GSH mediates EDB binding to Guengerich, 1983 hepatocytes volume omitted) activated by GSH DNA via an adduct tentatively 1 hour identified as S-[2-(N’~guany] ethyl] GSH. Plotnick et al., rats 15 mg/kg, oral Nonuniform preferential NR 1980 = intubation distribution in rat 24 hours liver nuclei may result in hepatic carcinogenicity Shih and Hill, rat 967.6 mg (total EDB bound to synthetic GSH conjugate binds to 198) hepatocytes volume unknown) polynucleotides polynucleotides 30 minutes Sundheimer et al., rat 3.76 mg/liter Binding of EDB to RNA GSH s-transferase catalyzes 1982 hepatocytes 2 hour incubation is greater than binding the bioactivation of EDB to to DNA reactive intermediates that bind to nucleic acids van Bladeren et al., rats 47 - 282 mg/kg 2-b romoacetaldehyde conjugation pathway respon-

1981b

stomach tube

not responsible for carcinogenicity

sible for carcinogenic activity

(continued on following page)

Table 26. (continued)

Dose Test Animal Route Author and Date or Species Duration Effect Proposed Mechanism White et al., Mouse 25-75 mg/kg IP. EDB caused DNA strand Result from lability of DNA

White et al., $983; = —~

hepatic nuclei

mice

3 hours

50 mg/kg IP. duration unknown

breaks

genotoxicity of EDB enhanced by deuterium substi tution

sites alkylated by EDB

probably GSH transferase catalyzed conjugation of EDB

results in its genotoxicity

MEDICAL/OCCUPATIONAL SURVEILLANCE

Summary

Exposure to an agent which has the potential for disease induction does not invariably produce disease. Acute toxicity is frequently self-evident whereas chronic toxicity requires time to develop. Disorders such as cancer and heritable changes in the genetic material--mutations--may require years to be manifest.

As with most exposure and any resultant health effect, there is not a single “test" which can be run at a specific point in time which will delineate effect. Rather, one must follow the rigorous principles of medical surveillance including history, physical examination, and laboratory study.

Medical surveillance of individuals and human populations for EDB effect is hampered by the fact that there have been no prior epidemiological investigations to guide examiners. Aggregate screening of heavily or uniformly exposed individuals by physicians who are cognizant of diseases which may be induced by EDB is likely to provide optimal surveillance.

The following section outlines known acute and chronic effects of EDB. If one assumes that EDB is a potential human carcinogen and has the potential to interfere with reproduction, careful search of each body system via history, physical examination, cytology, and other laboratory studies offers the best practical approach.

Clinical Presentation

Exposure to EDB occurs from inhalation, ingestion, and absorption through the skin (NIOSH, 1978a; Sittig, 1981). Signs and symptoms in clinical presentation are due to a) central nervous system depression, b)

pulmonary irritation, c) hepatic and renal damage. Symptoms include anorexia, nausea, headache, weakness, hematemesis, diarrhea, abdominal pain, and confusion, Signs include rapid pulse, heptomegaly, jaundice, pulmonary lesions and pulmonary edema (NIOSH, 1978a; Sittig, 1981; Hayes, 1982; Merck, 1983; OSHA, 1983; NLM, 1984), Laboratory findings include oliguria and albuminuria. EDB also causes severe eye and skin irritation Jeading to conjunctivitis, erythema, blistering, and skin ulcers (Gosselin, et al., 1976; NIOSH, 1978a; ACGIH, 1980; Sittig, 1981; Patty, 1982; Merck, 1983). Prolonged exposure causes lung, heart, liver and kidney damage (NIOSH, 1978a; Sittig, 1981; NIOSH, 1983c; OSHA, 1983) and EDB has the potential for inducing cancer, mutations, and reproductive abnormalities (NIOSH, 1978a; NIOSH, 1983c). One should refer to “Health Effects--Human" and "Health Effects--Non-Human" sections for a

description of studies that support and explain chronic effects and the long-term threat potential of EDB.

Poison Management

A person exposed to EDB by skin or eye contact should be removed from the exposure area. In the case of skin contact, clothing should be discarded, and the patient should have exposed parts of the body washed with soap and water. In the case of eye contact, one should copiously irrigate the eyes with water for at least 15 minutes. If irritation or pain persists after washing either the eyes or the skin, one should seek medical attention.

-105-

When an individual is exposed to EDB by ingestion or inhalation, again one should remove the person from the contaminated area to arrest further exposure. If the person develops respiratory problems, an artificial airway may be needed. In cases of oral ingestion, EDB absorption can be prevented by the following means: 1) initiate emesis (vomiting) with ipecac unless the individual is comatose, convulsing, or has lost the gag reflex; 2) have the exposed individual ingest activated charcoal (U.S.P.); 3) administer cathartics, such as magnesium sulfate, 50-percent solution (Arena, 1974).

General management, regardless of route of entry, includes several standard measures. Seizures may be treated with intravenous (I.V.} diazepam (Valium), administered according to standard prescriptive practices. One should monitor the urine for albumin content and watch for acute tubular necrosis and manage appropriately. Serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-py ruvic transaminase (SGPT), lactic dehydrogenase (LDH) and bilirubin should be monitored to detect and evaluate liver injury (NLM, 1984). The patient should be placed in a semi-recumbent position to delay or minimize pulmonary edema. If pulmonary edema develops, one should administer 100-percent oxygen (NLM, 1984). Administration of morphine, aminophylline and furosemide may help to control pulmonary edema (NLM, 1984; PDR, 1983). Antibiotics should be used in cases. of bronchopneumonia. Carbohydrates and vitamin B, when administered during recovery phase, may limit liver enzyme damage (NLM, 1984).

Toxicity of EOB may be enhanced in people taking disulfiram (Antabuse), which is used in some treatment regimens for alcoholics (Plotnick, 1978; Yodaiken, 1978). See “Mechanism of Action" section for discussion of the EDB/disulfiram synergism.

Medical Surveillance

A medical surveillance program should include, initially, a complete history and physical examination to detect any pre-existing conditions that place the worker at an increased risk. The medical examination should include evaluation of the nervous and respiratory systems, heart, liver, kidneys, and skin. Chest X-rays should be taken for evaluation of the lungs (NIOSH, 1978a). Pulmonary function tests (to include forced vital capacity (FYC) and forced expiratory volume (FEV) at one second) are utilized to determine whether the individual is capable of wearing a respirator, and to detect any existent lung disease (OSHA, 1983). Blood tests should be obtained as indicators of liver and kidney function, and should include protein, albumin, alkaline phosphatase, LDH, SGOT, SGPT, gamma glutamy] transpeptidase (GGTP) and cholesterol. Kidney function should be assessed by measuring calcium, phosphorus, blood urea nitrogen (BUN), uric acid and creatinine (OSHA, 1983). A urinalysis should also be performed (OSHA, 1983). An electrocardiogram should be performed on workers with cardiovascular disease to avoid additional risk (NIOSH, 1978a). Considering the strong reproductive effect in Israeli-Friesan bulls, one might consider obtaining sperm counts in men who have significant exposure to EOB (see “Health Effects--Non-Human" section).

-106-

FEDERAL AND STATE REGULATORY ACTIONS

Summary

EDB is regulated by the EPA as a pesticide under statutory authority of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), and as a toxic waste under the Resource Conservation and Recovery Act (RCRA); it is regulated by the Occupational Safety and Health Administration (OSHA) as an occupational hazard under the Occupational Safety and Health Act. Input into OSHA rulemaking is provided at the federal level by the National Institute of Occupational Safety and Health (NIOSH) and the American Conference of Governmental Industrial Hygienists (ACGIH), but their actions are purely advisory. Although EPA is in the process of cancelling the registration of EDB as a pesticide for its major applications, EPA's current + residue tolerance limits are purely advisory. EPA's and other agencies' guidelines are set forth in this section.

Most federal environmental regulatory programs contain provisions for state pre-emption of federal regulatory programs. To administer environmental regulatory programs, states usually must adopt programs that are either substantially equivalent to or more stringent than their federal counterparts. Envirologic Data has conducted a survey of state regulatory actions regarding EDB, and has found that some fifteen states engaged in rulemaking activities. The status of state regulations is set forth in this section.

Federal Regulatory Actions

FIFRA. EDB has been registered under the Federal Insecticide, Fungicide, and Rodenticide Act (79th Congress 1947) since 1948. Under FIFRA, the EPA registers a pesticide only if it performs its intended pesticidal function without causing "any unreasonable adverse effects on the environment, taking into account the economic, social and environmental costs and benefits of the use" [FIFRA Section 2(66)(73)]. Under Section 6 of FIFRA, the EPA can cancel the registration of a pesticide or modify the conditions under which it may be used if it is determined that the pesticide causes unreasonable adverse effects to man or the environment.

Major federal actions regarding EDB that have occurred under the authority of FIFRA between September 28, 1983 and April 1, 1984 have included notice of intent to cancel, emergency suspension order, use restrictions on minor uses, revocation of the exemption from tolerance of residues of EDB in fumigated grains and citrus fruit and papayas, establishing maximum permissible residue levels in grains, and establishment of interim tolerance level for citrus fruit and papayas. Federal actions are listed in Table 27.

-107-

Table 27,

Federal EDB Regulatory Actions

Action Use Effective Date Fed Register

Cancellation soil fumigation 10/11/84 10/11/83 (48FR46235)3 Emer. Sus. Order soil fumigation 10/11/84 10/11/83 (48FR46235) Cancellation spot fumigation 11/10/83 10/11/83 (48FR46235)@ Cancellation stored grain fumigation 11/10/83 10/11/83 (48FR46235)4 Cancellation felled log fumigation 11/10/83 10/11/83 (48FR46235)4 Cancellation quarantine fumigation, 9/1/84 10/11/83 (48FR46235)@

Restricted use

Emer. Sus. Order

Emer. Sus. Order

Rev. of Exemp. Prop. tolerances in grain, (max. residue levels)

Rev. of Exemp. Interim tolerance citrus and

papayas

Final tolerance grain

Final tolerance citrus

citrus, tropical fruits, vegetables

beehive supers*/ honeycombs, vault fumigation, Japanese beetle control

fumigation, stored grain

spot fumigation tolerances in grain

ready-to-eat-30 ppb intermed-150 ppb raw grain-900 ppb

tolerance in citrus edible portion citrus30 ppb; whole fruit citrus-250ppb

continued regis-

10/11/83 (48FR46235)

tration, label use restrictions; submission of monitoring

and use data

2/6/84

2/6/84 Proposed 2/22/84 Proposed 2/22/84

Proposed 3/2/84

Expected 4/5/84

Expected April

2/6/84 (49FR4452)b

2/6/84(49FR4452)> 2/22/84 (49FR6696)¢ 2/22/84 (49FR6696)°

3/6/84 (49FR8406)¢4

not announced

not announced

*supers-structure resting on top of beehive

Source:

4U.S. EPA, 1983b

by.S. EPA, 1984c CBNA, 1984 dy.s. EPA, 1984g

-108-

RCRA. EDB is regulated under the Resource Conservation and Recovery Act (94th Congress, 1976), under which it has been identified as a toxic waste under section 261.33(f), and given a hazardous waste number of U067. This regulation only applies when technical grade EDB in containers and the residues thereof are discarded. The producers or generators of EDB are subject to the small quantity exclusion defined in section 261.5(a) and (f). That is, if a small generator accumulates less than 1,000 kilograms in a calendar month, he is subject only to minimal requirements under RCRA, whereas a generator of over 1,000 kg per month is subject to notification requirements and transportation requirements.

Clean Air Act. Pesticides in the air are regulated under Section 112 of the Clean Air Act (91st Congress, 1970), which identifies which pollutants are hazardous. EDB is currently not regulated under Section 112. %It is not on the high priority list of chemicals for, regulatory action within the next two years by the EPA, Air Toxics Program*.

OSHA. EDB is regulated under the Occupational Safety and Health Act, (91st Congress, 1970). This act places regulation of toxic materials in the workplace under the authority of the Occupational Safety and Health Administration.

OSHA has established the following standards:

Permissable Exposure Limit (PEL) 8 hour time-weighted average (TWA): 20 ppm (20,000 ppb); in October, 1983 OSHA proposed a more stringent PEL of 0.1 ppm (100 ppb) (still pending).

The Short-Term Exposure Limit (STEL) is 30 ppm (30,000 ppb); in October, 1983 OSHA proposed a more stringent STEL of 0.5 ppm (50D ppb) (still pending).

OSHA also recommends a medical surveillance program to ensure worker safety. This program should be implemented for employees exposed to EDB at or above action level (0.05 ppm as an 8-hour time-weighted average) for 30 or more days per year, or for any employee who must wear a respirator (OSHA, 1983).

To prevent excessive exposure to EDB, respirators and protective clothing are required by OSHA. Respirators are worn to reduce exposure to or below the PEL. To limit dermal exposure, the wearing of special protective clothing designed to prevent penetration to skin for 480 minutes is required (OSHA, 1983). EDB penetrates most types of rubber and leather protective clothing (NIOSH, 1977b; NIOSH, 1978a). Viton and polyvinyl] alcohol are two resistant materials used for protective gear (Stampfer et al., undated; OSHA, 1983).

NIOSH. The National Institute of Occuational Health and Safety

(NIOSH) has reviewed OSHA's proposed rule and concluded that the exposure concentrations should be:

*Telephone communication, Karen Blanchard, Air Toxics Program, EPA, April 3, 1984.

-109-

8-hour TWA--0.045 ppm (45 ppb)

Ceiling limit--0.130 ppm (130 ppb) for 15 minute sampling period (NIOSH, 1983d)

ACGIH. American Conference of Governmental Industrial Hygienists - The “ACGIH developed a threshold limit value (TLV), which refers to airborne concentrations of EDB. The TLV is a recommendation and is not enforceable unless it is also made part of federal and/or state statutes. The ACGIH Committee, in 1978, voided the 20 ppm (TLV) for EDB and recategorized EDB as a human carcinogen having no TLY. . In 1980, the Committee changed the classification to suspect carcinogen potential for man, again, with no TLV assigned (NIOSH, 1981).

State Regulatory Actions

Envirologic Data conducted a survey of state regulatory acions regarding EDB. Individual states within the U.S. have reacted to regulatory actions undertaken by the EPA in different ways. Only those states that have adopted or are in the process of rulemaking are shown in Table 28.

The survey was conducted between February and March, 1984. During this time the states were in the process of reviewing related scientific material on EDB and conducting rule making procedures. It is important to note that due to the rapidly changing regulatory climate surrounding EDB, the data presented may not reflect the final action taken by a particular state.

Of the 50 states, 48 initially accepted EPA's recommended guideline levels for residues (30 ppb ready-to-eat, 150 ppb for intermediate, and 900 ppb for raw grain). Massachusetts established an emergency ban at 1 ppb for all grain-based products. New York recently established levels of 10 ppb for ready-to-eat and 50 ppb for intermediate grain products. Maine is in the process of rulemaking for 30 ppb ready-to-eat, 40 ppb for intermediates and 40 ppb for raw grain. Fifteen states have adopted a standard of non-detectable levels of EDB in grain-based baby foods or are in the process of rule making to develop baby food standards. Four states: Arizona, Colorado, New Mexico and Texas, have adopted the 30 ppb level for the edible pulp of citrus. On March 12, 1984 the Northeastern Regional States of Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, Rhode Island and Vermont, agreed on the fact that zero is, ultimately, the only acceptable level of EDB in food products; further, they agreed that EDB in baby food should be non-detectable; and that they would work to establish levels of no more than 10 ppb for ready-to-eat foods and 50 ppb for intermediate products (Northeastern Region, 1984). A food industry report is expected in April indicating the effect of compliance with U.S, EPA and regional interim levels on decreasing the amount of EDB in the food supply. The results will determine whether or not stricter regulatory actions are needed by the states.

-110-

Table 28. State EDB Regulatory Actions* Edible Intermediate ReadyEDB Portion Raw (Prepared to-eat Baby State Status of Fruit Grain Grain) Grain Food (ppb) (ppb) (ppb) (ppb) (ppb) Arizona state rule 30 California state rule il Connecticut rulemaking 50 10 1 Florida state rule 1 Idaho rulemaking <l Maine rulemaking 40 40 30 1 Massachusetts rulemaking { 1 1 1 Michigan rulemaking 30 1 New Hampshire rulemaking 50 10 1 New Jersey rulemaking 50 10 1 New Mexico state rule 30 New York state rule 50 10 2 Ohio rulemaking ‘ Oregon rulemaking 1 Texas state rule 30 1 Utah rulemaking 1 Vermont rulemaking 50 10 1

*Unlisted states have adopted EPA standards only.

States are addressing the problem of water contamination due to the use of EDB as a soil fumigant, and leakage of underground gasoline storage tanks. Twenty-one states have actively sampled water in areas where concentrated use

of EDB has occurred. towns in the Tobacco Valley to 18 towns. for EDB in water at 0.1 is 0.1 ppb and Florida is, however, proposing a new level of 0.02 ppb.

ppb.

Connecticut has expanded its original sampling of seven Connecticut has set an action level

Florida's current action level for EDB in water

The

State of Washington is conducting a water study with the EPA of agricultural areas which had used EDB.

-111-

LITERATURE CITED

ACGIH. 1980. Documentation of the Threshold Limit Values. Fourth Edition. Ethylene dibromide, American Conference of Governmental Industrial Hygienists, Inc., Cincinnati, Ohio. pp. 180-1;

Alumot (Olomucki), E. and £. Mandel. 1969. Gonadotropic hormones in hens treated with ethylene dibromide. Poultry Science. 48(3):957-60;

Alumot (Olomucki), E., E. Nachtomi, 0. Kempenich-Pinto, E. Mandel, and H. Schindler. 1968. The effect of ethylene dibromide in feed on the growth, sexual development, and fertility of chickens. Poultry Science. 47(4):1979-85;

American Petroleum Institute. 1982. Comments on OSHA advance notice of proposed pul eae on Sccupaetanat exposure to ethylene dibromide. Docket H-111. Comment No. 5-54. June 1;

Amir, D. 1973. The sites of the spermicidal action of ethylene dibromide in bulls. Journal of Reprod. Fert. 35:519-25;

ceeeee - 1975. Individual and age differences in the spermicidal effect of ethylene dibromide in bulls. Journal of Reprod. Fert. 44:561-5;

Amir, D. and &. Ben-David. 1973. The pattern of structural changes induced in bull spermatozoa by oral or injected ethylene dibromide {EDB). Ann. Biol. Anim. Bioch. Biophys. 13(2):165-70;

Amir, D., C. Esnault, J.C. Nicolle, and M. Courot. 1977. DNA and protein changes in the pleneator s of bulls treated orally with ethylene dibromide. Journal of Reprod. Fert. 51:453-6;

Amir, D. and U. Lavon. 1976. Changes in total nitrogen, lipoproteins, and amino acids in epididymal and ejaculated spermatozoa of bulls treated orally with ethylene dibromide. J. Repro. Fert. 47:73-6;

Amir, D. and A. Tadmor. 1981. The different labelling pattern of the genital tract of bulls and rams after a peritesticular injection of 34} ethylene dibromide. Comp. Biochem. Physiol. 69C:121-4;

Amir, D. and R. Volcani. 1967. The effect of dietary ethylene dibromide (EDB) on the testes of bulls. Fertility and Sterility. 18(1):144-8;

Aes ie and J.C. Livesey. 1980. Metabolism of 1,2-dihaloethanes. Banbury Report.

Arena, J.M. 1974. Poisoning. Toxicology Symptoms. Treatments. Third Edition Chas. C. Thomas, Publisher, Springfield, Illinois. 804 pp.;

Banerjee, S. and 8.L. van Duuren. 1979. Binding of carcinogenic halogenated hydrocarbons to cell macromolecules. Journal Nat. Cancer Inst. 63(3):707-11;

receee - 1983. Evidence for binding of metabolically activated 1,2-dibromoethane to chromatin of forestomach and liver. Life Sciences. 33:1763-70;

Banerjee, S., B.L. van Quuren, and S.A. Kline. 1979. Interaction of potential metabolites of the carcinogen ethylene dibromide with protein and DNA in vitro. Biochemical and Biophysical Research Communications. 90(4):1214-20;

Barber, E.D., H.H. Donish, and K.R. Mueller. 1981. A procedure for the quantitative measurement of the mutagenicity of volatile liquids in the Ames Salmonella/microsome assay. Mutation Research. 90:31-48;

Berk, B. 1974. Fumigant residues of carbon tetrachloride, ethylene dichloride, and ethylene dibromide in wheat, flour, bran, middlings, and bread. J. Agric. Fd. Chem. 6:977-84,

Bigler, W. 1983. Personal communication to S$. Cohen. Epidemiology Program, Department of Health and Rehabilitative Services, Jacksonville, Fla. (Cited in U.S. EPA, 1983a);

Brem, H., A.B. Stein, and H.S. Rosenkranz. 1974. The mutagenicity and ONA-modifying effect of haloalkanes. Cancer Research. 34:2576-9;

-112=

Brimer, P.A., E.L. Tan, and A.W. Hsie. 1982. Effect of metabolic activation on the vegas Seet and mutagenicity of 1,2-dibromoethane in the CHO/HGPRT system. Mutation Res. 95:377-88;

Broda, C., E. Nachtomi, and E. Alumot (Olomucki). 1976. Differences in liver morphology between rats and chicks treated with ethylene dibromide. Gen. Pharmac. 7:345-8;

Brown, S.L. et al. 1975. Research Program on Hazard Priority Ranking of Manufactured Chemicals (Chemical 1-20). Phase II-Final Report, by Stanford Research Inst. (Menlo Park, California) for National Science Foundation (Washington, D.C.) NSF/RA-E-75-190A (cited in Winzler and Kelly, 1982);

Brown, David R. 1983. Quantitative risk assessment for EDB. Northeastern University. Boston, Mass. 135 pp;

BNA. 1984. Bureau of National Affairs, Inc. Ethylene dibromide. Chemical dibromide. Chemical Regulation Reporter. 7(46):1662-3. Feb.17;

California Dept. of Health Services. (DHS). 1984. EDB risk assessment based upon Environmental Protection Agency report; Sacramento, Cal. 4 pp;

CAG. 1977. The Carcinogen Assessment Group's Preliminary Report on Ethylene Dibromide {EDB). 26 pp. U.S. EPA, Dec.2;

weeeee . 1978. Risk Assessment for Ethylene Dibromide (EDB). U.S. EPA, April 21;

-. 1980. Risk estimate utilizing the revised non-dietary exposure to ethylene orm de (EDB). Memorandum to Peter McGrath, Hazard Evaluation Division U.S. EPA, May 16;

Castro, C.E., and N.0. Belser. 1968. Biodehalogenation. Reductive dehalogenation of the biocides ethylene dibromide, 1,2-dibromo-3-chloropropane, and 2,3-dibromobutane in soil. Environ. Sci. Technol. 2:779-83. (cited in U.S, EPA 1983a);

Centaur Associates, Inc. 1983. Analysis of four industry segments which would be

affected by proposed EDB standard. Prepared for the Occupational Safety and Health Administration. pp. 60-69;

CDC. 1984. 1,2-dibromoethane (ethylene dibromide); Center for Disease Control, Atlanta, Ga. i.p.;

Clive, D. 1973. Recent developments with the L5178Y TK heterozygote mutagen assay system. Environmental Health Perspectives. 6:119-25;

Clive, D., K. O.Johnson, J.F.S. Spector, A.G. Batson, and M.M.M. Brown. 1979. and Validation characterization of the L5178Y/TK+/-mouse lymphoma mutagen assay system. Mutation Research, 59:61-108;

Clower, M. Jr. 1980a. Analysis for residues of ethylene dibromide in citrus. Laboratory Information Bulletin, 10 pp. July;

wennee « 1980b. Modification of the AOAC method for determination of fumigants in wheat. J. Assoc. Off. Anal. Chem. 63(3):539-45;

79th Congress. 1947. FIFRA - Federal Insecticide, Fungicide, and Rodenticide Act, 7 U.S.C. 135 et seq., as amended by the Federal Environmental Pesticide Control Act (FEPCA) October, 1972 (Public Law 92-516, 86 Stat. 973, 21, October, 1972, and the FIFRA amendments of 1975 (Public Law 94-140, 28 November) 1978 (Public Law 95-396, 92 Stat. 819, 30 September), and 1980 (Public Law 96-531, 94 Stat. 3194, 17 December);

9ist Congress. 1970. Clean Air Act Amendments of 1970. P.L. 91-604; 91st Congress. 1970. Occupational Safety and Health Act of 1970. P.L. 91-596;

94th Congress. 1976. Resource Conservation and Recovery Act of 1976. P.L.94-5B0. 42 U.S.C. 6901 et seq.;

Courtens, J.L., 0. Amir, and J. Durand, 1980. Abnormal spermiogenesis in bulls treated with ethylene dibromide: an ultrastructural and ultracytochemical study. Journal of Ultrastructure Research. 71:103-15;

deSerres, F.J. and H.¥. Malling. 1983. The role of Neurospora in evaluating environmental chemicals for mutagenic activity. Annals N-Y. Acad. of Sci. 407:177-85;

Doull, J., C.0, Klaassen, M. Amdur, eds. 1980. In: Casarett and Doull's Toxicology. The Basic Science of Poisons. Second Edition. Macmillan, New York. 778 pp;

-113-

Oumas, T. 1973. Inorganic and organic bromide residues in foodstuffs fumigated with methyl bromide and ethylene dibromide at Jow temperatures. J. Agr. Food Chem. 21(3):433-6;

Dumas, T., and E.J. Bond. 1975. Bromide residues in apples fumigated with ethylene dibromide. J. Agri. Food Chem, 23(1):95-8;

Dynamac Corporation. 1983. Quantitative risk assessment for ethylene dibromide in its use as a post-harvest fumigant for citrus fruit. Submitted to United Fresh Fruit and Vegetable Association, Virginia. Rockville, Md. 10 pp;

Edwards, K., H. Jackson, and A.R. Jones. 1970. Studies with alkylating esters - II. A chemical interpretation through metabolic studies of the anti-fertility effects of ethylene dimethanesulphonate and ethylene dibromide. Biochem. Pharma. 19:1783-9;

Elliott, B.M., and J. Ashby. 1980. Ethylene dibromide and disulfiram: studies in vivo and in vitro on the mechanism of the observed synergistic carcinogenic response. Carcinogenesis. 1(12):1049-57;

Epstein, S.S., E. Arnold, J. Andrea, W. Bass, and Y. Bishop. 1972. Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. and Applied Phar. 23:288-325;

Epstein, S.S. 1979. The Politics of Cancer. N.Y., Anchor Press/Doubleday. 628 pp;

Fanini, D., M.S. Legator, and P.M. Adams. 1984. Effects of paternal ethylene dibromide exposure on Fl generation behavior in the rat. Mutation Research. 139:133-8;

Fishbein. 1980. Production, uses, and environmental fate of ethylene dichloride and ethylene dibromide. Banbury Report. 5:227-38;

Geddes, A.D. 1982. Sister chromatid exchange (SCE) induction in human lymphocytes in vitro by 1,2-dibromoethane with and without metabolic activation. Mutation Research. O7:TEB-9 (Abstract);

Going, J. and S. Long. 1975. Sampling and analysis of selected toxic substances, task II[-ethylene dibromide. Midwest Research Institute. Prepared for U.S. Environmental Protection Agency. EPA 560/6-75-001;

Gosselin, R.E., H.C. Hodge, R.P. Smith, and M.N. Gleason. 1976. 1, 2-dibromoethane. Clinical Toxicology of Commercial Products. Williams & Wilkins, Baltimore, Md. i.p.;

Grocery Manufacturers of America, Inc. 1984. Report of ethylene dibromide residues in food produced from grain. A compilation and analysis of data obtained from laboratory analyses of raw grains, flour, mixes, meals, uncooked products, and foods as eaten. Washington, D.C. 33 pp;

Hardin, B.O., G.P. Bond, M.R. Sikov, F.D. Andrew, R.P. Beliles, and R.W. Niemeier. 1981. Testing of selected workplace chemicals for teratogenic potential. Scand. J. Work Environ. Health 7 (Supp1.4):66-75;

Hayes, W.d. Jr. 1982. 1,2-dibromoethane. Pesticides Studied in Man. Williams & Wilkins, Baltimore, Md. pp 149-50;

Hemminki, K., K. Falck, and H. Vainio. 1980. Comparison of alkylation rates and mutagenicity of directly acting industrial and laboratory chemicals: epoxides, glycidyl ethers, methylating and ethylating agents, halogenated hydrocarbons, hydrazine derivatives, aldehydes, thiuram, and dithiocarbanate derivatives. Arch. Toxicol, 46:277-85;

Hill, E.I., T.W. Shih, T.P. Hohnston, and R.F. Stuck. 1978. Macromolecular binding and metabolism of the carcinogen 1,2-dibromoethane. Cancer Research. 38:2438-42;

Huff, J.£. 1983. 1,2-dibromoethane (ethylene dibromide). Environmental Health Perspectives. 47:359-63;

TARC. 1977. Ethylene dibromide. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. United Nations International Agency for Research on Cancer. 15:195-209;

ICRP. 1975. International Commission for Radiological Protection. Physiological data

for reference man. In: Report of the task group on reference man, No. 23. Pergamon Press, New York. pp. 335-65;

~114-

Jacobs, E.S. 1980. Use and air quality impact of ethylene dichloride and ethylene dibromide scavengers in leaded gasoline. Banbury Report. 5:239-55;

Jakobson, I., J.E. Wahlberg, 8. Holmberg, and G. Johansson. 1982. Uptake via the blood and elimination of 10 organic solvents following epicutaneous exposure of anesthetized guinea pigs. Toxicology and Applied Pharmacology. 63:181-7;

Johns, R. 1976. Air pollution assessment of ethylene dibromide. Mitre Corp., Va. MTR-7222. Prepared for U.S. Environmental Protection Agency. 36 pp;

Jovanovich, A.P. 1983. Monitoring groundwater in Georgia for ethylene dibromide (EDB). A preliminary reconnaissance in Seminole County, Georgia. Washington, D.C. U.S. EPA Office of Pesticide Programs. October;

dungclauss, G., R. Blair, and S.Z. Cohen. In progress. Kinetics and Mechanism of ee of 1,2-dibromoethane. Midwest Research Institute and EPA (cited in U.S. EPA 1983a);

Kale, P., and J.W. Baum. 1979a. Sensitivity of Drosophila melanogaster to low concentrations of the gaseous 1,2-dibromoethane: T. Acute exposures. Environmental Mutagenesis. 1:15-8;

------ - 1979b. Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens: Il. Chronic exposures. utation Research. 68:59-68;

a - 1981. Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens: III. Dose-rate effects. Environmental Mutagenesis. 3:65-70;

------ . 1983. Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens. IY. Mutations in embryonic ReraRTAgORTS. Mutation Research. 113:135-43;

Kim, N. 1984. A risk assessment for ethylene dibromide. New York State. Dept. of Health, Bureau of Toxic Substances Assessment, i.p., Feb. 29;

King, J.R., D.L. von Windeguth, and A.K. Burditt, Jr. 1980, An electron capture gas chromatographic method for determination of residues of 1,2-dibromoethane in fumigated grapefruit. J. Agric. Food Chem. 28:1049-52;

Kirby, K.W., H. Tremmel, and J.E. Keisert. 1980. Determination and metabolism of organohalide compounds on renal tubular cells in vivo and in vitro. The Journal of Pharmacology and Experimental Therapeutics. 220(3):597-603,

Kluwe, W.M., F.W. Harrington, and S.£. Cooper. 1982. Toxic effects of organohalide compounds on renal tubular cells in vivo and in vitro. The Journal of Pharmacology and Experimental Therapeutics. 22013):597-603

Kluwe, W.M., R. McNish, J.B. Hook. 1981. Acute nephrotoxicities and hepatotoxicities of 1,2-dibromo-3-chlorapropane and 1,2-dibromoethane in male and female F344 rats. Toxicology Letters. 8:317-21;

Kluwe, W.M., R. McNish, J.B. Hook, and K. Smithson. 1981. Depletion by 1,2-dibromoethane, 1,2-dibromo-3-chloropropane, tris (2,3-dibromopropy]) phosphate, and hexachloro-1, 3-butadine of reduced non-protein sulfhydryl groups in target and non-target organs. Biochem. Pharmacol. 30(16):2265-71;

Kochmann, M. 1978. Possible industrial poisonings with ethylene dibromide. Munch ed Wochenscher. 75:1334-6 (cited in 8rown, 1983);

Kronevi, T., J.E. Walhberg, and B. Holmberg. 1981. Skin pathology following epicutaneous exposure to seven organic solvents. Int. J. Tiss. Reac. III(1):21-80;

Lash, J., and K. Gillman. 1984. The politics of pesticides. How the Reagan Administration failed to control E0B. The Boston Globe Magazine. March 18;

Leinster, P., R. Perry, and R.J. Young. 1978. Ethylene dibromide in urban air. Atmos. Envy. 12:2383-7;

Ma, T.H., A.H. Sparrow, L.A. Schairer, and A.F. Nauman. 1978. Effect of 1,2-dibromoethane (DBE) on meiotic chromosomes of Tradescantia. Mutation Research. 58:251-8;

Malling, H.V. 1969. Ethylene dibromide: a potent pesticide with high mutagenic activity. Genetics 61: S39;

-115-

Markel, H.L. Jr., and D. Peter. 1981. National Institute for Occupational Safety and Health (NIOSH) Health Hazard Evaluation Report #TA80-017-1017 undertaken for the U.S. Department of Agriculture, Rio Grande Yalley, Texas. 6 pp. + tables. December;

Merck. 1983. Ethylene dibromide. The Merck Index. Edited by M. Windholz and S. Budavari. Merck & Co., Inc., Rahway, N.J. i.p.;

Milks, M.M., S.R. Wilt, I. Ali, M.A. Pereira, and D. Couri. 1982. Dibromoethane effects on the induction of gamma-glutamyi-transpeptidase positive foci in rat liver. Arch. Toxicol. 51:27-35;

Mink, J.F. 1981. DBCP and EDB in Soil and Water at Kunia, Oahy, Hawaii. (In addition to several personal communications to $. Cohen in 1980-81.) For Del Monte Corp., 48 pages and appendices (cited in U.S. EPA 1983a);

Moody, D.E., J.L. James, and E.A. Smuckler. 1980. Cytochrome P-450 lowering effect of alkyl halides, correlation with decrease in arachidonic acid. Biochemical and Biophysical Research Communications. 97(2}:673-9;

Moody, D.E., G.A. Clawson, C.H. Woo, and E.A. Smuckler. 1982. Cellular distribution of cytochrome P-450 loss in rats of different ages treated with alkyl halides. Toxicology and Applied Pharmacology. 66:278-89;

Moriya, M., T. Ohta, K. Watanabe, T. Miyazawa, K. Kato, and Y. Shirasu. 1983. Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mut. Research. 116:185-216;

Nachtomi, E. 1970. The metabolism of ethylene dibromide in the rat; the enzymic reaction with glutathione in vitro and in vivo. Biochem. Pharmacol. 19:2853-60;

------ . 1980. Modulation of the mitotic action of ethylene dibromide. Chem. Biol. Interactions. 32:311-9;

Nachtomi, E. and D.S.R. Sarma. 1977. Repair of rat liver DNA in vivo damaged by ethylene dibromide. Biochem. Phar. 26:1941-5; =

Nachtomi, E., Alumot, E., and A. Bondi. 1966. The metabolism of ethylene dibromide in the rat. I. Identification of detoxification products in urine. Israel J. of Chemistry. 4:239-46;

weon-- . 1968. Biochemical changes in organs of chicks and rats poisoned with ethylene dibromide and carbon tetrachloride. Israel J. of Chemistry. 6:803-811;

NCI. 1978. Bioassay of 1,2-dibromoethane for possible carcinogenicity. National Cancer Institute. Tech. report series No. 86. Bethesda, MD. 64 pp plus appendices;

NCI. 1979. Technical background information report on carcinogenesis bioassay of 1,2-dibromoethane (EDB). Bethesda, MD. National Cancer Institute. Clinical Toxicology. 14(4}:473-8;

NIOSH. 1977a. Criteria for a recommended standard: Occupational exposure to ethylene dibromide. Doc. #DHEW(NIOSH) Publ.#77-221. 2D8 pp. U.S. Govt. Printing Office, Wash., D.C. August;

------ . 1977b, 1,2-dibromoethane. Occupational diseases: A guide to their recognition. Publication No. 77-181, pp. 198-9. U.S. Govt. Printing Office, Wash., D.C.;

veeene . 19784. Occupational health guideline for ethylene dibromide. National Institute for Occupational Safety and Health (NIOSH). U.S. Dept. of Labor. Occupational Safety and Health Administration. September;

an--- . 1978b. 1,2-dibromoethane. NIOSH Manual of Analytical Methods, 2nd edition. NIOSH, Cincinnatti, Ohio. 4:260-1-260-8;

. 1981. Ethylene dibromide (EDB) revised. NIOSH, Cincinnatti, Ohio. Current ligence Bulletin 37, pub. no. 82-105. 13 pp. October 26;

------ . 1983a. 1,2-dibromoethane (EDB). Third Annual Report on Carcinogens. National Toxicology Program Public Information Office. NTP82~-330;

~116-

oe . 1983b. Ethane, 1,2-dibromo. Registry of Toxic Effects of Chemical Substances: 1981-2, Vol. II. NIOSH Pub. #83-107. Cincinnati, Ohio. p.222;

weeeee . 1983c. NIOSH recommendations for occupational health standards. Centers for Disease Control Morbidity and Mortality Weekly Report. Supplement 32(15):135;

------. 1983d. NIOSH comments on the occupational safety and health administration proposed standard: occupational exposure to ethylene dibromide. Docket No. H-111. Cincinnatti, Ohio. 7 pp;

w----- . 1984a. Centers for Disease Control, Interim report no. 1 HETA83-375. Federal Grain Inspection Service. Portland, Oregon. 6 pp. plus appendices. Feb.;

Nitschke, K.D., R.J. Kociba, 0.G. Keyes, and M.J. McKenna. 1981. A thirteen week repeated inhalation study of ethylene dibromide in rats. Fundamental and Applied Toxicology. 1:437-42;

NLM. 1984. Toxicology Database (TDB). National Library of Medicine. Feb.;

Northeastern Region. 1984. Statement of agreement. EDB meeting, Boston, Mass. March tas

NTP. 1982a. Carcinogenesis bioassay of 1,2-dibromoethane (CAS #106-93-4) in F344 rats and B6C3F1 mice (inhalation study). National Toxicology Program. Technical report series No. 210. 163 pp. Research Triangle Park, N.C. March;

a----- - 1983. Occupational exposure to ethylene dibromide; notice of proposed rule-making. Dept. of Labor. Federal Register. 48(196):45956-6003. Oct. 7;

Olmstead, E.¥. 1960. Pathological changes in ethylene dibromide poisoning. AMA Archives of Industrial Health. 21:525-9;

Olson, W.A., R.T. Habermann, £.K. Weisburger, J.M. Ward, and J.H. Weisburger. 1973. Brief communication: induction of stomach cancer in rats and mice by halogenated aliphatic fumigants. Journal of Nat. Cancer Inst. 51(6):1993-5;

OTA. 1981. U.S. Congress. Office of Technology Assessment, Washington, D.C. Assessment of Technologies for Determining Cancer Risks from the Environment. Report OTA-H-138. 240 pp. June;

Ott, M.G., H.C. Scharnweber, and R.R. Langner. 1980. Mortality experience of 161 employees exposed to ethylene dibromide in two production units. British Journal of Industrial Medicine. 37:163-8;

Ozawa, N. and F.P. Guengerich. 1983. Evidence for formation of an S-[2-(N?-quanyl )ethy]] glutathione adduct in glutathione-mediated binding of the carcinogen 1 ,2-dibromoethane to DNA. Proc. Nati. Acad. Sci. 80:5266-70;

Page, G.W. 1981. Comparison of groundwater and surfacewater for patterns and levels of contamination by toxic substances. Envir. Sci. and Tech. 15(12):1475-81;

Patty. 1982. Patty's Industrial Hygiene and Toxicology, 3rd edition. John Wiley and Sons, Inc. New York. ¥ol.1IB:2901-2, 3497-502;

PDR. 1983. Physician's Desk Reference. 37th edition. Medical Economics Company, Inc. Oradell, N.J. 3068 pp;

Pflessor, G. 1938. Skin damaging effects of ethylene dibromide. A constituent of the liquid Me ial water gauges. Arch. Gewerbepathol Gewerbehyg. 8:591-600 (cited in Brown, 1983);

Perocco, P. and G. Prodi. 1981. DNA damage by haloalkanes in human lymphocytes cultured in vitro. Cancer Letters. 13:213-8;

Plotnick, H.B. 1978. Carcinogenesis in rats of combined ethylene dibromide and disulfiram. Journal of the American Medical Association. 239(16):1609;

Plotnick, H.B., and W.T. Conmer. 1976. Tissue distribution of 14C-labelled ethylene dibromide in the guinea pig. NIOSH. Cincinnatti, Ohio;

Plotnick, H.B., W.W.Weigel, D.E. Richards, and K.L. Cheever. 1979. The effect of dietary disulfiram upon the tissue distribution and excretion of 14C-1,2-dibromoethane in the rat. Research Communications in Chemical Pathology and Pharmacology. 26(3):535-45;

=liz=

Plotnick, H.B., W.W.Weigel, D.E. Richards, K.L. Cheever, and C. Kommineni. 1980. Dietary disulfiram enhancement of the toxicity of ethylene dibromide. Banbury Report 5:279-86;

Principe, P., E. Dogliotti, M. Bignami, R. Crebelli, E. Falcone, M. Fabrizi, G. Conti, and P. Comba. 1981. Mutagenicity of chemicals of industrial and agricultural relevance in Salmonella, Streptomyces, and Aspergillus. J. Sci. Food Agric. 32:826-32;

Rains, D.M., and J.W. Holder. 1981. Ethylene dibromide residues in biscuits and commercial flour. J. Assoc. Off. Anal. Chem. 64(5):1252-4;

Ramsey, J.C., C.N. Park, M.G. Ott, and P.J. Gehring. 1979. Carcinogenic risk assessment: ethylene dibromide. Toxicology and Applied Pharmacology. 47:411-4;

Rannug, U. and B. Beije. 1979. The mutagenic effect of 1,2-dichloroethane on

Salmonella typhimurium. II. Activation by the isolated perfused rat liver. Chem. Biol. Tnteree 24(3): 265-85;

Rannug, U., A. Sundvall, and C. Ramel. 1978. The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. I. Activation through conjugation with glutathion in vitro. Them. Biol. Interactions. 20:1-16;

Rowe, ¥.K., H.C. Spencer, D.D. McCollister, R.L. Hollingsworth, and E.M. Adams. 1952. Toxicity of ethylene dibromide determined on experimental animals. Arch. Ind. Hyg. Occ. Med. 6:158-73;

Ruckelshaus, W.D. 1984. Open letter on EDB to users of grain fumigants. U.S. EPA. Washington D.C. 2 pp. February 3;

Rumsey, D.W., and R.K. Tanita. 1978. An industrywide industrial hygiene study of ethylene dibromide. DHEW (NIOSH) Pub. Mo. 79-112. Prepared by Stanford Research Institute for the U.S. Dept. of Health, Education and Welfare under Contract No. 210-76-0162. Cincinnati, NIOSH, 36 pp;

Sax, I.N. 1975, Ethylene dibromide. In: Dangerous Properties of Industrial Materials. Fourth edition. Van Nostrand Reinhold, New York. p. 736;

Schairer, L.A., J. Van't Hof, C.G. Hayes, R.M. Burton, and F.J. de Serres. 1978. Exploratory monitoring of air pollutants for mutagenicity activity with the Tradescantia stamen hair system. Environmental Health Perspectives. 27:51-60;

Schlinke, J.C. 1969. Toxicologic effects of five soil nematocides in cattle and sheep. J.A.¥.M.A, 155(8):1364-1366;

cornea e--- « 1970. Toxicologic effects of five soil nematocides in chickens. Am. J. Vet. Res. 31(6):1119-21;

Scott, B.R., A.H. Sparrow, S.S. Schwemmer, and L.A. Schairer. 1978. Plant metabolic activation of 1,2-dibromoethane (EDB) to a mutagen of greater potency. Mutation Research. 49:203-12;

Shiau, S.Y., R.A. Huff, B.C. Wells, and I.C. Felkner. 1980. Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants. Mutation Research. 71:169-79;

Shih, T.W., and D.L. Hill. 1981. Metabolic activation of 1,2-dibromoethane by glutathione transferase and by microsomal mixed function oxidase: further evidence for formation of two reactive metabolites. Research Communications in Chemical Pathology and Pharmacology. 33(3)449-61;

Short, R.D., J.L. Minor, 8. Ferguson, T. Unger, C.C. Lee. 1976. The developmental toxicity of ethylene dibromide inhaled by rats and mice during organogenesis. EPA-560/6-76-018. 11 pages;

Short, R.D., J.L. Minor, J.M. Winston, J. Seifter, and C.C. Lee. 1978. Inhalation of ethylene dibromide during gestation by rats and mice. Toxicology and Applied Pharmacology. 46:173-182;

Short, R.D., J.M. Winston, C.B. Hong, J.L. Minor, C.C. Lee, and J. Seifter. 1979.

Effects of ethylene dibromide on reproduction in male and female rats. Toxicology and Applied Pharmacology. 49:97-105;

~118-

Sigsby, J.E. Jr., D.L. Dropkin, R.L. Bradow, and J.M. Lang. 1982. Automotive emissions of ethylene dibromide, SAE Technical Paper Series no, 820786. Warrendale, Pa. 17 pp;

Singh, H.B., L.J. Salas, A.J. Smith, and H. Shigeishi. 1981. Measurements of some potentially hazardous organic chemicals in urban environments. Atmospheric Environment. 15:601-12;

Singh, H.B., L.J. Salas, and R.E. Stiles. 1982. Distribution of selected gaseous organic mutagens and suspect carcinogens in ambient air. Environ. Sci. Techno. 16(12):872-80;

Sittig, M. 1981. Ethylene dibromide. In: Handbook of Toxic and Hazardous Chemicals. Noyes Publications, Park Ridge, N.J. pp.311-3;

Smith, R.F., and L, Goldman, 1983. Behavioral effects of prenatal exposure to ethylene dibromide. Neurobehavioral Toxicology and Teratology. 5:579-85;

Sparrow, A.H., L.A. Schairer, and R. Villalobos-Pietrini. 1974. Comparison of somatic mutation rates induced in Tradescantia by chemical and physical mutagens. Mutation Research. 26:265-76;

SRI International. 1982. Quantitative cancer risk assessment for occupational exposure to ethylene dibromide. Prepared for NIOSH. June. i.p.;

Stampfer, J.F., M.J. McLeod, M.R. Betts, A.M. Martinez, and S.P. Beraidinelli. Undated. The permeation of eleven protective garment materials by four organic solvents;

Stinson, S.F., G. Reznik, and J.M. Ward. 1981. Characteristics of proliferative lesions in the nasal cavities of mice following chronic inhalation of 1,2-dibromoethane. Cancer Letters. 12:121-9;

Stolzenberg, S.J., and C.H. Hine. 1980. Mutagenicity of 2 - and 3 - carbon halogenated compounds in the Salmonella/mammalian-microsome test, Environmental Mutagenesis. 2:59-66;

Storer, R.D., and R.B. Conolly. 1983. Comparative in vivo genotoxicity and acute hepatotoxicity of three 1,2-dihaloethanes. Carcinogenesis. 4(11):1491-4;

Sun, M. 1984. EDB contamination kindles federal action. Science. 223:464-6;

Sundheimer, D.W., R.D. White, K. Brendel, and 1.C. Sipes. 1982. The bioactivation of 1,2-dibromoethane in hepatocytes: covalent binding to nucleic acids. Carcinogenesis. 3(10):1129-33;

Tan, E.L., and A.W. Hsie. 1981. Mutagenicity and cytotoxicity of haloethanes as studied in the CHO/HGPRT system. Mutation Research. 90:183-91;

Teramoto, S., R. Saito, H. Aoyama, and Y. Shirasu. 1980. Dominant lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane (DBCP). Mutation Research. 77:71 -8;

Ter Haar, G. 1980. An investigation of possible sterility and health effects from exposure to ethylene dibromide. Banbury Report. 5:167-8B (cited in Brown, 1983);

Tezuka, H., N. Ando, R. Suzuki, M. Terahata, M. Moriya, and Y. Shirasu. 1980. Sister-chromatid exchanges and chromosomal aberrations in cultured Chinese hamster cells treated with pesticides positive in microbial reversion assays. Mutation Research. 78:177-91;

Townshend, J.L., V.A. Dirks, and C.F. Marks. 1980. Temperature, moisture, and action and their effects on the diffusion of ethylene dibromide in three Ontario soils. Can. J. Soil Sci. 60:177-84; U.S. EPA. Undated. Master list grain fumigation and spot fumigation;

--. 1980a. Ethylene dibromide. 121 pp. Position Doc. 2/3;

re . 1983a. Ethylene dibromide (EDB). Office of Pesticide Programs. Position Document 4. 286 pp. Sept. 27;

nd + 1983b. Ethylene dibromide; decision and emergency order suspending registrations of pesticide products containing ethylene dibromide for use as a soil fumigant. Federal Register. 48(197): 46228-48;

ao---- . 19844, Additional residue data for EDB in grain products. Jan;

pee, . 1984b, EDB Facts. 4 pp. Washington, D.C. Feb.3.

-119-

o----- . 1984c. Ethylene dibromide, decision and emergency order suspending

registrations of pesticide products containing EDB; Notice. Federal Register. Feb. 6. 49(25):4452-7;

weene- . 1984d. Ethylene dibromide (EDB) - Scientific support and decision document for grain and prety milling fumigation uses. Washington, D.C. Office of Pesticide Programs. 118 pp. Feb. 8;

nao--- . 1984e, EPA proposed revocation of exemption, tolerances, new tolerances for ethylene dibromide in food, animal feed. 49FR6696, Feb. 22; Chemical Regulation Reporter. 7(47):1683-8;

w----- - 1984f. Ethylene dibromide (EDB) - Technical support document for quarantine fumigation uses, Washington, D.C. Office of Pesticide Programs. 106 pp. March 2;

------ . 19849. Pesticides; ethylene dibromide; proposed revocation of tolerances. Federal Register. 49(45):8406-9. March 6;

ooeee= . 19845. EDB Facts. Washington, D.C. 4 pp. March.

Valencia, R. 1981. Mutagenesis screening of pesticides using Drosophila. WARF Institute Inc., Madison, Wisc. Feb. EPA-600/1-81-017. 71 pp;

van Bladeren, P.J., D.D. Breimer, G.M.T. Rotteveel-Smijs, R.A.W. DeJong, W. Buijs, A. van der Gen, and G.R. Mohn. 1980. The role of glutathione conjugation in the mutagenicity of 1,2-dibromoethane. B8iochem. Phar. 29:2975-82;

van Bladeren, P.J., 0.0. Breimer, J.A.T.C.M. van Huijgevoort, M.P.E£. Vermeulen, and A. van der gen. 198la. The metabolic formation of N-acetyl-S-2-hydroxyethyl-1-cystine from tetradeutero-] ,2-dibromoethane. Relative importance of oxidation and glutathione conjugation in vivo. Biochem. Phar. 30(17):2499-502;

van Bladeren, P.J., J.J. Hoogeterp, D.0. Sreimer, and A. van der gen. 1981b. The influence of disulfiram and other inhibitors of oxidative metabolism on the formation oF ee ee reerne acid from 1,2-dibromoethane by the rat. Biochem. Phar. 30(21): 2983-7;

van Durren, B.L., B.M. Goldschmidt, G. Loewengart, A.C. Smith, S. Melchionne, I. Seldman, and D. Roth, 1979. Carcinogenicity of halogenated olefinic and aliphatic hydrocarbons in mice. Journal of the Nationa? Cancer Institute. 63(6):1433-9;

Vogel, E., and J.L.R. Chandler. 1974. Mutagenicity testing of cyclamate and some pesticides in Drosophila melanogaster. Experientia. 30:621-3;

Wade, R.L., and J.D. Sakura. 1982. Qualitative and quantitative assessment of the toxicity of ethylene dibromide (unpublished communication cited in Brown, 1983).

Waldott, G.L. 1978. Health Effects of Environmental Pollutants. The C.V¥. Mosby Co., Saint Louis. 350 pp;

Ward, J.M., and R.T, Habermann. 1974. Pathology of stomach cancer in rats and mice induced with the agricultural chemicals ethylene dibromide and dibromochloropropane. The Bull. of the Society of Pharmacological and Environ. Pathologists. 2(2):10-11;

Watanabe, P.G., J.D. Young, M.M. Schlachter, J.A. Zempel, and R.J. Karbowski. 1978. Fate of inhaled ethylene dibromide in rats. Toxicology and Applied Pharmacology. 45(1):224. (Abstr. );

Weisburger, E.K. 1977. Carcinogenicity studies on halogenated hydrocarbons. Environmental Health Perspectives. 21:7-16;

Westlake, G.E., P.J. Bunyan, P.I. Stanley, and C.H. Walker. 1981, A study on the toxicity and the biochemical effects of ethylene dibromide in the Japanese quail. British Poultry Science. 22:355-64;

White, G.L., and J. Lybarger. 1978. Health hazard evaluation determination. Report 77-119-481 for Houston Chemical Co., Beaumont, Texas. 22 pp. April;

White, R.D., [.G. Sipes, A.J. Gandolfi, and G.T. Bowden. 1981. Characterization of the

hepatic DNA esi caused by 1,2-dibromoethane using the alkaline elution technique. Carcinogenesis. 2(9):839-44;

-120-

1983. Deuterium isotope effect on the metabolism and toxicity of -dibromoethane. Toxicology and Applied Pharmacology. 69:170-8;

Winzler and Kelly. 1982. Environmental implications of changes in the brominated chemicals industry. Final report prepared for U.S. EPA. Eureka, Ca. pp.20-31;

Wit, S., A. Besemer, H. Das, W. Goedkoop, F. Loosjes, and E. Meppeling. 1969. Results of an investigation on the regression of three fumigants (carbon tetrachloride, ethylene dibromide, and ethylene dichloride) in wheat during processing to bread. Report No. 36/39 Tox, National Institute of Public Health. Bilthoven, Netherlands. 21 PP;

Wong, O0., Utidjian, H.M.D., and ¥.S. Karten. 1979. Retrospective evaluation of reproductive performance of workers exposed to ethylene dibromide (EDB). J. Occup. Medicine. 21(2):98-102;

Wong, L.C.K., J.M. Winston, C.B. Hong, and J. Hagensen. 1980. Chronic inhalation toxicity of 1,2-dibromoethane in rats with and without dietary disulfiram. Contract #210-76-0131 for U.S. Dept. of Health and Human Services, Public Health Service, Centers for Disease Control, National Institute for Occupational Safety and Health, division of Biomedical and Behavioral Science, Cincinatti, Ohio. In cooperation with Midwest Research Institute, Kansas City, Missouri. 36 pp. Nov;

Wong, L.C.K., J.M. Winston, C.B. Hong, and q. Plotnick. 1982. Carcinogenicity and toxicity of 1,2-dibromoethane in the rat. Toxicology and Applied Pharmacology. 63:155-65;

Yodaiken, R.E. 1978. Ethylene dibromide and disulfiram - a lethal combination. Journal of the American Medical Association. 239(26):2783;

Zalkin, F., R.J. Oshima, R. Sava, and M. Wilkerson. In progress. Pesticide Movement to Groundwater. (Volumes I and II are in draft form; Volume III not completed.) California pena tent of Food and Agriculture, Sacramento, California (cited in U.S.EPA 1983a).

=T21=

ABOUT THE PRINCIPAL AUTHORS

Frank H. Lawrence, M.D.: Dr. Lawrence's prior work in the area of risk assessment and environmental health forms the basis for Envirologic Data's development. Over the past three years he has helped create a scientific data production and analysis methodology for pesticide assessment and regulation. Dr. Lawrence has twice been appointed by Maine's governor as the physician member of the State's Pesticide Control Board, and has been appointed by the State Medical Association as Chairman of its Enviromental Health Committee. He has provided consulting services for state agencies and law firms on toxicological hazards and has developed occupational surveillance programs for the Maine Department of Environmental Protection and for hazardous waste site investigators of the engineering and pollution control firms.

Education: M.D., Indiana University, 1971. B.A., University of Delaware, 1965.

Robert A. Michaels, Ph.D.: Dr. Michaels is an Environmental Toxicologist with consulting and teaching experience. His expertise is in assessing risks from hazardous waste and toxic substances in commerce and determining reasonable strategies for risk reduction.

4 Major clients of Dr. Michaels have included the Office of Technology Assessment, U.S. Congress; and the Governor's Office of Appropriate Technology, State of California. Project experience includes evaluating the Environmental Protection Agency's §5.4 million environmental monitoring program at Love Canal with respect to its design, implementation, results, statistical methods, and conclusions concerning levels of contamination in the surrounding evacuated area. He has investigated hazardous chemicals used in daily commerce in California and explored safe substitutes and policy options for industry and government for reduction of risk. Additionally, Dr. Michaels recommended regulations for carcinogens and other biohazards based upon toxicology, persistence, mobility, and other predictors of environmental behavior.

Education: Ph.D., Environmental Ecology/Toxicology, SUNY, Stony Brook, 1979. M.S., Ecology, University of Georgia, 1971. B.S., Biology, City College of CUNY, 1967.

-122-

FENviRoLoGic data Analysis & Risk Assessment P.O. Box 1438 Portland, Maine 04104

Publication citation

Lawrence, Frank H.; and RA Michaels. Ethylene Dibromide (EDB) – A Guide for Decision Makers. Portland, Maine, EnviroLogic Data, 122 pages, March 1984;

Robert A. Michaels on ResearchGate