PB86-122702
Health Assessment Document for
1,2-Dichloroethane (Ethylene Dichloride)
Final Report
(U.S.) Environmental Protection Agency
Research Triangle Park, NC
Sep 85
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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&EPA
United States,
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA 600/8-84, 006F
September 1 985
Final Report
Research and Development
PE86-122702
Health Assessment
Document for
1,2-Dichloroethane
(Ethylene Dichloride)
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• ICHNICAL REPORT DATA
Plfssc read ln^r.n -ions on ;he re\ er'.c it tore comr!:tnif.
EPA/600/8-84/Q06F
3 lECIP'ENT'S ACCESSIO"»NO
1227C? ,'3T
- TITLE
HEALTH ASSESSMENT DOCUMENT FOR 1,2-OICHLOROETHANE
(Ethylene Dichloride): Final Report
REPORT DATE
September 1985
6 PERFORMING ORGANIZATION CODE
AL7HCR.3)
i PERFORMING ORGANIZATION REPORT NO
. See List of Consultants, Reviewers, and Authors
9>ERFORM~lNG ORGANISATION NAME AND ADDRESS
10 PROGRAM ELEMENT NO
11 CONTRACT,GRANT NO
Same as box 1 2.
12 SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Health & Environmental Assessment
Environmental Criteria & Assessment Office (MD-52)
Research Trianole Park. NC 27711
13 TVPE OF REPORT AND PERIOD COVERED
Final
14 SPONSORING AGENCY CODE
EPA/600/23
IS SUPPLEMENTARY NOTES
16 ABSTRACT
Ethylene Dichloride (EDC), a clear, colorless volatile liquid, is principally emitted
to the environment during manufacturing. Monitoring data, including ambient urban
areas, indicate a concentration of equal to or less than 0.5 ppb for most locations.
EDC is rapidly absorbed, metabolized, and eliminated. Unmetabolized EDC is eliminated
almost exclusively via the lungs. In humans, the symptoms of acute toxicity from
repeated exposures exceeding 60 ppm are irritation of the respiratory tract and eyes
and CNS depression. According to available evidence EDC does not adversely affect the
reproductive or development process in animals except at maternally toxic levels.
Additional human epidemiologic studies are needed to establish conclusively that EDC
is not a teratogen and does not cause adverse reproductive effects. Positive response;
in different text systems indicate that EDC is a weak, direct-acting mutagen; however,
several of its metabolites, formed in animals, are more potent mutagens than EDC.
As a carcinogen, EDC induces tumors in rats and mice by various routes of exposure
(gavage, intraperitoneally, dermally). However, lifetime inhalation exposure
conditions did not produce tumors in rats or mice. Results from animal carcinogen
studies, when considered with the positive evidence of mutagenicity and the presence
of reactive metabolites and covalent bonding to DNA, suggest that EDC is a potential
human carcinogen.
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DISCLAIMER
This document has been reviewed in accordance with the U.S. Environmental
Protection Agency's peer and administrative review policies and approved for
publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
ii
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PREFACE
The Office of Health and Environmental Assessment has prepared this health
assessment to serve as a "source document" for EPA use. The health assessment
document was originally developed for use by the Office of Air Quality Planning
and Standards to support decision-making regarding possible regulation of
ethylene dichloride as a hazardous air pollutant. However, the scope of this
document has since been expanded to address multimedia aspects.
In the development of the assessment document, the scientific literature
has been inventoried, key studies have been evaluated and summary/conclusions
have been prepared so that the chemical's toxicity and related characteristics
are qualitatively identified. Observed effect levels and other measures of dose-
response relationships are discussed, where appropriate, so that the nature of
the adverse health responses is placed in perspective with observed
environmental levels.
Any information regarding sources, emissions, ambient air concentrations,
and public exposure has been included only to give the reader a preliminary
indication of the potential presence of this substance in the ambient air. While
the available information is presented as accurately as possible, it is
acknowledged to be limited and dependent in many instances on assumption rather
than specific data. This information is not intended, nor should it be used, to
support any conclusions regarding risks to public health.
If a review of the health information indicates that the Agency should
consider regulatory action for this substance, a considerable effort will be
undertaken to obtain appropriate information regarding sources, emissions, and
ambient air concentrations. Such data will provide additional information for
drawing regulatory conclusions regarding the extent and significance of public
exposure to this substance.
iii
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AUTHORS AND REVIEWERS
AUTHORS
The EPA's Office of Health and Environmental Assessment (OHEA) is
responsible for the preparation of this health assessment document. The OHEA
Environmental Criteria and Assessment Office (ECAO-RTP) had overall
responsibility for coordination and direction of the document (Dr. Robert M.
Bruce, Project Manager). The chapters addressing physical and chemical
properties, sampling and analysis and toxic effects were either rewritten or
revised by Syracuse Research Corporation. The pharmacokinetics chapter was
written by Dr. I.W.F. Davidson of the Bowman Gray School of Medicine, Wake Forest
University and Dr. Jean C. Parker, Carcinogen Assessment Group, U.S.
Environmental Protection Agency. The air quality chapters addressing sources,
emissions and ambient concentrations were originally written by Syracuse
Research Corporation and revised by Radian Corporation under a contract with the
Office of Air Quality Planning and Standards.
The principal authors of the chapters prepared by Syracuse Research
Corporation are:
Stephen Bosch
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York 13210-4080
D. Anthony Gray, Ph.D.
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York 13210-4080
Joseph Santodonato, Ph.D., C.I.H.
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York 13210-4080
iv
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The OHEA Carcinogen Assessment Group (CAG) was responsible for preparation
of the sections on carcinogenicity. Participating members of the CAG are listed
below (principal authors of present carcinogenicity materials are designated by
an asterisk [*]).
Roy Albert, M.D. (Chairman)
Elizabeth L. Anderson, Ph.D.
Steven Bayard, Ph.D.
David L. Bayliss, M.S.
Robert P. Beliles, Ph.D.
Chao W. Chen, Ph.D.*
Margaret M.L. Chu, Ph.D.
I.W.F. Davidson (consultant)
Herman J. Gibb, M.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.*
Robert E. McGaughy, Ph.D.
Jean C. Parker, Ph.D.*
Charles H. Ris, M.S., P.E.
Dharm V. Singh, D.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
The OHEA Reproductive Effects Assessment Group (REAG) was responsible for
the preparation of sections on mutagenicity, teratogenicity and reproductive
effects. Participating members of REAG are listed below (principal authors of
present sections are indicated by an asterisk [*]). The Environmental Mutagen
Information Center (EMIC), in Oak Ridge, TN, identified literature bearing on the
mutagenicity of EDC.
Eric D. Clegg, Ph.D.
John R. Fowle III, Ph.D.*
David Jacobson-Kram, Ph.D.
K.S. Lavappa, Ph.D.
Sheila L. Rosenthal, Ph.D.
Carol N. Sakai, Ph.D.*
Lawrence R. Valcovic, Ph.D.
Vicki L. Vaughan-Dellarco, Ph.D.
Peter E. Voytek, Ph.D. (Director)
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REVIEWERS
The following individuals provided peer review of this draft and/or earlier
drafts of this document:
U.S. Environmental Protection Agency
Karen Blanchard
Office of Air, Noise and Radiation
Office of Air Quality Planning and Standards
Research Triangle Park, NC
Robert M. Bruce, Ph.D.
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC
Larry T. Cuppitt, Ph.D.
Office of Research and Development
Environmental Sciences Research Laboratory
Research Triangle Park, NC
James W. Falco, Ph.D.
Office of Health and Environmental Assessment
Exposure Assessment Group
Washington, DC
Lester D. Grant, Ph.D.
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC
Thomas G. McLaughlin, Ph.D.
Office of Health and Environmental Assessment
Exposure Assessment Group
Washington, DC
David Patrick
Office of Air, Noise and Radiation
Office of Air Quality Planning and Standards
Research Triangle Park, NC
David J. Reisman
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, OH
Jerry F. Stara, D.V.M.
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, OH
vi
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CONSULTANTS, REVIEWERS AND CONTRIBUTING AUTHORS:
Joseph F. Borzelleca, Ph.D.
Virginia Commonwealth University
Richmond, VA
Mildred Christian, Ph.D.
Argus Research Laboratories, Inc.
Perkasia, PA
Herbert H. Cornish, Ph.D.
The University of Michigan
Ypsilanti, MI
I.W.F. Davidson, Ph.D.
Bowman Gray School of Medicine
Wake Forest University
Winston Salem, NC
Larry Fishbein, Ph.D.
National Center for Toxicological Research
Jefferson, AR
Mark M. Greenberg
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC
Derek Hodgson, Ph.D.
University of North Carolina
Chapel Hill, NC
George R. Hoffman, Ph.D.
Holy Cross College
Worcester, MA
Rudolph J. Jaeger, Ph.D.
Consultant Toxicologist
7 Bolgert Place
Westwood, NJ
Marshall Johnson, Ph.D.
Thomas Jefferson Medical College
Philadelphia, PA
Bruce Kleinstein, Ph.D.
1500 Locust Street, Suite 3504
Philadelphia, PA
John L. Laseter, Ph.D.
Environmental Affairs, Inc.
New Orleans, LA
vii
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Edmond J. LaVoie, Ph.D.
American Health Foundation
Valhalla, NY
Marvin S. Legator, Ph.D.
University of Texas Medical Branch
Calveston, TX
P.D. Lotilaker, Ph.D.
Pels Research Institute
Temple University Medical Center
Philadelphia, PA
Bernard Schwetz, Ph.D.
National Institute of Environmental Health Sciences
Research Triangle Park, NC
Sam Shibko, Ph.D.
Health and Human Services
Division of Toxicology
Washington, DC
Charles M. Sparacino, Ph.D.
Research Triangle Institute
Research Triangle Park, NC
Daniel S. Straus, Ph.D.
University of California
Riverside, CA
Darrell D. Sumner, Ph.D.
CIBA GEIGY Corporation
High Point, NC
Robert Tardiff, Ph.D.
1i»23 Trapline Court
Vienna, VA
Norman M. Trieff, Ph.D.
University of Texas Medical Branch
Department of Pathology, UTMB
Galveston, TX
Benjamin Van Duuren, Ph.D.
New York University Medical Center
550 First Avenue
New York, NY
James Withey, Ph.D.
Room 315C
Environmental Health Center
Division of Environmental and Occupational Toxicology
DeLa Colonbine
Tunney's Pasture
Ottawa, Ontario
CANADA K1A OL2
viii
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SCIENCE ADVISORY BOARD
ENVIRONMENTAL HEALTH COMMITTEE
CHLORINATED ORGANICS SUBCOMMITTEE
The content of this health assessment document on ethylene dichloride was
independently peer-reviewed in public session by the Environmental Health
Committee of the Environmental Protection Agency's Science Advisory Board.
ACTING CHAIRMAN. ENVIRONMENTAL HEALTH COMMITTEE
Dr. Seymour Abrahamson, Professor of Zoology and Genetics, Department of
Zoology, University of Wisconsin, Madison, Wisconsin 53706.
EXECUTIVE SECRETARY. SCIENCE ADVISORY BOARD
Dr. Daniel Byrd III, Executive Secretary, Science Advisory Board, A-101 F, U.S.
Environmental Protection Agency, Washington, DC 20460.
MEMBERS
Dr. Ahmed E. Ahmed, Associate Professor of Pathology, Pharmacology and
Toxicology, the University of Texas Medical Branch at Galveston.
Dr. George T. Bryan, Professor of Human Oncology, K4/528 C.S.C. Clinical
Sciences, University of Wisconsin, 500 Highland Avenue, Madison, Wisconsin
53792.
Dr. Ronald D. Hood, Professor and Coordinator, Cell and Developmental Biology
Section, Department of Biology, The University of Alabama, and Principal
Associate, R.D. Hood and Associates, Consulting Toxicologists, P.O. Box
1927, University, Alabama 35486.
Dr. K. Roger. Hornbrook, Department of Pharmacology, P.O. Box 26901, University of
Oklahoma, Oklahoma City, Oklahoma 73190.
ix
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TABLE OF CONTENTS
DISCLAIMER ii
PREFACE iii
AUTHORS AND REVIEWERS iv
LIST OF TABLES xii
LIST OF FIGURES xvii
1. SUMMARY AND CONCLUSIONS.. ^ T'1
2. INTRODUCTION 2~1
3. PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.1. NAME •. 3-1
3.2. CAS REGISTRY, RTECS, AND STORET NUMBERS 3-1
3.3. DESCRIPTION 3-1
3.4. STRUCTURE 3-1
3.5. PHYSICAL PROPERTIES OF PURE ETHYLENE DICHLORIDE 3-1
4. SAMPLING AND ANALYSIS OF ETHYLENE DICHLORIDE 1-1
1.1. SAMPLING 1*-1
1.2. ANALYSIS 4-1
4.2.1. Ethylene Dichloride in Air 4-1
4.2.2. Ethylene Dichloride in Water 1-2
1.2.3. Ethylene Dichloride in Solid Samples 1-3
4.2.4. Ethylene Dichloride in Blood 4-3
4.2.5. Ethylene Dichloride in Urine 4-3
4.2.6. Ethylene Dichloride in Tissue 4-4
5. SOURCES IN THE ENVIRONMENT 5-1
5.1. PRODUCTION PROCESSES 5-1
5.2. ETHYLENE DICHLORIDE PRODUCERS 5-2
5.3. ETHYLENE DICHLORIDE PRODUCTION AND TRENDS 5-2
5.4. ETHYLENE DICHLORIDE USES 5-5
5.5. SOURCES OF EMISSIONS 5-9
5.5.1. Production and Related Facilities 5-12
5.5.2. Dispersive Uses 5-13
5.5.3. Conclusions 5-14
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TABLE OF CONTENTS (cont.)
Page
6. TRANSPORT AND FATE IN THE ENVIRONMENT 6-1
6.1. ATMOSPHERE 6-1
6.2. AQUATIC MEDIA 6-7
6.3- SOIL 6-9
6.4. SUMMARY 6-10
7. ENVIRONMENTAL LEVELS AND EXPOSURE 7-1
7.1. ENVIRONMENTAL LEVELS 7-1
7.1.1. Atmospheric Levels 7-1
7.1.2. Ground and Surface Water Levels 7-9
7.1.3. Soil and Sediment Levels 7-14
7.2. ENVIRONMENTAL EXPOSURE 7-14
7.2.1. Exposure from Air 7-18
7.2.2. Exposure from Water 7-20
7.2.3* Exposure from Food 7-20
7.3. CONCLUSIONS 7-20
8. ECOLOGICAL EFFECTS 8-1
9. BIOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS 9-1
9.1. PHARMACOKINETICS 9-1
9.1.1. Absorption and Distribution 9-1
9.1.2. Excretion 9-18
9.1.3. Metabolism 9-32
9.1.4. Summary and Conclusions 9-55
9.2. ACUTE, SUBCHRONIC AND CHRONIC TOXICITY 9-57
9.2.1. Effects in Humans 9-57
9.2.2. Effects in Animals 9-86
9.2.3. Summary of Acute, Subchronic and Chronic Toxicity 9-115
9.3- REPRODUCTIVE AND TERATOGENIC EFFECTS 9-122
9.3.1. Summary and Conclusions 9-129
9.4. MUTAGENICITY 9-131
9.4.1. Gene Mutation Studies 9-131
9.4.2. Chromosome Aberration Studies 9-152
9.4.3. Other Evidence of DNA Damage 9-157
9.4.4. Summary and Conclusions 9-162
XI
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TABLE OF CONTENTS (cont.)
Page
9.5. CARCINOGENICITY 9-164
9.5.1. Animal Studies 9-164
9.5.2. Epidemiologic Studies 9-204
9.5.3. Risk Estimation from Animal Data 9-205
9.5.4. Summary 9-257
9.5.5. Conclusions 9-260
Appendix A. Comparison Among Different Extrapolation Models 9-263
Appendix B. Risk Calculation Based on Time-to-Event Data 9-265
10. REFERENCES 10-1
xii
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LIST OF TABLES
Table Page
5-1 Major Manufacturers of Ethylene Bichloride 5-3
5-2 U.S. Production and Sales of Ethylene Dichloride 5-*4
5-3 Ethylene Dichloride Uses 5-6
5-H Consumption of Ethylene Dichloride in 1979 and 197U • 5-7
5-5 Uses of Ethylene Dichloride for Intermediate Purposes 5-8
5-6 Estimated Environmental Releases of Ethylene
Dichloride in 1979 5-10
6-1 Rate Constants for a Few Chlorinated Ethanes at
Room Temperature 6-3
7-1 Ambient Atmospheric Levels of Ethylene Dichloride 7-2
7-2 Ambient Concentrations of Ethylene Dichloride in Urban Areas... 7-8
7-3 Reported Occurrence of Ethylene Dichloride in Groundwater
Systems — Combined Federal Data (NORS, NOMS, NSP, CWSS, GWSS). 7-15
7-1 Reported Occurrence of Ethylene Dichloride in Surface Water
Systems — Combined Federal Data (NORS, NOMS, NSP, CWSS) 7-16
7-5 State Data on Ethylene Dichloride in Groundwater 7-17
7-6 Estimated Respiratory Intake of Ethylene Dichloride by
Adults and Infants 7-19
7-7 Maximum Concentration Level in the Vicinity of Various
Emission Sources 7-19
7-8 Total Estimated Population (in Thousands) Exposed to
Ethylene Dichloride in Drinking Water at the Indicated
Concentration Ranges 7-21
7-9 Estimated Drinking Water Intake of Ethylene Dichloride
by Adults and Infants 7-21
9-1 Physical Properties of Ethylene Dichloride and
Other Chloroethanes 9-3
9-2 Partition Coefficients for Ethylene Dichloride 9-4
9-3 Fate of 14C-EDC in Rats 18 Hours After Oral (150 mg/kg)
or Inhalation (150 ppm, 6-hr) Exposure 9-6
xiii
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LIST OF TABLES (cont.)
Table Page
9-1 Peak Blood and Tissue Levels After Single Oral Dosage
of EDC in Male Rats 9-8
9-5 The Absorption Rate Constants (k ) and Area Under Curve
(AUC) for Rats After Single Oral Doses of EDC in Oil
and in Water as Vehicle and After Intravenous
Administration • 9-9
9-6 EDC Tissue Levels After 50 ppm Inhalation Exposure 9-14
9-7 EDC Tissue Levels After 250 ppm Inhalation Exposure 9-15
9-8 Blood Tissue Levels of EDC in Male Rats 2 Hours After
Single Oral Doses in Corn Oil 9-17
9-9 Percent Distribution of Radioactivity Excreted (48-hr) by
ill
Mice Receiving 1,2-Dichloroethane- C 9-20
9-10 Pharmacokinetic Parameters of EDC Administered as Single Bolus
Intravenous Injections in Saline to Sprague-Dawley Male Rats.
Parameters Calculated from a 2-Compartment Open Model 9-24
9-11 Pharmacokinetic Parameters of EDC Administered as Single Oral
Doses in Corn Oil and Water to Sprague-Dawley Male Rats.
Parameters Calculated from a 2-Compartment Open Model 9-26
9-12 Pharmacokinetic Parameters of EDC Following Termination of
Steady State Inhalation Conditions (5-hr Exposure) of 50
and 250 ppm to Male Sprague-Dawley Rats 9-29
9-13 Pharmacokinetic Parameters of EDC Following Termination of
Steady State Inhalation Conditions; 6-Hr Exposure, 150 ppm
to Male Osborne-Mendel Rats. Parameters Calculated from a
Two-Compartment Model 9-30
9-14 Identified Metabolites of Ethylene Dichloride and
Ethylene Bromide 9-34
9-15 Percent Distribution of Radioactivity Excreted (48-hr) as^
Urinary Metabolites by Mice Receiving 1,2-Dichloroethane- C
or 2-Chloroacetic Acid-^C 9-37
9-16 Total Macromolecular Binding and DNA Binding in Selected
14
Tissues of Rats After Exposure to C-EDC by Oral or
Inhalation Routes 9-52
xiv
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LIST OF TABLES (cont.)
Table
9-17
9-18
9-19
9-20
9-21
9-22
9-23
9-24
9-25
9-26
9-27
9-28
9-29
9-30
9-31
9-32
9-33
9-31
9-35
14
Effect of Dietary Disulfiram Upon the C Content of
Liver Nuclei Isolated 21 or 48 Hours After Administration
of a Single Oral Dose of 15 mg/kg [U-14C]EDB
Effects Associated with Acute Lethal Oral Doses of
Effects of Acute Oral Ingestion of 1 ,2-Dichloroethane
Effect of Ethylene Dichloride Exposure on Eye
Morbidity and Lost Workdays of Aircraft Industry Gluers
Concentrations of Ethylene Dichloride in Oil Refinery
Mineral Oil Purification Process Air
Effects Observed in Polish Oil Refinery Workers
Effects of Ethylene Dichloride on the Cornea
Summary of Mutagenicity Testing of EDC: Gene Mutations
Summary of Mutagenicity Testing of EDC: Gene Mutation
Tests in Insects
Summary of Mutagenicity Testing of EDC: Mammalian Systems....
Summary of Mutagenicity Testing of EDC: Chromosomal
Summary of Mutagenicity Testing of EDC: DMA Binding
Studies
Design Summary for 1 ,2-Dichloroethane (EDC) Gavage
Terminal Survival of Osborne-Mendel Rats Treated With
Page
9-54
9-58
9-65
9-72
9-78
9-80
9-81
9-87
9-95
9-102
9-132
9-143
. 9-145
9-151
9-153
9-158
9-161
9-166
9-170
XV
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LIST OF TABLES (cont.)
Table Page
9-36 Squamous Cell Carcinomas of the Forestomach in Osborne-
Mendel Rats Treated With 1,2-Dichloroethane (EDC) 9-171
9-37 Hemangiosarcomas in Osborne-Mendel Rats Treated With
1,2-Dichloroethane (EDC) 9-172
9-38 Adenocarcinomas of the Mammary Gland in Female Osborne-
Mendel Rats Treated With 1,2-Dichloroethane (EDC) 9-173
9-39 Design Summary for 1,2-Dichloroethane (EDC) Gavage
Experiment in B6C3F1 Mice 9-175
9-10 Terminal Survival of B6C3F1 Mice Treated With
1,2-Dichloroethane (EDC) 9-176
9-11 Hepatocellular Carcinomas in B6C3F1 Mice Treated With
1,2-Dichloroethane (EDC) 9-179
9-12 Alveolar/Bronchiolar Adenomas in B6C3F1 Mice Treated
With 1,2-Dichloroethane (EDC) 9-180
9-13 Squamous Cell Carcinomas of the Forestomach in B6C3F1 Mice
9-11
9-15
9-16
9-17
9-18
9-19
9-50
9-51
Adenocarcinomas of the Mammary Gland in Female B6C3F1 Mice
Treated With 1 , 2-Dichloroethane (EDC)
Endometrial Polyp or Endometrial Stromal Sarcomas in Female
B6C3F1 Mice Treated With 1 , 2-Dichloroethane (EDC) ,
Characterization of 1 , 2-Dichloroethane (EDC) Inhalation
Design Summary for 1, 2-Dichloroethane (EDC) Experiment
Survival of Sprague-Dawley Rats Exposed to EDC at 52 and
1 01 Weeks ,
Tumor Incidence in Sprague-Dawley Rats Exposed to
Mammary Tumors in Sprague-Dawley Rats Exposed to
Design Summary for 1 ,2-Dichloroethane (EDC) Experiment
9-182
9-183
9-181
9-185
9-186
9-188
9-192
9-195
xv i
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LIST OF TABLES (cont.)
Table Page
9-52
9-53
9-51
9-55
9-56
9-57
9-58
9-59
9-60
9-61
9-62
9-63
9-61
9-65
9-66
Survival of Swiss Mice Exposed to 1 , 2-Dichloroethane (EDC)
at 52 and 78 Weeks
Tumor Incidence in Swiss Mice Exposed to 1 , 2-Dichloroethane
(EDO
Pulmonary Tumor Response in Strain A/st Mice Injected
with 1 , 2-Dichloroethane (EDC)
Mouse Skin Bioassay of 1, 2-Dichloroethane (EDC) and
Chloroacetaldehyde
Incidence Rates of Hemangiosarcomas in the Circulatory
Systems of Male Osborne-Mendel Rats and of Hepatocellular
Disposition of 1 C-EDC in Rats After Single Administrations
of 150 mg/kg Orally and 150 ppm for 6 Hours by Inhalation
Comparison of the Metabolism and Pharmacokinetics of the
"High" Doses in Rats of the Maltoni et al. Inhalation and
Evidence of Nonlinear 'Kinetics for EDC in Rats After
1U
DNA Covalent Binding in Rats Exposed to C-EDC
Lifetime Average Human Equivalent Doses Based on the NCI
Lifetime Average Human Equivalent Doses Based on the NCI
Rat Bioassay
Incidence Rates of Hemangiosarcomas in the Circulatory
System of Male Osborne-Mendel Rats
Incidence Rates of Hepatocellular Carcinomas in Male
B6C3F1 Mice
The 95$ Upper-Bound Estimate of Risk at 1 mg/kg/day Using
Relative Carcinogenic Potencies Among 51 Chemicals
Evaluated by the Carcinogen Assessment Group as Suspect
Human Carcinogens
9-196
9-197
9-201
9-203
9-207
9-210
9-211
9-219
9-227
9-233
9-235
9-215
9-215
9-216
9-253
xvii
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LIST OF FIGURES
Figure I2E§
9-1 EDC levels after single oral administration in rats 9-7
9-2 Top: Blood levels of EDC observed during and following a
6-hour inhalation exposure to 150 ppm EDC. Bottom: Semi-
logarithmic plot of EDC blood levels vs. time after
exposure termination 9-12
9-3 Blood levels of EDC in rats after i.v. administration 9-23
9-1 Levels of EDC in rats after inhalation exposure to 50 ppm
(top) and 250 ppm (bottom). Levels were measured at
termination of 5-hour exposure period 9-27
9-5 Postulated microsomal oxidative metabolism of 1,2-dihaloethanes. 9-39
9-6 Further metabolism of 2-chloroacetaldehyde and 1-chloroso-
2-chloroethane from miorosomal oxidation 9-11
9-7 Postulated cytosolic metabolism of ethylene dichloride 9-14
9-8 Growth curves for male and female Osborne-Mendel rats
administered EDG by gavage 9-168
9-9 Survival comparisons for male and female Osborne-Mendel rats
administered EDC by gavage 9-169
9-10 Growth curves for male and female B6C3F1 mice administered
EDC by gavage 9-177
9-11 Survival comparisons for male and female B6C3F1 mice
administered EDC by gavage 9-178
9-12 Relationship in mice between dose of EDC (mg/kg) and the amount
of EDC exhaled unchanged and the amount of the dose metabolized
to urinary metabolites plus C02 9-211
9-13 Relationship in Sprague-Dawley rats between the area under the
blood EDC concentration-time curve (AUC) and the gavage dose
of EDC in corn oil (mg/kg) or inhaled doses of EDC (ppm for
6 hours) 9-218
9-lU Linear relationship for EDC between the logarithm of the area
under the blood C,t curve (AUC) and the logarithm of gavage
dose in corn oil given to Sprague-Dawley rats 9-221
9-15 Relationship in Sprague-Dawley rats between gavage doses of EDC
in corn oil and the ratio from the area under the blood EDC
concentration-time curve (AUC) divided by the administered dose. 9-223
xviii
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LIST OF FIGURES
Figure Page
9-16 Blood levels of EDC following exposure to EDC by inhalation
(150 ppm, 6 hours) or gavage (150 mg/kg) 9-225
9-17 Histogram representing the frequency distribution of the
potency indices of the 54 suspect carcinogens evaluated by
the Carcinogen Assessment Group 9-252
xix
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1. SUMMARY AND CONCLUSIONS
Cthylene dichoride (EDC) is a clear, colorless, volatile liquid with a
pleasant odor. EDC has a molecular weight of 98.96, a boiling point of 83-7°C
and a vapor pressure of 64 torr at 20°C. Unlike more highly chlorinated
hydrocarbons, EDC has a flashpoint (17°C closed cup), an autoignition tempera-
ture (413°C) and explosive limits (6.2-15.6$ by volume in air). It has a water
solubility of 8820 mg/2. and a log octanol/water partition coefficient of 1.48.
EDC is analyzed best by gas chromatography using either an electron capture
or halogen specific (microcoulometric or electrolytic conductivity) detector.
Alternatively, a gas chromatograph/mass spectrometer may be used. EPA methods
502.1, 601, 624 and 8010 detail the analysis of EDC from water, wastewater and
solid samples. Variations in these methods provide for analysis of EDC in
biological media (e.g., blood, urine and tissue). EDC in air is .analyzed by
condensing the volatiles in a previously collected air sample in a GC column,
followed by temperature programming the column.
EDC is produced commercially by the direct chlorination or oxychlorination
of ethylene. Most of the production capacity, which exceeds nine million metric
tons annually, is located in Texas and Louisiana. Roughly 90$ of the production
is captively consumed by the producers. Production during 1980, 1981 and 1982
totalled 5.037, 1.523 and 3-155 million metric tons, respectively. A major
portion (84$) of the United States EDC production is consumed to make vinyl
chloride monomer. It is also used to make chlorinated solvents, vinylidene
chloride and ethyleneamines.
The major source of EDC emissions to the environment is from vent gas
streams during the manufacture of EDC and its derivatives. Significant amounts
are also emitted to the environment from dispersive uses in gasoline, paints and
1-1
-------
cleaning agents. In 1979, an estimated 6696 metric tons of EDC were released to
the atmosphere from EDC and derivative production, while WW metric tons were
released from dispersive uses.
The most likely removal mechanism for EDC from the atmosphere is reaction
with hydroxyl free radicals. Based on available kinetic data and average
tropospheric hydroxyl free radical concentrations, the half-life of EDC has been
estimated to range from 36-127 days. Due to different factors that may cause
variations in *OH concentrations in the troposphere, the persistence of EDC could
vary somewhat from the estimated value. Chloroacetyl chloride is probably the
principal product resulting from the reaction of EDC with >OH radicals.
EDC is not expected to play a significant role in stratospheric ozone
destruction reactions due to its relatively short tropospheric half-life.
However, Chloroacetyl chloride may have sufficient stability to diffuse to the
stratosphere and may participate in UV reactions, producing chlorine atoms.
In the aquatic environment, volatilization appears to be the most
significant removal mechanism. The half-life for this process has been estimated
to be -4 hours.
From the little information that is available regarding the fate of EDC in
soil, it can be surmised that both volatilization from and leaching through soil
may be two significant removal mechanisms for EDC.
The highest atmospheric concentrations that have been monitored for EDC
have been detected near production and use facilities. Mean levels as high as
27.5 ppb have been monitored near production-use facilities in Lake Charles, LA.
However, atmospheric exposure appears to vary greatly from one location to
another. The available monitoring data, which includes levels detected in
general ambient urban areas, indicate that most locations have concentrations of
<0.5 ppb. The data are not sufficient to determine regional variations in
1-2
-------
exposure levels. Using Federal Reporting Data System information, it has been
projected that EDC levels in all groundwater and surface water systems in the
United States fall below 10 ug/S,, and that most are <1.0 ug/fc. No data are
available indicating the presence of EDC in finished foods.
Pharmacokinetic studies in animals indicate that EDC is rapidly and
extensively absorbed following oral and inhalation exposure. Dermal absorption
is negligible in most vapor exposure situations, although absorption by this
route may be significant with direct liquid contact. Complete tissue
distribution of EDC is consistent with its lipophilic nature; the chemical
crosses the blood/brain and placental barriers and concentrates in breast milk.
Up to 90? of low oral or inhalation doses are metabolized by rats and mice, with
biotransformation occurring by multiple pathways; EDC is metabolized to
2-chloroacetaldehyde, S-(2-chloroethyl)-glutathione, and other putative
reactive metabolites capable of covalent binding to cellular macromolecules.
Metabolism is dose-related; as the dose increases, the percent metabolized
decreases, although the absolute amount of EDC metabolized increases until
saturation of metabolic pathways occur. Elimination of unmetabolized EDC occurs
almost exclusively via the lung, with a greater percentage being exhaled at
higher doses. Total body elimination of EDC is relatively rapid and is
consistent with a two-compartment system and Michalis-Menten kinetics.
Significant bioaccuraulation of EDC in blood and tissues is not expected to occur.
The effects of acute inhalation exposure to EDC are similar in humans and
animals. Immediate symptoms of toxicity are CNS depression and irritation of the
respiratory tract and eyes. Death was usually ascribed to respiratory and
circulatory failure, and pathologic examinations typically revealed congestion,
degeneration, necrosis and hemorrhagic lesions of most internal organs (e.g.,
liver, kidneys, spleen, lungs, respiratory tract, gastrointestinal tract).
1-3
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Several papers describing human health surveys appear in the literature;
adverse effects are largely associated with the gastrointestinal and nervous
systems. The exposure information in these studies is not well documented, but
taken together indicate that adverse effects have likely occurred in humans at
EDO levels below 100 ppm, although probably not as low as 10 ppm. Subtle
neurological effects (e.g., fatigue, irritability, sleeplessness) may be more
prevalent than overt symptoms of CNS toxicity at lower concentrations. Studies
with multiple species of animals have shown that subchronic or chronic exposure
to EDC at vapor concentrations of £100 ppm did not produce treatment-related
adverse effects on survival, growth, hematology, clinical chemistry, organ
weight or histology. Toxic effects were apparent at higher concentrations and
were exposure-related; exposure to 400-500 ppm produced high mortality and
histopathological alterations in rodents within a few exposures.
Limited data indicate that the toxic response to acute oral exposure is
similar to that of inhalation exposure in humans and animals. Subchronic/chronic
oral administration of EDC at daily dosages of =200 mg/kg and higher caused
decreased growth rate and mortality in mice, which may have been tumor-related.
Chronic oral administration of lower dosages of EDC (=34 mg/kg/day) to rats
produced mortality that appeared to be due to non-neoplastic lesions including
bronchiopneumonia and endocardial thrombosis.
The available evidence suggests that EDC does not adversely affect the
reproductive or development process in laboratory animals except at maternally
toxic levels. However, additional laboratory testing is needed, as well as
epidemiological studies, to conclusively establish that EDC is not a human
teratogen and does not cause adverse reproductive effects.
Positive responses in different test systems representing a wide range of
organisms indicate that EPC is capable of causing gene mutations in prokaryotes
1-14
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and eukaryotes. Several of its putative metabolites, thought to be formed in
rats and mice, are judged to be more potent mutagens than EDO. EDC has not been
adequately tested for its ability to cause chromosomal aberrations or heritable
effects. Further testing is needed to assess its ability to cause these effects.
EDC was shown to be carcinogenic in a lifetime gavage bioassay that was
conducted by the National Cancer Institute, producing tumors in both rats
(forestomach carcinomas, cirulatory system hemangiosarcomas, subcutaneous
fibromas, mammary gland adenocarcinomas) and mice (hepatocellular carcinomas,
alveolar/bronchiolar adenomas, mammary carcinomas, endometrial tumors). EDC
produced an elevated but not a statistically significant increase in the
incidence of lung adenomas in strain A mice when administered intraperitoneally.
EDC induced statistically significant benign lung tumors in mice when applied to
the skin, but statistically significant skin tumors were not observed. No
statistically significant increases in tumors occurred in rats or mice following
lifetime inhalation exposure. No case reports or epidemiologic studies
concerning EDC were available in the published literature for analysis.
From a weight-of-evidenee approach, the direct and supporting evidence for
carcinogenicity includes: 1} multiple tumor types in an oral rat bioassay and an
oral mouse bioassay, 2} suggestive evidence in two other animal bioassays,
3) demonstrated evidence of reactive metabolites of EDC and formation of a DNA
adduct, and 4) evidence that EDC is also a mutagen.
The U.S. Environmental Protection Agency is using, on an interim basis, a
proposed classification scheme for evaluating the weight of evidence for
carcinogenicity. Using this classification, the positive findings in the oral
rat and mouse studies would be considered a sufficient level of evidence in
experimental animals. The sufficient level of animal evidence, together with an
absence of epidemiologic data, provides a basis for an overall weight-of-
1-5
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evidence ranking of Group B2, meaning that EDC is a "probable" carcinogen in
humans.
Applying the International Agency for Research on Cancer (IARC)
classification scheme, which EPA has used in the past, the positive finding in
the experimental animals and the absence of epidemiologic data provide an overall
weight-of-evidence ranking of Group 2B, meaning that EDC is a "probable"
carcinogen in humans.
While the "probable" carcinogenicity conclusion obviously applies to
ingestion exposure since the positive evidence comes from gavage experiments, it
is likely that EDC is a "probable" carcinogen for humans via inhalation exposure.
In view of the evidence, it is prudent to consider EDC to be carcinogenic via
inhalation exposure although the potency may vary from that estimated from gavage
studies.
Assuming that EDC is carcinogenic for humans, data from gavage studies using
both rats and mice can be used to estimate the carcinogenic potency and unit
risks. The development of the potency and unit risk values is for the purpose of
providing an estimator to assess the possible magnitude of the public health
impact if EDC is carcinogenic for humans. The carcinogenic potencies estimated
on the basis of hemangiosarcomas in rats and hepatocellular carcinomas in mice
are comparable when the linearized multistage model is used. The upper-bound
_2
estimate of EDC potency is 9.1 x 10 /mg/kg/day based on the hemangiosarcoma
response in rats using a time-to-death adjustment and an adjusted dose derived
from the metabolism/kinetic evaluation. The upper-bound estimate of the
incremental cancer risk due to 1 ng/i of EDC in drinking water is 2.6 x 10~ .
Two upper-bound estimates of the incremental cancer risk for the inhalation of
1 jig/nr of EDC are calculated: 2.6 x 10 on the basis of the gavage potency
value, and 1.0 x 10~ on the basis of a negative inhalation study. The
1-6
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inhalation unit risks are presented as two values in recognition of the
uncertainties associated with the different data sets used, there being no
certain reasoning that one value has more scientific merit than the other value.
The upper-bound nature of the unit-risk estimates is such that the true risk is
not likely to exceed these values and may be lower.
Expressed in terms of relative potency, EDC ranks in the fourth quartile
among 51 carcinogens evaluated by EPA's Carcinogen Assessment Group.
1-7
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2. INTRODUCTION
EPA's Office of Research and Development has prepared this health assess-
ment to serve as a "source document" for Agency use. This health assessment was
originally developed for use by the Office of Air Quality Planning and Standards
to support decision-making regarding possible regulations of ethylene dichloride
under Section 112 of the Clean Air Act. However, based on the expressed interest
of other agency offices, the scope of this document was expanded to address
ethylene dichloride in relation to sectors of the environment outside of air. It
is fully expected that this document will serve the information needs of many
government agencies and private groups that may be involved in decision-making
activities related to ethylene dichloride.
In the development of the assessment document, existing scientific litera-
ture has been surveyed in detail. Key studies have been evaluated and summary
and conclusions have been prepared so that the chemical's toxicity and related
characteristics are qualitatively identified.
The document considers all sources of ethylene dichloride in the envi-
ronment, the likelihood for its exposure to humans, and the possible effect on
man and lower organisms from absorption. The information found in the document
is integrated into a format designed as the basis for performing unit risk
assessments. When appropriate, the authors of the document have attempted to
identify gaps in current knowledge that limit risk evaluation capabilities.
The basic literature search for this document is complete through 1983;
however, selected publications have been included in the sections on
mutagenicity and carcinogenicity through 198H.
2-1
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3. PHYSICAL AND CHEMICAL PROPERTIES
3.1. NAME
Ethylene dichloride (EDC) is the common name for 1,2-dichloroethane. It is
also commonly referred to as ethylene chloride and S-dichloroethane (Archer,
1979).
3.2. CAS REGISTRY, RTECS, AND STORET NUMBERS
CAS Registry: 107-06-2
RTECS: KI05250000
STORET: 3^531
3.3. DESCRIPTION
Ethylene dichloride is a clear, colorless, volatile liquid with a pleasant
odor, and is stable at ordinary temperatures (Archer, 1979).
3.U. STRUCTURE
C1CH2CH2C1
3.5. PHYSICAL PROPERTIES OF PURE ETHYLENE DICHLORIDE
The physical properties of pure ethylene dichloride were taken from Archer
(1979) and Weast (1980), unless otherwise stated.
Molecular weight: 98.96
Melting point: -35.3°C
Boiling point: 83.7°C
Density at 20°C: 1.2529 g/mfi.
Viscosity: 1.U*»51 m Pa S (cP)
Flashpoint:
Closed cup 17°C
Open cup 21°C
Explosive limits in
air at 25°C: 6.2-15.6* by volume
3-1
-------
Autoignition tempera-
ture in air:
Vapor pressure:
413°C
^C
10
20
30
kPa
5.3
8.5
13-3
Torr
40
64
100
Solubility in water: 0.0891 M (Valvani et al., 1981)
8,820 mg/JZ,
Log Octanol/Water
Partition Coefficient: 1.48 (Valvani et al., 1981)
Henry's Law Constant a
atm mj mol : 9.14 x 10 (Mabey et al., 1981)
Blood/Air Partition
Coefficient (37°C): 19.5 + 0.5 (Sato and Nakajima, 1979)
3-2
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4. SAMPLING AND ANALYSIS OF ETHYLENE DICHLORIDE
4.1. SAMPLING
Talcing environmental samples representative of the area in question is a
complex task beyond the scope of this document. A number of documents either
devoted exclusively to sampling or containing considerable information relating
to the subject are listed below and should be consulted prior to initiating
environmental sampling. For air, Singh et al. (1979, 1983b), Pellizzari (1978),
and Pellizzari et al. (1979) have detailed sampling methodologies that describe
and/or show equipment and strategies. Water and wastewater sampling is described
by Berg (1982) and solids sampling by U.S. EPA (19&2b).
4.2. ANALYSIS
4.2.1. Ethylene Bichloride in Air. Ethylene dichloride in air can be analyzed
by a number of methods; however, the method of Singh et al. (1980) appears to be
substantially free of artifact problems and completely quantitative. In this
method, an air sample in a stainless steel canister at 32 psig is connected to a
preconcentration trap consisting of a 4" x 1/16" ID stainless steel tube
containing glass beads, glass wool, or 3% SE-30 on acid washed 100/120 mesh
Chromosorb W. The sampling line and trap, maintained at 90°C, are flushed with
air from the canister; then the trap is immersed in liquid 0_ and air is passed
through the trap, the initial and final pressure being noted (usually between 30
and 20 psig) on a high-precision pressure gauge. The ideal gas law can be used to
estimate the volume of air passed through the trap. The contents of the trap are
desorbed onto a chromatography column by backflushing it with an inert gas while
holding the trap at boiling water temperature. Suitable columns include 10$ SP-
1000 on Supelcoport (100/120 mesh, 15' x 1/8" stainless steel) and 0.2$ CW-
-------
1500 on Carbopack C (80/100 mesh, 10' x 1/8" Ni). Both columns can be operated at
45°C with a carrier gas flow of 25 mil/minute on the former column and 10 mil/min-
ute on the latter. An electron capture detector operating at 330°C was found to
be optimum. It should be noted that the above authors found Tenax to be unsuit-
able for air analyses because of the presence of artifacts in the spectrum from
oxidation of the Tenax monomer. In addition, when Tenax is used as a sorbent,
safe sampling volumes (i.e., that volume of air which, if sampled over a variety
of circumstances, will not cause significant breakthrough) should be used. Brown
and Purnell (1979). determined the safe volume for ethylene dichloride per gram
Tenax to be 27 I (flow rate 5-600 mi/minute; ethylene dichloride cone.
<250 mg/nr; temp, up to 20°C) with a safe desorption temperature of 90°C.
The detection limits of this method were not specified and are dependent on
the volume of air sampled. Analyses as low as 33 ppt have been reported using
this method (Singh et al., 1980).
4.2.2. Ethylene Dichloride in Hater. Ethylene dichloride in water can be
analyzed by the purge-and-trap method (Method 502.1) as recommended by the
Environmental Monitoring and Support Laboratory of the U.S. EPA (1981). In this
method, an inert gas is bubbled through 5 ml of water at a rate of 40 mil/minute
for 11 minutes, allowing the purgable organic compounds to partition into the
gas. The gas is passed through a column containing 3% OV-1 on Chromosorb W,
Tenax GC silica gel, and coconut charcoal at 22°C, which traps most of the
organics removed from the water. The adsorption column is then heated rapidly to
1dO°C and backflushed with helium (20-60 mi/minute, 4 minutes) to desorb the
trapped organics. The effluent of the column is passed into an analytical gas
chromatography column packed with 1$ SP-1000 on Carbopack-B (60/80 mesh,
8' x 0.1" ID) maintained at 40°C. The column is then temperature programmed
starting at 45°C for 3 minutes and increasing at 8°C/minute until 220°C is
4-2
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reached; it is then held there for 15 minutes or until all compounds have eluted.
A halogen-specific detector (or GC-MS) having a sensitivity of 0.10 \ig/l with a
relative standard deviation of <10$ must be used. This method is similar to EPA
method 601,621 for use with wastewater (Longbottom, 1982).
U.2.3. Ethylene Dichloride in Solid Samples. Ethylene dichloride in solid
samples may be analyzed by EPA method 8010 "Halogenated Volatile Organ!cs" (U.S.
EPA, 1982b). With this method, a portion of the sample is dispersed in poly-
ethylene glycol or methanol. The dispersion is then mixed with water and purged
in a manner identical to the analysis of EDC in water. The trap contains 3% OV-1
on Chromosorb W (60/80 mesh), Tenax GC (60/80 mesh), Grade 15 silica gel (35/60
mesh), and activated coconut charcoal (6/10 mesh); desorption is performed at
180°C for 1 minutes with a gas flow of 20 to 60 mi/minute. Gas chromatographic
conditions are identical to the analysis of ethylene dichloride in water.
1.2.1. Ethylene Dichloride in Blood. Ethylene dichloride in blood can be
analyzed by using a modified purge-and-trap method (Pellizzari et al., 1979).
This method involves diluting an aliquot of whole blood (with anticoagulant) to
=50 mil with prepurged, distilled water. The mixture is placed in a 100 mi 3-neck
round bottom flask along with a Teflon-lined magnetic stirring bar. The necks of
the flask are equipped with a helium inlet, a Tenax trap, and a thermometer. The
Tenax trap is a 10 cm x 1.5 cm ID glass tube containing pre-extracted (soxhlet,
methanol, 21 hrs) and conditioned (270°C, 30 mil/minute helium flow, 20 minutes)
35/60 mesh Tenax (=1.6 g, 6 cm). The sample is then heated to 50°C and purged
with a helium flow rate of 25 mi/min for 90 minutes. Analysis can be performed as
indicated in Section 1.2.2.
1.2.5. Ethylene Dichloride in Urine. Ethylene dichloride in urine can be
analyzed by using an apparatus identical to the one described in Section 1.2.1,
using 25 mH of urine, diluted to 50 ml, instead of blood.
4-3
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U.2.6. Ethylene Dichloride in Tissue. Ethylene dichloride in tissue can be
analyzed by using an apparatus identical to the one described in Section U.2.U,
using 5 g of tissue, diluted to 50 md, instead of blood and macerated in an ice
bath. The purge time is reduced to 30 minutes.
-------
5. SOURCES IN THE ENVIRONMENT
5.1. PRODUCTION PROCESSES
Two commercial processes account for virtually all of the EDC produced
currently. One is the direct chlorination of ethylene with chlorine. The other
is an oxychlorination process in which ethylene, hydrogen chloride and oxygen are
reacted to form EDC. Currently, most production centers around large manufac-
turing plants employing a balanced combination of both of these two processes.
Over 80$ of the EDC produced is used to make vinyl chloride monomer via
dehydrohalogenation of EDC. Most EDC producers have nearby vinyl chloride
production facilities. The balanced plants use the hydrogen chloride recovered
when the EDC is dehydrohalogenated to vinyl chloride as feed to the
oxychlorination reactor. This requires roughly a 50-50 split between direct
chlorination and oxychlorination for a completely balanced operation assuming
all EDC is cycled to the vinyl chloride facilities. In this manner, there will
be no net production of hydrogen chloride. As of 1971, =58? of the total EDC
production capacity was based upon direct chlorination and =42$ was based upon
oxychlorination (Pervier et al., 1974). Current capacities are judged to be
roughly the same.
In the direct chlorination process, EDC is produced by the catalytic vapor-
or liquid-phase chlorination of ethylene as follows (Archer, 1979):
FeCl.
CH, = CH_ + Cl- (5$ air) ^ » C1CH,CH-C1
* 40-50°C * 2
Most liquid-phase processes use ferric chloride as the catalyst. The chlori-
nation is carried out at 40-50°C with 5% air added to prevent substitution
chlorination of the product.
5-1
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The oxychlorination process is usually incorporated into an integrated
vinyl chloride plant in which hydrogen chloride (which is recovered from cracking
the EDC to vinyl chloride) is recycled to an oxychlorination unit (Archer, 1979).
The hydrogen chloride by-product is used as the chlorine source in the chlorina-
tion of ethylene in the presence of oxygen and copper chloride catalyst as
follows:
CuCl
2CH- = CH_ + 4HC1 + 0- =-» 2C1CH_CH_C1 + H,0
22 2 270oc 22 2
5.2. ETHYLENE DICHLORIDE PRODUCERS
The producers of EDC in the United States are listed in Table 5-1 along with
their respective production capacities. As can be noted from Table 5-1, most of
the production capacity is located in Texas and Louisiana. These production
facilities also captively consume the major portion of the EDC produced, as in
the manufacture of vinyl chloride and, to a much lesser extent, in the
manufacture of vinylidene chloride and various chlorinated solvents. From Table
5-2, it can be noted that only =10$ of the EDC production has been sold on the
open market in most recent years.
5.3* ETHYLENE DICHLORIDE PRODUCTION AND TRENDS
U.S. production volumes and sales of EDC are listed in Table 5-2. Blackford
(1974) and the U.S. International Trade Commission (1974) have stated that the
production volumes as listed may be somewhat smaller than the actual production
because some EDC may be produced, but not separated or accurately measured (and
therefore not reported) by some producers. Blackford (1971) has estimated actual
productions on the order of 10$ higher than the reported productions.
The demand for EDC for the years 1982 and 1983 has been estimated to be
4.127 and 4.716 million metric tons, respectively, while the demand for the year
1987 is expected to be 5.487 million metric tons (CMR, 1983). Production of EDC
5-2
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TABLE b-T
Major Manufacturers of Ethylene Dichloride"
Manufacture
Plant Sites
Annual Capacity
(Millions of Metric Tons)
Atlantic Richfield (ARCO)
Borden, Inc.
Dow Chemical USA
E.I. duPont (Conoco)
Port Arthur, TX
Geismar, LA
Freeport, TX
Oyster Creek, TX
Plaquemine, LA
Lake Charles, LA
0.200
0.230
0.725
0.500
0.840
0.525
Ethyl Corp.
Formosa Plastics Corp.
Georgia-Pacific Corp.
B.F. Goodrich Co.
PPG Industries
Shell Chem. Co.
Union Carbide Corp.
Vulcan Materials Co.
Baton Rouge, LA
Pasadena, TX
Baton Rouge, LA
Point Comfort, TX
Plaquemine, LA
Deer Park, TX
La Porte, TX
Calvert City, KY
Convent , LA
Lake Charles, LA
Deer Park. TX
Norco, LA
Taft, LAd
Texas City, TX
Geismar, LA
0.320
0.110
0.240
0.385
0.735
0.110
0.720
O.J»50
0.360
1.230
0.620
0.540
0.070
0.070
0.160
Total 9.110
Note: Capacities are flexible depending on finishing capacities for
vinyl chloride and chlorinated solvents.
a
Source: SRI, 1983; CMR, 1983
Operated under a toll agreement with Diamond Shamrock Corp.
«i
"Closed for an indefinite time period because of the temporary closing
of Shell's vinyl chloride monomer plant located there.
Captive use only.
5-3
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TABLE 5-2
U.S. Production and Sales of Ethylene Dichloride3
Year
1982
1981
1980
1979
1978
1977
1976
1975
1974
1973
1972
1971
1970
1965
1960
1950
Millions of Metric
Production
3- ^55
4.523
5.037
5.349
4.989
4.987
3.617
3.617
4.156
14.214
3.541
3.428
3-383
1.113
0.575
0.138
Tons
Sales
0.640
0.401
0.510
0.633
0.469
0.692
0.617
0.345
0.596
0.613
0.656
0.595
0.596
0.140
0.198
0.021
aSource: USITC, USTC, 1952-1982
Production totals may be understated in some years because some
ethylene dichloride is produced but not separated or accurately
measured (and therefore not reported) by some producers.
5-4
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in 1982 was sharply lower than in preceding years (see Table 5-2). This was
largely due to a recession in the vinyl chloride market and a decision of
producers to use their EDC inventories during the winter of 1982 (Chemical and
Engineering News, 1982, 1983). Production of EDC is expected to rebound during
1983. Historically, the demand for EDC grew at a rate of =1% per year during the
period from 1973-1982. Demand is anticipated to grow at a rate of 1$ per year
through 1987 (CMR, 1983).
5.4. ETHYLENE DICHLORIDE USES
A major portion (84J) of U.S. EDC production is converted to vinyl chloride
monomer. The major uses of EDC for the years 1983, 1980, 1977 and 1974 are given
in Table 5-3* Consumption of EDC for specific end uses is given in Table 5-4.
In addition to vinyl chloride, EDC is used as a starting material in the
production of chlorinated solvents such as 1,1,1-trichloroethane, trichloro-
ethylene and perchloroethylene and as intermediate in the production of
ethyleneamines and vinylidene chloride (Archer, 1979; Blackford, 1974). Table
5-5 lists the users of EDC for these purposes. In general, these users are also
producers of EDC.
EDC is used as an additive (lead scavenger) in tetraethyl lead antiknock
mixtures for gasolines. Scavenging agents are used in gasolines to transform the
combustion products of lead alkyls to forms that are more likely to be vaporized
from the engine surfaces. The most important lead scavengers used for this
purpose are EDC and ethylene dibromide (McCormack et al., 1981; Blackford, 1974).
The sale of tetraalkyl lead compounds is always in admixture with EDC and/or
ethylene dibromide. Conventional motor-mix formulations contain 1 raol of EDC and
1/2 mol of ethylene dibromide per mol of tetraethyl lead (McCormack et al.,
1981). This use of EDC is expected to decline in future years due to EPA
regulations concerning the use of leaded gasolines. CMR (1982) projects that the
5-5
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TABLE 5-3
Ethylene Bichloride Uses
a
Percent of Total
Use
Vinyl chloride monomer
Chlorinated solvents
Vinylidene chloride
Exports
Lead scavenger
Amines
Miscellaneous
1983
en
4
2
9
—
—
1b
1980
84
7
2
5
—
--
2b
1977
80
10
—
—
•3
—
7°
1974
78
8
—
~
3
2
9°
aSource: CMR, 1974, 1977, 1980, 1983
Includes lead scavenger
Includes exports
5-6
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TABLE 5-t
Consumption of Ethylene Dichloride in 1979 and 1974a
Consumption (Thousands of Metric Tons)
Vinyl chloride monomer
1,1,1 -Trichloroe thane
Trichloroethylene
Perchloroe thy lene
Vinylidene chloride
Ethyleneamines
Lead scavenger
Exports
Preparation of pclysulfides
Paints, coatings and adhesives
Extraction solvents
Cleaning of fabric and polyvinyl
chloride equipment
Grain fumigation
Other uses
Total
1979 • %
4420 85.0
—
89.1 1.7
—
118.7 2.3
168.1 3.2
81.7 1.6
179.0 3.4
0.5
1.36
1.05
0.91
0.46
0.46
5199. W 99.8
1974
3891.2
153-3
132.8
124.7
97.0
131.5
97.0
167.3
—
—
—
—
—
6.8
14804.4
%
81.0
3.2
2.8
2.6
2.0
2.7
2.0
3.5
—
—
—
—
—
0.1
99.9
aSource: Seufert et al., 1980; Blackford, 1974
5-7
-------
TABLE 5-5
Users of Ethylene Dichloride for Intermediate Purposes
a
i
00
Vinylidene
1,1,1-Trichloroethane Trichloroethylene Perchloroethylene Ethyleneamines Chloride
Dow Chem. USA
Freeport, TX
Plaqueraine, LA
X
X
X X
X
X X
X
PPG Industries
Lake Charles, LA
Union Carbide Co.
Taft, LA
Texas City, TX
Diamond Shamrock Corp.
Deer Park, TX
E.I. duPont
Corpus Christ!, TX
X
X
aSource: SRI, 1983; Blackford, 1971
X = produces this chemical from ethylene dichloride
-------
demand for lead in gasoline will decrease at a rate of 11$ per year (average)
through 1986.
Exports of EDO have been increasing in recent years (see Table 5-6) and may
grow by as much as 8$ per year through the mid 1980s (CMR, 1983)* The Japanese
are expected to become major importers of EDC as they close down significant
amounts of chlorine capacity (mercury cell method) because of environmental
regulations (some of the chlorine made by this method is used to make EDC).
However, part of that demand will be filled by Shell's one billion pounds/year
EDC plant in Jubail, Saudi Arabia, which is due on stream in 1985.
EDC has a variety of relatively small miscellaneous uses. It is used as a
solvent in applications that include textile and PVC cleaning, metal degreasing,
extractions, and use in paints, coatings and adhesives. As an intermediate, EDC
is used to produce polysulfide elastomers and ethylenimine (aziridine). Dow
chemical captively consumes several million pounds per year of EDC at their
Freeport, TX, facility to produce ethylenimine. EDC is used as a grain fumigant;
Auerbach Associates (1978) estimated grain fumigant uses; Metcalf (1981) lists
EDC as a fumigant for use in household and soil applications. Other uses
mentioned for EDC include varnish and finish removers, soaps and scouring
compounds, wetting and penetrating agents and ore flotation (Hawley, 1981).
5.5. SOURCES OF EMISSIONS
EDC can enter the environment through atmospheric emissions, waste
effluents to waterways, and land disposals of liquid and solid wastes. EDC in
liquid and solid waters evaporates rapidly because of its high volatility;
consequently, releases to land and water can be expected to enter predominantly
into the atmosphere. The sources of EDC emissions are during EDC manufacture,
intermediate use of EDC in production of other chemicals, and dispersive uses of
EDC in end-point product applications.
5-9
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TABLE 5-6
Estimated Environmental Releases of Ethylene Dichloride in 1979a
Application
Production of 1 ,2-dichloroethane
Indirect production
Feedstock uses
Trichloroethylene
Tetrachloroethylene
1 , 1-Dichloroethylene
Ethyleneamines
Preparation of polysulfides
Vinyl chloride
Exports
Dispersive uses
Lead scavenging
Fabric and PVC equipment cleaning
Paints, coatings, adhesives
Extraction solvents
Grain fumigation
Miscellaneous uses
Total
Releases (metric
Air Water
6,151 61
65 191
73
138
86
104
5
136
180
956
864
1,364
1,000
460
300
11,885 252
tons)
Solid waste
5
46
50
101
aSource: Seufert et al., 1980
5-10
-------
A number of estimates have been generated that predict the amount of EDO
that ±a released to the environment. These estimates are based, in large part,
on a sampling of monitoring data and a variety of engineering estimates. In many
instances, the impact of current control technology may not have been adequately
assessed. Therefore, the estimates that have been generated for EDC releases
should not be interpreted as exact measurements, but are best regarded as order
of magnitude estimates. Estimates of releases are in Table 5-6.
Table 5-6 lists sources of EDC emissions in 1979 and estimates of the
amounts released in air, water, and solid waste. Total 1979 emissions to the
atmosphere, water, and solid waste amounted to 12,238 metric tons. The data in
Table 5-6 indicate that gaseous emissions from EDC production amounted to 52.3$
of total atmospheric releases, while feedstock uses, dispersive applications,
and exports accounted for 12.6, 33-6, and 1.5$, respectively (Seufert et al.,
1980).
Utilizing data from EDC producers, state and local emissions control
agencies and the open literature, Hobbs and Key (1978) estimated 1978 emissions
of EDC at =11,000 metric tons. The degree of current emissions control on
domestic processes involved in the primary production of EDC was assessed by
Hobbs and Key (1978).
Estimates by SRI International (U.S. EPA, 1979a) indicated that total
domestic emissions of EDC from primary production, fugitive sources, and tank
storage are 44,000 metric tons or =0.8$ of the amount produced. The impact of
current control technology was not addressed in the report.
Eimutis and Quill (1977) estimated process emissions of EDC for 1977 at
50,000 metric tons. Presumably, this estimate did not take into account current
control technology.
5-11
-------
For 197*4, Patterson et al. (1975) estimated total domestic emissions at
71,000 metric tons. Total domestic emissions for 1973 were reported at 54,000
metric tons by Sharael et al. (1975). The sources of EDC releases are discussed
below.
5.5.1. Production and Related Facilities. In general, EDC is produced commer-
cially at an integrated manufacturing facility that uses some .or all of the
produced EDC to make vinyl chloride and, in some cases, other derivatives. In a
typical vinyl chloride manufacturing facility, waste streams are generated by
the three distinct processes: direct chlorination of ethylene, oxychlorination
of ethylene, and dehydrochlorination to vinyl chloride; these are typically
combined at a given facility for recovery, treatment and disposal. The specific
number of point sources of releases at a manufacturing site is a function of
plant design. Point sources of EDC loss from an integrated vinyl chloride plant
include direct and oxychlorination reactor vent streams, light-ends distillation
column vent, heavy-ends from the EDC recovery tower, wastewater from drying
columns and scrubbers, and fugitive emissions from storage, pumps, seals, etc.
(Catalytic, 1979; Drury and Mammons, ?979). Releases from these manufacturing
sites are mostly to the atmosphere and arise largely from vent gas streams (Drury
and Hammons, 1979). Control devices used to limit EDC escape to the atmosphere
include thermal oxidizers, catalytic oxidizers, vent condensers, scrubbers, and
vent gas post-reactors (Hobbs and Key, 1978).
Combined wastewaters that are generated from EDC manufacture (vent gas
scrubbers, water produced during oxychlorination, and washwater) are treated in
several ways depending upon the plant (Catalytic, 1979). Treatments include pre-
treatment and steam stripping prior to biological treatment, incineration of a
portion of the waste stream, neutralization and chemical treatment, secondary
treatments and final discharges to surface waters, to public owned treatment
5-12
-------
works, or to deep-well injection. Estimates of the amounts of EDC released to
the surface waters are dependent upon engineering estimates of the overall
success of treatments and the volume of wastewater flow.
Solid wastes generated at an integrated vinyl chloride plant are usually
treated to recover organic compounds present. Wastes are subsequently disposed
of in a landfill or incinerated, recovering chlorine as hydrogen chloride
(McPherson et al., 1979). Some solid wastes, possibly tars and heavy-ends, may
be a suitable feedstock for tetrachloroethylene/carbon tetrachloride via a
chlorination process.
5.5.2. Dispersive Uses.
5.5.2.1. LEAD SCAVENGING — Seufert et al. (1980) estimated that 956 metric
tons of EDC were released to the environment in 1979 from lead scavenging
applications. Releases occur during blending of the gasolines, refueling of
automoblies, filling and-evaporation from gasoline storage tanks, and combustion
of the gasoline. Approximately 1Jt of the EDC used for lead scavenging is
estimated to be released into the environment; the remainder is converted to
hydrogen chloride and then to lead chloride during combustion.
5.5.2.2. PAINTS, COATINGS, ADHESIVES — In this application, EDC is used as
a solvent that is allowed to evaporate. Therefore, all of the EDC is emitted to
the environment.
5.5.2.3. GRAIN FUMIGATION — Fumigants are defined as gaseous pesticides.
They must remain in the gas or vapor state and in sufficient concentration to be
lethal to the target pest species. Therefore, all of the EDC used for this
purpose will be vented to the atmosphere, as there are no control methods used.
5.5.2.4. FABRIC AND PVC CLEANING — It is presumed that most EDC used for
cleaning purposes will eventually be emitted to the atmosphere. Some cleaning
wastes from PVC reactors may be drummed for landfill disposal.
5-13
-------
5.5.2.5. OTHER DISPERSIVE USES — In other dispersive uses, it has been
assumed that most EDC will eventually be emitted to the atmosphere. Some may be
landfill, as with extraction solvents, and some may be incinerated.
5.5.3. Conclusions. The major sources of EDC emissions are from vent gas
streams released during the manufacture of EDC and subsequent integrated produc-
tion of vinyl chloride monomer, and other EDC derivatives. Significant amounts
of EDC are also released to the environment from dispersive uses. In 1979, an
estimated 6,696 metric tons of EDC were released to the atmosphere from EDC
production and feedstock uses, while an estimated 4,944 metric tons were released
to the atmosphere from dispersive uses (see Table 5-6).
5-14
-------
6. TRANSPORT AND FATE IN THE ENVIRONMENT
The transport and fate of EDC in the environment depend on the medium in
which it is present. The fate and transport of EDC in three environmental media
(atmosphere, water and soil) are discussed below. Since a large percent of EDC
emitted into the environment is in the atmospheric medium (see Chapter 5), the
fate and transport of the chemical in this medium deserve special attention.
6.1. ATMOSPHERE
The fate of EDC in the atmosphere is dictated by its ability to undergo
chemical and physical removal processes in this medium. Reaction with *OH
radicals is the principal chemical process by which many organic compounds,
including EDC, are removed from the atmosphere (Crutzen and Fishman, 1977; Singh,
1977; Altshuller, 1979; Cupitt, 1980). Photolysis of 0 in the troposphere
produces singlet atomic oxygen [01Q] that then reacts with water vapor to produce
•OH radicals. The tropospheric half-life (t1/?) of a compound is related to the
•OH radical concentration according to the expression t 1/2 = [0.693] [K(-OH)]~1
where K is the rate constant of the reaction and [«OH] is the concentration of
•OH radicals.
It is obvious from the above equation that an estimation of the tropospheric
half-life for EDC due to -OH radical reaction requires that the values of both K
and [-OH] be known. There are only two measurements of EDC reaction rate
constant with -OH radicals. The absolute rate constant for EDC reaction with -OH
radicals was determined by Howard and Evenson (1976) in a conventional discharge
flow system. The value of the rate constant obtained in the system at pressures
ranging from 0.7 to 7 mm Hg and at a temperature of 23°C was 22 + 5 x 10~14
(standard deviation of average) cm molecule' sec~ .
6-1
-------
The rate constant for the reaction of «OH radicals with EDC in the presence
of 02 and Ng was determined by Butler et al. (1978). At 29.5°C and a total
pressure of 400 mm Hg, the reaction rate constant was determined to have a
probable value of 6.5 x 10~14 cm3 molecule'1 sec~1, with an upper limit value of
29 x 10~11* cm3 molecule'1 sec~1. Although Butler et al. (1978) argued that the
presence of oxygen and nitrogen at a pressure of 400 mm Hg was more represen-
tative of actual tropospheric conditions, the presence of 02 and N? actually
complicated the reaction scheme through side reactions and made the extraction of
kinetic data more difficult. It is for this reason that Butler et al. (1978)
failed to determine the precision of the determined rate constant value.
_1H 3 _!
Therefore, the measured rate constant value of 22.0+5 x 10 cm molecule
sec'1 for EDC reaction with -OH radicals as reported by Howard and Evenson (1976)
appears to be more accurate than the value of 6.5 x 10~ cm molecule sec
given by Butler et al. (1978).
In Table 6-1, the rate constants for a number of chlorinated ethanes,
including EDC, at room temperature have been shown. It seems clear from this
table that a value of 22.0 x 10~ cm molecule" sec~ is more consistent with
-11
the measured K values for other chlorinated ethanes than a value of 6.5 x 10
cm molecule sec
On the basis of the above discussion, it is reasonable to accept a value of
22 x 10~ cm molecule' sec' as the rate constant value for EDC reaction with
•OH radicals at 296°K (room temperature).
It should be recognized that the value of K is dependent on temperature.
The temperature in the troposphere is dependent on latitude, altitude and
seasonal variations. The average annual tropospheric temperature weighted over
6-2
-------
TABLE 6-1
Rate Constants for a Few Chlorinated Ethanes
at Room Temperature
Compound
1U
Rate Constant x 10
cnr molecule*" sec"
Reference
CH.CH2C1
CH2C1CH2C1
39, W
26
22, 6.5
33
Howard and Evenson,
1976; Butler et al.,
1978
Howard and Evenson,
1976
Howard and Evenson,
1976; Butler et al.,
1978
Singh et al., 19&1
6-3
-------
those variables is close to 265°K, or -8°C (Altshuller, 1979). Therefore, the
evaluation of reaction rate at this temperature representing an average
tropospheric reaction rate must incorporate the rate constant value at 265°K.
When the rate constant is not known, Altshuller (1979) estimated it by dividing
the K value at 298°K by 1.75. This is an empirical relationship and is
applicable only to saturated organic compounds. Therefore, the rate constant for
x 1
i—
1 7"5
_
22 x 10 ^ -1 -1
EDC reaction with -OH at 265°K can be estimated as - — i— ^ cm^ molecule sec ,
or 12.6 x 10 car molecule' sec" "
The second factor required for the evaluation of tropospheric half-life for
EDC reaction with «OH radicals is the average -OH concentration. Besides
hemispheric difference, the concentration of -OH radicals in the troposphere is
dependent on latitude, altitude and seasonal variations. The concentration of
tropospheric »OH radicals in the Northern Hemisphere across the latitudes of the
continental United States has been estimated by different investigators to range
from 0.2 to 0.9 x 10 molecules cm"3 annually (Logan et al.f 1981; Crutzen and
Fishman, 1977; Neely and Plonka, 1978). Singh et al. (1983a) have recently
estimated a mean hydroxyl radical concentration of 0.1-0.6 x 10 molecules cm"
over the troposphere of the continental United States. Therefore, a value of 0.5
x 10 molecules cm is a good estimate for the average tropospheric hydroxyl
radical concentration over the continental United States. It should be
emphasized that the «OH concentration can vary substantially from the average
value of 0.5 x 10 molecules cm . Calvert (1976) estimated the average ambient
level of »OH radicals in the morning hours in Los Angeles to be =2.6 + 2 x 10
molecules cm . Assuming equal periods of daylight and darkness, the average
fi T
concentration for a 2^-hour day should be =1 x 10 molecules cm" .
The calculations of tropospheric half-life for EDC reaction with -OH
radical can be made under two scenarios. In the first case, a rate constant
value at ambient temperature (22 x 10~ cm molecule" sec" ) and an «OH
6-4
-------
concentration of 1 x 10 molecule cm~^ can be used to derive the half-life value
of 36 days. In the second case, a rate constant value of 265°K corresponding to
annual average tropospheric temperature over the continental United States (12.6
x 10~ cm3 molecule' sec ) and a value of -OH concentration of 0.5 x 10
molecules cm"^ corresponding to the annual average tropospheric concentration of
•OH radical over the continental United States can be used to derive a half-life
value of 127 days. Therefore, the tropospheric half-life for EDC reaction with
•OH may vary from 36 to 127 days.
The reactivity of EDC with chlorine atoms was studied by Spence and Hanst
(1978). These investigators found that the chlorine-sensitized photooxidation
of 10 ppm EDC with 4 ppm C12 at 22.5°C and irradiated by UV lamp (365 nm
wavelength max.) and sun lamp (310 nm wavelength max.) produced a number of
reaction products. Although hydrochloric acid (6.5 ppm), carbon monoxide
(1 ppm), formyl chloride (3.5 ppm), C02 (2 ppm) and chloroacetyl chloride (0.5
ppm) were formed, no phosgene could be detected as a reaction product. EDC was
found to have a relatively low reactivity toward Cl atoms compared to other
chlorinated ethanes.
That a chlorine-sensitized photooxidation is representative of actual
tropospheric conditions has been contested by Singh (1978). This investigator
estimated that chlorine atom concentrations in simulation chambers can be 10^ to
10 times larger than in the troposphere. Thus, the role of chlorine atoms would
be minimal under actual tropospheric conditions where -OH radicals are
predominant.
The role of halocarbons in the catalytic destruction of ozone (0,) is well
documented (Altshuller, 1979; Altshuller and Hanst, 1975). Any halogenated
compound with a sufficiently long lifetime may be transported to the stratosphere
where photolytic mechanisms may release halogen atoms to participate in the
6-5
-------
destruction of ozone. The rate of reaction of -OH radical with halocarbons is
one mechanism that competes with stratospheric photolysis as a halocarbon sink.
Since transport to the stratosphere is a relatively slow process, the
reported half-life for EDC suggests that only a small fraction released in the
troposphere will reach the stratosphere. Thus, emissions of EDC would not be
expected to play a significant role in the catalytic destruction of ozone.
However, in the view of Altshuller and Hanst (1975), EDC should be considered a
threat in this regard since the production volume of EDC is so large. These
investigators cite chloroacetyl chloride as the sole product of EDC reaction with
•OH. The possibility that this reaction product may have sufficient stability to
travel to the stratosphere was raised. If formed in the troposphere, most of the
chloroacetyl chloride probably will be washed out by rain. If it travels to the
stratosphere, however, it may undergo photodissociation to form atomic chlorine.
The three most likely physical processes that may participate in the removal
of EDC from the atmosphere were studied by Cupitt (1980). The half-life for EDC
removal by rain droplets was estimated to be 390 years. EDC in the vapor phase
may be adsorbed on aerosol particles and removed from the atmosphere with the
aerosol. However, the half-life of this removal process was estimated to be =13
years (Cupitt, 1980). It has been estimated by Cupitt (1980) that compounds with
_7
saturation vapor pressures of <10 mm Hg are likely to be adsorbed on
atmospheric aerosols. Since the vapor pressure of EDC is much higher, it is not
expected to be adsorbed by aerosol, and the probability of its removal by this
process seems to be remote.
The third physical removal mechanism, dry deposition of EDC, was also
studied by Cupitt (1980). He estimated a half-life value of 25 years for this
process. Therefore, it is not expected to be a significant EDC removal mechanism
from the atmosphere.
6-6
-------
It is apparent from the above discussion that physical processes are not
significant for the removal of EDO from the atmosphere. Of the chemical
processes, the most significant reaction for removal of EDO from the atmosphere
is its reaction with «OH radicals. The half-life for this reaction has been
estimated to be -36 to 127 days. Therefore, it is anticipated that EDC will
persist for a sufficiently long time in the atmosphere that it may participate in
intramedia transport from its source of emissions, but not sufficiently long to
allow for transport to the stratosphere.
6.2. AQUATIC MEDIA
The fate of EDC in aquatic media is expected to be determined by its
chemical, physical and microbiological removal mechanisms. The fate of EDC with
respect to its possible chemical reactions has been reported by Callahan et al.
(1979). Both photolysis and hydrolysis of EDC in aquatic media are expected to
be insignificant fate processes (Callahan et al., 1979). Based on the oxidation
rate constant data given by Mabey et al. (1981) and the concentration of free
radicals in aquatic media given by Mill et al. (1982), the oxidation of EDC by
singlet oxygen (K«360 M~ hr~ ) and peroxy radicals (1 M~ hr~ ) can be
predicted to be environmentally insignificant processes.
The degradation possibilities of EDC by microorganisms in aquatic media
have been studied by several authors. From the results of the laboratory tests
conducted by several investigators with activated sludge or sewage seed as
microbial inoculum in aquatic media, it can be concluded that EDC may biodegrade
slowly under these conditions (Price et al., 1974; Tabak et al., 1981; Ludzack
and Etlinger, 1960; Henckelikian and Rand, 1955; Stover and Kincannon, 1983).
Under methanogenic conditions, Bouwer and McCarty (1983) observed up to 50$
degradation of EDC to C02 over a 25 week period. In a river die-away test, Mudder
et al. (1982) reported no degradation of EDC at concentrations of =1 ppm and
6-7
-------
above. Stuck! et al. (1981, 1983) isolated an organism from soil capable of
utilizing EDC as a sole source of carbon. The bacterium could not grow on solid
media but grew in liquid culture. No degradation studies were performed in soil
so that no assessment can be made of the ability of this organism to degrade EDC
in the environment. It can be concluded from these discussions that
biodegradation will be an insignificant fate process for EDC in ambient aquatic
media.
The two likely physical processes that may remove EDC from aquatic media are
sorption and volatilization. The removal of EDC by sorption on sediments and the
subsequent sedimentation is probably not a significant process (Callahan et al.,
1979; Mabey et al., 1981). A calculation based on EXAMS modeling by the method
of Burns et al. (1982) with the input parameters given in Section 3 also shows
that EDC is not likely to be significantly sorbed onto particulate matter in
water.
The evaporation half-life for EDC from water at a depth of 1.5 cm, a
concentration of 2 ppra, and a temperature of 24°C was estimated to be 8 minutes
under stirring (Chiou et al., 1980). Under somewhat more realistic conditions of
a wind speed of 3 m/sec, water current of 1 m/sec, and a water depth of 1 m, Lyman
et al. (1982) calculated the evaporation half-life of EDC as U hours. Both of
these methods, however, neglect a number of important environmental variables
such as sorption, advection, and diffusion. These are particularly important in
bodies of water deeper than one mile.
The bioconcentration factor (BCF) for EDC in aquatic organisms was
estimated as 6.03 by Kenaga (1980) from the water solubility and a linear
regression equation. In a 14-day exposure experiment with bluegill sunfish
(Lepomis macrochirus). Barrows et al. (1980) determined a BCF of 2.0 for this
compound. Therefore, EDC is not likely to bioconcentrate in aquatic organisms.
6-8
-------
It can be concluded from the discussion above that the primary removal
mechanism of EDC from aquatic media is probably volatilization. The half-life
for volatilization is such that the compound may persist in aquatic media for a
reasonable period and may participate in transport in aquatic bodies.
6.3- SOIL
Data pertaining to the fate of EDC in soil are very limited. If one
considers the chemical reactivity of EDC in aquatic media (see Section 6.2), it
is possible to speculate that chemical reactions of EDC may be faster in soil
than in water, particularly with soil acclimatized with EDC. However, Wilson
et al. (1981) found virtually no biodegradation of EDC with soils collected near
Ada, OK, that contained an average sand of 92$ and organic carbon of 0.09$.
The sorption constant (K ) of EDC on soils was predicted by Kenaga (1980)
oc
to be 43 (standardized with respect to soil organic content) from regression
equation and solubility of this chemical in water. Chiou et al. (1979) estimated
a value of 19 for K with Willamette silt loam containing 1.6$ organic matter,
26$ clay, 3-3$ sand and 69$ silt. Therefore, the compound is not expected to be
sorbed strongly onto soils, particularly onto soils containing low organic
matter, and may percolate through the soil column. This has been confirmed by
Wilson et al. (1980), who found *50$ percolation of EDC through a column of 140
cm depth containing the previously described Oklahoma soil. The fact that Page
(1981) reported the detection of EDC in 10$ groundwater samples in New Jersey
also indicates that the compound may transport downwards through some soils.
The volatilization of EDC from soil may be an important removal mechanism.
However, very few investigative data are available on this subject. Again, if
one considers the investigation of Wilson et al. (1981) that reported the loss of
50$ EDC from the soil column through volatilization, one can conclude that
volatilization may be one of the most significant physical removal processes for
EDC from soil.
6-9
-------
6.4. SUMMARY
The most likely removal mechanism for EDC from the atmosphere is reaction
with hydroxyl free radicals. Based on available kinetic data and average
tropospheric hydroxyl free radical concentrations, the half-life of EDC has been
estimated to range from 36-127 days.
EDC is not expected to play a significant role in stratospheric ozone
destruction reactions due to its relatively short tropospheric half-life.
However, chloroacetyl chloride may have suffficient stability to diffuse to the
stratosphere and may participate in UV reactions producing chlorine atoms.
In the aquatic environment, the most significant removal mechanism appears
to be its volatilization. The half-life for this process has been estimated to
be =4 hours. Therefore, EDC may persist in the aquatic media for a reasonable
period and may participate in its transport in water bodies.
From the little information that is available regarding the fate of EDC in
soil, it has been concluded that both volatilization from and leaching through
soil are the two significant removal mechanisms for EDC.
6-10
-------
7. ENVIRONMENTAL LEVELS AND EXPOSURE
7.1. ENVIRONMENTAL LEVELS
EDC contamination of the environment can result from the manufacture, use
and disposal of the chemical. Sources of environmental release and quantitative
estimates of releases are discussed in Section 5 of this report. This section is
concerned with the levels of EDC that have been detected in various areas and
monitoring sites. Sizable quantities of EDC were judged to be released to the
air during manufacture and conversion to vinyl chloride at an integrated manufac-
turing site. This judgment is borne out by the high ambient concentrations of
EDC found near several of these sites.
7.1.1. Atmospheric Levels. In addition to atmospheric releases of EDC from
manufacture and feedstock uses, EDC can be released to the air from dispersive
uses such as grain fumigation, solvent uses in paints, coating and adhesives,
cleaning applications, and gasoline uses related to lead scavenging.
Recent air monitoring data for EDC are listed in Table 7-1. These data
indicate that EDC is often present in ambient air in urban and industrial areas,
especially near plants manufacturing or using the chemical. Measurements made at
sites in three geographical areas central to EDC production and user facilities
indicated EDC concentrations as high as 184 ppb (Elfers, 1979). These levels
were associated with industrial sources and occurred during atmospheric
conditions of calm or low wind speeds. Measurements were made during 10- to 13-
day periods at Lake Charles and New Orleans, LA, and at Calvert City, KY. The
highest concentrations were reported for the Lake Charles area, with the highest
single concentration being 184 ppb and the average detectable concentration
being 27.5 ppb. In Calvert City, ambient concentrations ranged from <0.12 to
7-1
-------
TABLE 7-1
Ambient Atmospheric Levels of Ethylene Dichlonde
Location
ALABAMA
Birmingham
Hirmingham
ARIZONA
Phoenix
Grand Canyon
CALIFORNIA
borcinguez
Loa Angelea
Oakland
71 Ri'/erside
w Upland
Upland
COLORADO
Denver
ILLINOIS
Ch i cago
KENTUCKY
Calvert City
LOUISIANA
Baton Rouge
Baton Rouge
GeLsmar
[berville Pariah
Lake Charles
Type or Si'e
urban
urban
urban
rural
urban
urban
urban
urban
urban
urban
urban
urban
12 cities near
chera. plant
urban
urban
Industrial
urban
urban
Date
April, 1977
April, 1977
April-Hay, 1979
Nov-Dec, 1977
May, 1976
April, 1979
June-July, 1979
July, 1980
Aug-Sept, 1977
June, 19BO
April, 1981
Aug-Sept, 1978
March. 1977
March, 1977
Feb. Mar, 1977
Jan, Feb. 1977
June-Oct, 1978
Detection
Method
GC/MS
GC/MS
GC/ECD
GC/MS
CC/ECD
CC/ECD
GC/ECD
CC/MS
CC/MS
GC/ECD
GC/ECD
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
Number of
Samples
2
7 (1 ND)
NR
7 (7 ND)
1
102
B
16 (11 ND)
95
NR
88
25 (9 ND)
18 (2 ND)
10 (1 ND)
11
98
Concentration (ppt. v/v)
Max. Mln. Avg.
98.6
99
1451.1
—
—
1351.8
803. 0
580
212.9
210
480
2820
17800
630
2556.5
2551.5
11614.2
61000
50.7
0
38.8
—
—
173.0
38.3
89
61.8
0
100
22
<120
0
19.3
21.7
. 2.2
150
7U.7
35
216.3
—
3662.3
519.2
82.5
360
110.3
27
210
195
5000
270
129.1
760.1
320.9
27000
Reference
Pellizzan and
Bunch, 1979
Pelllzzan, 1979a
Singh et al., I960
Pellizzan, 1979a
Pellizzari and
Bunch, 1979
Singh et al., 1980
Singh et al., I960
Singh et al., 1982
Pellizzan and
Bunch, 1979
Pellizzari, 1979a
Singh et al., 1980
Singh et al., 1982
Elfers, 1979
Pellizzari et al . , 1979
Pellizzari, 1978a
Pellizzari, 1978a
PeHlzzan, 1978a
Pellizzari, 1979b
-------
TABLE 7-1 (cont.)
Location
LOUISIANA (cont.)
Lake Charles
Lake Charles
New Orleans
Plaquemine
MISSOURI
St. Louis
NEW JERSEY
^ Bats to
<•»» Bound Brook
Bridgeport
Bridgewater
Burlington
Caraden
Carlstadt
Clifton
Deepuater
East Brunswick
Edison
Edison
Type of Site
urban
12 sites near
chera. plant
near chem.
plant
urban
urban
rural
off highway
urban
urban
urban
near chem.
plant
marina
urban
near waste
disposal area
Date
June, 1978
Sept-Oct, 1978
October, 1978
Jan-Feb, 1977
May- June, 1980
Feb-Dec, 1979
March, 1976
September, 1977
July-Aug, 1978
September, 1977
Apr-Oct, 1979
September, 1979
March, 1976
June, 1977
July, 1976
Mar-July, 1976
Mar-July, 1976
Detection
Method
GC/MS
GC/MS
GC/MS
GC/MS
GC/ECD
GC/FID-ECD-MS
GC/MS
GC/MS
NR
GC/MS
GC/FID-EDC-MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
Number of
Samples
6
110
91
11
90
42 (40 ND)
1
2
22 (18 ND)
1
23 (21 ND)
16 (7 ND)
1
6 (3 traces)
2
33 (20 ND)
16 (1 trace)
Concentration (ppt. v/v)a
Max. Min. Avg.
307.3
184000
Hi 700
921.1
260
—
—
—
380
—
—
998.0
—
13.1
85.8
3400
14091.5
5.2
<120
<120
2.2
65
—
—
—
0
—
33-4
—
5.9
37.1
8.6
53.6
86.5
27500
2900
337.2
120
Trace
(2 samples)
Trace
Trace
170
Trace
Trace
(2 samples)
537.2
15950
7.4
61.6
1600
2712
Reference
Pellizzarl and
Bunch, 1979
Elfers, 1979
Elfers, 1979
Pellizzari and
Bunch, 1979
Singh et al.,
Bozzelli et al
Pellizzarl and
Bunch, 1979
Pellizzari and
Bunch, 1979
Bozzelli et al
Pellizzarl and
Bunch, 1979
Bozzelli et al
1980
., 1980
., 1979
., 1980
Pellizzarl, 1978b
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
-------
TABLE 7-1 (cent.)
Location
NEW JERSEY Ccont.]
Elizabeth
Elizabeth
Fords
Hoboken
Linden
Linden
Middlesex
Newark
Newark
Newark
Hdssaic
Haterson
Rahuay
Rutherford
Rutherford
Sayreville
Somerset
South Amboy
New YORK
Niagara Falls
Staten Island
Staten Island
Type of Site
urban
near indus-
trial plant
urban
urban
Industrial
urban
suburban
urban
urban
urban
suburban
suburban
industrial
residential
(Love Canal)
urban
urban
Date
Sept 1978-Dec, 1979
Jan-Dec, 1979
March, 1976
March, 1976
June, Nov. 1977
June, 1977
July, 1978 '
Jan -Dec, 1979
March, 1976
Mar, 1976-Dec, 1979
March, 1976
March, 1976
September, 1978
May, 1978-Dec, 1979
May, 1979
July, 1976
July, 1978
Jan-Dec, 1979
February, 1978
November, 1976
Mar-Apr, 1981
Detection
Method
GC/FID-ECD-MS
GC/FID-ECD-MS
CC/MS
GC/MS
GC/MS
GC/MS
NR
GC/FID-ECD-MS
GC/MS
GC/FID-ECD-MS
GC/MS
GC/MS
GC/MS
GC/FID-ECD-MS
GC/FID-ECD-MS
GC/MS
NR
GC/FID-ECD-MS
GC/MS
GC/MS
GC/ECD
Number of
Samples
71 (53 ND)
54 (7 trace, 16 ND)
1
2 (2 ND)
%
16 (1 trace)
11 (1 ND)
IB (13 ND)
37 (9 trace, 28 ND)
1
160 (112 ND)
1
1
16
196 (141 ND)
46 (3 trace, 42 ND)
1
29 (17 ND)
48 <4 trace, 44 ND]
9 (2 trace, 7 ND)
3
NR
Concentration (ppt, v/v)a
Max. Mln. Avg.
2200
—
—
0
48.2
1.9
290
—
—
5800
—
—
77-3.5
3200
—
—
1800
—
—
50.7
4312
0
—
—
0
2.0
1.9
28
—
—
0
—
—
165.4
0
—
—
0
—
46.0
55
220
2202.7
Trace
0
11.3
1.9
110
Trace
Trace
450
Trace
Trace
525. B
370
593-3
9372.8
490
Trace
Trace
48.2
256
Reference
Bozzelli et al., 1980
Bozzelli et al., I960
Pellizzari and
Bunch, 1979
Pellizzari, )977a;
Pellizzari et al., 1979
Pellizzari and
Bunch, 1979
Pellizzari, 1978c
Bozzelli et al., 1979
Bozzelli et al., 1980
Pellizzari and
Bunch, 1979
Bozzelli and
Kebbekus, 1979;
Bozzelli et al., I960;
Pellizzari, 1977
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari, 197Bb,d
Bozzelli et al.,
1979, 1980
Bozzelli et al., 1980
Pellizzari and
Bunch, 1979
Bozzelli et al., 1979
Bozzelli et al., 1980
Pellizzari, 1978c,d
Pellizzari and
Bunch, 1979
Singh et al., 1982
-------
TABLE 7-1 (cont.)
Location
NORTH CAROLINA
Chapel Hill
OKLAHOMA
Liberty Mound
Tulsa
Vera
PENNSYLVANIA
Bristol
Marcus Hook
N. Philadelphia
Pittsburgh
TEXAS
Aldine
Beaumont
Deer Park
El Paso
Freeport
Houston
Houston
Houston
La Porte
Pasadena
UTAH
Magna
Type of Site
rur.il
urban
urban
urban
urban
urban
industrial
urban
industrial
urban
urban
streets,
parks, rural
highway
urban
rural
Detection
Date Method
June, I960
July-Sept, 1977
July-Sept, 1977
July, 1977
August, 1977
August, 1977
August, 1977
April, 1981
June-Oct, 1977
March, 1980
August, 1977
Apr-May, 1978
August, 1977
July 1976-May, 1980
NR
July, 1976; June-
July, 1978
August, 1976
July, 1976
Oct-Nov, 1977
NR
GC/MS
GC/MS
GC/MS
GC/KS
GC/MS
GC/MS
GC/ECD
GC/MS
NR
GC/MS
CC/MS
GC/MS
CC/ECD
GC/MS
GC/MS
GC/MS
GC/HS
GC/HS
Number of
Samples
6
2 (2 ND)
2 (2 ND)
1 (1 ND)
2
2
4
NR
3 (3 ND)
11
6 (3 trace)
22 (19 ND)
2
99 (5 NO)
30
10 (1 trace)
1
1
9 (9 ND)
Concentration (ppt. v/v)a
Max. Kin. Hvg.
110
0
0
0
—
—
238.6
237
0
670
16390.6
29
1112.5
3000
16390.6
109.8
—
• 39
0
110
0
0
0
—
—
11.3
66
0
670
1002.5
0
815.8
50
73.1
30.1
—
39
0
no
0
0
0
63.8
IB. 2
139.2
121
0
670
6353.5
1.3
961.2
1300
893-0
63.0
192.3
39
0
Reference
Wallace, 1981
Pellizzari, 197Se
Pellizzari, 1978e
Pellizzari, 1978e
Pellizzarl and
Bunch, 1979
Pellizzarl and
Bunch, 1979
Pellizzarl and
Bunch, 1979
Singh et al., 1982
Pellizzari et al., 1979
Hal lace, 1981
Pellizzari and
Bunch, 1979
Pellizzarl, 1979a
Pellizzari and
Bunch, 1979
PeUlzzarl et al., 1979
Singh et al., 1980
Pellizzari, 1978b
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari et al., 1979
Pellizzari, 1979a
-------
TABLE 7-1 (cent.)
Location
VIRGINIA
Front Royal
WEST VIRGINIA
Charleston
Charleston
Institute
Institute
Nitro
Nitro
St. Albans
71 St. Albans
O* S. Charleston
5. Charleston
W. Belle
W. Belle
WASHINGTON
Pullman
Pullman
Type of Site
industrlrl
industrial
industrial
industrial
industrial
Industrial
industrial
industrial
industrial
industrial
industrial
industrial
industrial
rural
rural
Detection
Date Method
Oct-Nov, 1977
Sept, Nov, 1977
Sept-Nov, 1977
November, 1977
November, 1977
Oct. Nov, 1977
Sept-Oct, 1977
October, 1977
Sept-Nov, 1977
Har, Sept-Nov, 1977
Mar-Nov, 1977
Sept-Nov, 1977
Sept-Nov, 1977
Dec, 1974-Feb, 1975
November, 1975
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/MS
GC/ECD
Number of
Samples
16
3
M (4 ND)
2 (2 ND)
3
M
6 (6 ND)
1
M (4 ND)
6
16 (15 ND)
6 (6 ND)
4
NR
1
Concentration (ppt. v/v)a
Max. Min. Avg.
86.0
0
0
—
63.8
0
—
0
63.8
37
0
61.8
...
——-
37.3
—
0
0
—
37.3
0
—
0
37.3
0
0
37.3
<5
10
48.2
57.1
0
0
37.3
46.7
0
48.2
0
52.4
2.3
0
47.0
™-
Reference
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari, 1978e
Pellizzari, 1978e
Pellizzari and
Bunch, 1979
Pellizzari and
Bunch, 1979
Pellizzari, 197Be
Pellizzari and
Bunch, 1979
Pellizzari, 1978e
Pellizzari and
Bunch, 1979
Pellizzari, 1979a;
Pellizzari, 1978e
Pellizzari, 1978e
Pellizzari and
Bunch, 1979
Grimsrud and
Rassmusaen, 1975
Harsch et al., 1979
Assume ambient temperature of 25°C and atmospheric pressure of 760 mmHg.
ND = Not detected
NR = Not reported
ECD : Electron capture detection
FID - Flame lonization detection
GC = Gas chromatography
MS = Mass spectronetry
-------
17.8 ppb. Levels recorded in the New Orleans study area ranged from <0.12 to
Ul.7 ppb.
Analyses done by Elfers (1979) were performed by collecting ambient air on
charcoal tubes followed by desorption by carbon disulfide. Detection and quanti-
tation was made by gas-chromatograph-mass spectroraetry. Quality control check
sample analysis indicated that recovery of standard concentrations was highly
variable. The detection limit of the gas chromatographic method was reported as
1 jig. The relative standard deviation of replicate standard solutions was found
to be 3$. The precision of the analytical and sampling methods together was 6%.
The accuracy of the analytical method varied between 72 and 97$.
The highest reported reading for a single sample listed in Table 7-1 is
191.5 ppb, which was recorded in Lake Charles, LA, the site for several EDC-vinyl
chloride manufacturing facilities. Concentrations in excess of 15 ppb for a
single sample were also reported near manufacturing facilities in Calvert City,
KY; New Orleans, LA; Deer Park and Houston, TX.
Recent studies have measured general urban ambient concentrations of EDC in
ten cities (Singh et al., 1980, 1981, 1982). The results of these studies are
summarized in Table 7-2. The averaging time for the mean concentrations in these
studies was 2 weeks. Electron capture detector-gas chromatography was the
primary means of analysis.
Low levels of EDC were recorded in a year-long monitoring program of five
cities in industrial northern New Jersey (Bozzelli et al., 1980). Samples were
collected at the five sites regularly throughout 1979. Only two of the 208
samples analyzed had EDC concentrations higher than trace (*I1 ng/nr or 10 ppt).
In a survey of air contaminants in the rural northwest, Grimsrud and
Rasmussen (1975) found EDC concentration of <5 ppt.
7-7
-------
TABLE 7-2
Ambient Concentrations of Ethylene Dichloride in Urban Areas
City
Los Angeles, CA
Phoenix, AZ .
Oakland , CA
Houston, TX
St. Louis, MO
Denver , CO
Riverside, CA
Staten Island, NY
Pittsburgh, PA
Chicago , IL
Ethylene
Mean
519
216
83
1512
124
241
357
256
121
195
Dichloride Concentrations (ppt)
Maximum
1353
1450
842
7300
607
2089
2505
4312
237
2820
Minimum
173
39
38
50
45
56
63
55
66
22
^Source: Singh et al., 1980, 1981, 1982
7-8
-------
7.1.2. Ground and Surface Water Levels. EDC can be released to the water
environment via wastewaters generated during production of ethylene dichloride
and its derivatives. It is also possible that EDC may be inadvertently produced
in the water environment due to chlorination processes of public water supplies
or chlorination of sewage or wastewaters (Versar, 1975; Seufert et al., 1980).
One chemical reaction that could lead to EDC production during water treatment is
the reaction of alkenes with hypochlorite. However, it is expected that
industrial discharges to surface water and leaching from solid waste are the
primary causes of EDC contamination in drinking water (Letkiewicz et al., 1982).
Most discharges of EDC are judged to ultimately reach the atmosphere because of
its high volatility (Letkiewicz et al., 1982). The identification and levels of
EDC that have been found in the ground and surface water of the United States are
discussed below.
EDC was reported to be a principal contaminant in finished water in 1975 by
Dowty et al. (1975). Because the gas chromatographic peak could not be resolved,
it was not possible to quantitate EDC. It was indicated that EDC levels could be
increased as a result of the water treatment processes and could pass the treat-
ment plant without removal. The water analyzed in this study was obtained from
the Mississippi River.
Deinzer et al. (1978), citing the results of a 1975 national drinking water
survey, reported EDC concentrations ranging from 0 to 6 ppb.
While sampling for pollutants in 14 heavily-industrialized river basins in
the United States, Ewing et al. (1977) found that EDC was present in 53 of the 204
samples purged for volatile organic analysis. The majority of the 81 purgeable
organic compounds detected were C to C, halogenated hydrocarbons. Only chloro-
form, trichloroethylene and tetrachloroethylene were found with greater
7-9
-------
frequencies. Identification was made by a GC-MS procedure. The limit of sensi-
tivity was <1 ppb.
In a compilation of pollutants found in water, Shackleford and Keith (1976)
identified EDC as having been found in industrial effluents, in finished drinking
water, and in river waters.
Letkiewicz et al. (1982), in a recent study for the U.S. EPA, discuss the
results of six federal surveys in which a number of public water supplies were
selected for analysis of chemical contaminants, including EDC. The six Federal
drinking water surveys providing data on EDC include the National Organics
Reconnaissance Survey (NORS), the National Organics Monitoring Survey (NOMS),
the National Screening Program for Organics in Drinking Water (NSP), the 1978
Community Water Supply Survey (CWSS), the Groundwater Supply Survey (GWSS), and
the Rural Water Survey (RWS). All surveys sampled both ground and surface waters
except for the GWSS. The scope and methodology of each survey is outlined below,
along with the identification of EDC presence.
The National Organics Reconnaissance Survey (NORS) was conducted in 1975 to
determine the extent of the presence of EDC, carbon tetrachloride, and four
trihalomethanes in drinking water supplies from 80 cities across the country
(Symons et al., 1975). Another stated objective of the study was to determine
the effect of raw water source and treatment practices on the formation of these
compounds. The water samples were collected in 50 ml sealed vials, with vola-
tilization prevented by the absence of head space. Samples were subsequently
shipped on ice to the EPA Water Supply Research Laboratory in Cincinnati for
analysis. Analyses were performed by purge-and-trap gas chromatography with an
electrolytic conductivity detector. A population base of 36 million from 80
cities across the country was covered during the study.
7-10
-------
In the NORS study, 16 groundwater systems were analyzed for EDC contamina-
tion. None of the 16 systems contain detectable levels. Of the 64 surface water
samples analyzed for CDC, six were found to contain quantifiable levels of EDC at
0.2-6.0 |ig/&.
The National Organics Monitoring Survey (MOMS) was instituted to identify
contaminant sources, to determine the frequency of occurrence of specific
drinking water contaminants, and to provide data for the establishment of maximum
contaminant levels (MCL's) for various organic compounds in drinking water (U.S.
EPA, 1977). The NOMS was conducted in three phases, March-April 1976, May-July
1976, and November 1976-January 1977. Drinking water samples from 113 communi-
ties were analyzed for 21 different compounds by purge-and-trap gas chromato-
graphy with electrolytic conductivity detectors. During Phase I the samples were
collected in 25- or 40-mi open-top screw cap septum vials with no head space, a
feature that prevented volatilization of the compounds of interest. These
samples were then stored at 2-8°C for 1-2 weeks prior to analysis. Samples were
collected in a similar fashion for Phases II and III, but were held at 20°C for
periods up to 6 weeks. Sodium thiosulfate was added to a number of samples from
Phases II and III to reduce any residual chlorine present.
Of 18 groundwater systems analyzed for EDC during Phase I of the NOMS study
(March to April 1976), none contained quantifiable levels. When these systems
were sampled again during Phase II (May to July 1976), one system was found to
contain EDC, at 0.02 |jg/
-------
During the third phase of the NOMS (November 1976 to January 1977), analyses
revealed EDC contamination in one system, at 1.25 ng/i.
In the National Screening Program for Organics in Drinking Water (NSP),
conducted from June 1977 to March 1981, both raw and finished drinking water
samples from 169 water systems in 33 states were analyzed for 51 organic chemical
contaminants (SRI, 1981). Analyses were carried out by gas chroraatography with
various detection methods, including the mass spectrometer, halogen-specific
detector, and flame ionization detector.
Thirteen groundwater supplies were tested for EDC contamination during the
NSP study. Of these systems, one was found to be contaminated with EDC at
0.2 ug/£. Surface water samples for 107 drinking water systems were analyzed for
EDC. Of these, only one system was found to be contaminated with EDC, at
3.8 ug/fc.
In the Community Water Supply Survey (CWSS), carried out in 1978, 110
surface water and 330 groundwater supplies were examined for contamination by
volatile organic chemicals (U.S. EPA, 198la). Fourteen purgeable organic
compounds were analyzed, and total organic carbon levels were determined. At the
time of analysis, the samples were 1 to 2 years old. Long storage periods may
have resulted in the loss of some aromatics and' unsaturated halocarbons due to
biological action. The analytical method of choice for the CWSS was purge-and-
trap gas chromatography with an electrolytic conductivity detector for halo-
carbons and a flame ionization detector for aromatic analysis.
The CWSS survey provided information on EDC levels in finished groundwater
supplies from 312 systems. Of the samples taken from these systems, three
contained detectable quantities of EDC with levels of 0.57, 1.06 and 1.1 jig/*.-
Of the 110 surface water systems tested, only one contained quantifiable EDC,
with a concentration of 0.62
7-12
-------
The Groundwater Supply Survey of 1980 (GWSS) was initiated to provide a
clearer picture of the extent of contamination of groundwater supplies with
volatile organic compounds (U.S. EPA, 1982a). A total of 945 systems was
sampled, of which 466 were chosen at random and 479 were picked from locations
near potential sources of contamination. Samples were collected at points close
to the actual distribution source, with mercuric chloride added as a preservative
to prevent biodegradation of aromatics. Sodium thiosulfate was also added to the
vials to reduce any residual chlorine, thus preventing its reaction with organic
matter. Analyses were made for 37 volatile organic compounds by purge-and-trap
gas chromatography with electrolytic conductivity detectors. Of the 166
randomly chosen water systems, seven were contaminated with EDO, at concentra-
tions ranging from 0.29-0.57 ug/i. Of the seven positive samples, six were from
systems serving populations in excess of 10,000 people. The average for all
randomly chosen systems was 0.5 ug/H. Of the 479 nonrandom locations sampled,
nine were contaminated with EDC, at concentrations between 0.33-9.8 |ag/i. Of the
nine positive samples, four were from systems serving populations in excess of
10,000 people. The average EDC level for the positive nonrandom systems was 2.7
ug/l.
The Rural Water Survey (RWS), conducted in 1978 (Brass, 1981), was carried
out in response to Section 3 of the Safe Drinking Water Act, which mandated that
EPA "conduct a survey of the quantity, quality and availability of rural drinking
water supplies." A total of 800 of the 2655 samples collected in the RWS was
analyzed for volatile organic chemicals. Three hundred of the samples were
selected by a random generation procedure and 500 were picked randomly by state
(10 per state). There were 633 samples from groundwater, 47 samples from surface
water, and 81 samples from other water source categories.
Of 633 groundwater samples, five were found to have EDC present (minimum
quantification limit generally 0.5-1.0 \ig/l). The range of concentrations was
7-13
-------
0.5-18 jig/A; the mean and median concentrations of the positive values were 5.6
and 1.7 ug/2., respectively. Both the mean and median values of all samples were
<0.5 ug/fc. Of the 47 surface water samples, only one (2.1?) was found to have EDC
present (minimum quantification limit generally 0.5-1.0 iig/J,). The concentra-
tion of the one positive sample was 19 ug/fc. The mean and median values of all
samples were both <0.5 |ig/&.
The combined EDC groundwater data and surface water data from the above
Federal studies are summarized in Tables 7-3 and 7-1, respectively. The RWS
study was deleted from these summaries because the RWS data were recorded by a
number of service connections rather than population served. From the data
presented in Tables 7-3 and 7-4, Letkiewicz et al. (1982) have projected that EDC
levels in all groundwater and surface water systems in the United States fall
below 10 ug/&, and that most are below 1.0 ug/Jl.
In addition to the six Federal surveys discussed by Letkiewicz et al.
(1982), six states (California, Connecticut, Deleware, Indiana, Massachussetts
and New Jersey) provided the U.S. EPA with information concerning EDC contamina-
tion in groundwater supplies. These data are listed in Table 7-5. The only
state-supplied surface water information on EDC was from New York; one sample
from Poughkeepsie assayed positive at 5.9 iig/& (Letkiewicz et al., 1982).
7.1.3. Soil and Sediment Levels. No data were available on monitoring of EDC
concentrations in soils, sediments or solid wastes in the sources consulted for
this report.
7.2. ENVIRONMENTAL EXPOSURE
The general population can be exposed to EDC from three sources, air
emissions, drinking water and consumed foods. Some individuals may be exposed to
EDC from sources other than the three considered here, such as in occupational
settings and in the use of consumer products containing EDC. However, this
-------
TABLE 7-3
Reported Occurrence of Ethylene Dichloride in Groundwater Systems
Combined Federal Data (NORS, NOMS, NSP, CWSS, GWSS)a
System size
(population
served )
<100
101-500
501-1,000
i
^ 1,001-2,500
2,501-3,300
3,301-5,000
5,001-10,000
10,001-50,000
50,001-75,000
75,001-100,000
>100,000
Number of
systems
in U.S.
19,632
15,634
4,909
1,331
881
1,065
1,159
1,101
68
16
58
Number of
positive
systems
Number of
systems
sampled
1/175
0/220
0/114
3/151
0/40
2/79
1/114
11/296
1/37
0/12
1/43
Positive
systems
(*)
0.6
0.0
0.0
2.0
0.0
2.5
0.9
3.7
2.7
0.0
2.3
Number
undetected
174
220
114
148
40
77
113
285
36
12
42
Number of systems with
measured concentration
(ug/Jl) of:
<1.0
1
0
e
1
0
1
0
6
1
0
1
1.0-5
0
0
0
2
0
1
1
4
0
0
0
>5-10
0
0
0
0
0
0
0
1
0
0
0
>,0
0
0
0
0
0
0
0
0
0
0
0
Source: Letkiewicz et al., 1982
^Positive systems are those with quantified levels of ethylene dichloride.
-------
TABLE 7-4
Reported Occurrence of Ethylene Dichloride in Surface Water Systems —
Combined Federal Data (NORS, NOMS, NSP, CWSS)a
System size
(population
served)
<100
101-500
501 -1 , 000
1 ,001-2,500
2,501-3,300
3,301-5,000
5,001-13,000
10,001-50,000
50,001 -75,000
75,001-100,000
MOO.OOO
Number of
systems
in U.S.
1,412
2,383
1,341
1,911
51 4
720
912
1,306
156
85
21 8
Number of
positive
systems
Number of
systems
sampled
0/4
0/19
0/13
1/22
0/6
1/15
0/10
1/38
2/22
0/14
5/102
Positive
systems
(*)
0.0
0.0
0.0
4.5
0.0
6.7
0.0
2.6
9.0
0.0
4.9
Number
undetected
<1 . 0 jig/A
4
19
13
21
6
14
10
37
20
14
97
Number of systems with
measured concentration
( u«/£) of:
0.0
0
0
o
1
0
0
0
1
1
0
3
1.0-5
0
0
0
D
0
1
0
0
1
0
1
>S-10
0
0
0
0
0
0
0
0
0
0
1
>,0
0
0
0
0
0
0
0
0
0
0
0
Four systems reported as undetected with the unusually high detection limit of 2.0 Mg/? were deleted.
Positive systems are those with quantified levels of ethylene dichloride.
Source: Letkiewicz et al., 1932
-------
TABLE 7-5
State Data on Ethylene Dichloride in Groundwaterc
Location
CALIFORNIA
Baldwin Park
Morada
Unspecified
Unspecified
CONNECTICUT
Colchester
Danbury
DELAWARE
Collins Park
Midvale
Newark
(North and South)
INDIANA
Elkhart
Granger
MASSACHUSETTS
Acton
Belchertown
Dartmouth
Rowley
NEW JERSEY
Bergen County
Essex County
Fair Lawn
Morris County
Passaic County
9 counties
12 counties
Water
type
N/S
N/S
N/S
N/S
D
D
F
F
F
F, N/S
N/S
F
F
F
F
N/S
N/S
F, N/S
N/S
N/S
N/S
N/S
N/S
N/S
N/S
Mean
(ug/Jl)
21
12
1.2
ND
7.8
ND
ND
ND
111
ND
14.5
ND
ND
ND
1.3
ND
ND
ND
Number
Range of
(ug/&) samples
1
1
2 (1 ND)
1.2, 5.3 2
1
1
1
1
2
30-2,100 11 (4 ND)
19, 160 15 (13 ND)
4
10.1-19.1 10 (7 ND)
11.6, 18.2 2
1
4
7
1.1-1.9 15
2.0-2.1 13 (11 ND)
1
154
228
0.1-0.9 4
1-10 4
10-100 1
Source: Letkiewicz et al., 1982
ND = Not detected; N/S = Not specified; F
= Finished; D r Distribution
7.17
-------
section is limited to air, drinking water and food since these are considered to
be general sources common to most individuals. It must be noted that individual
exposure will vary widely depending upon factors such as where the individual
lives, works or travels, or what the individual may eat or drink. Individuals
living in the same neighborhood can experience vastly different exposure
patterns.
Unfortunately, methods for estimating the exposure of identifiable popula-
tion subgroups from all sources simultaneously have not yet been developed
(Letkiewicz et al., 1982). Exposure from the three sources considered here is
discussed below.
7.2.1. Exposure from Air. Atmospheric exposure to EDC appears to vary greatly
from one location to another. Mean levels as high as 27.5 ppb have been
monitored near production and use facilities in Lake Charles, LA (Elfers, 1979).
However, the available monitoring data (see Section 7.1) indicate.that most
locations have EDC concentrations of 0.5 ppb or less. The monitoring data
presented are not sufficient to determine regional variations in exposure levels
for EDC. The majority of high values reported were for samples taken near
production and use facilities.
Letkiewicz et al. (1982) have estimated respiratory intake of ethylene
dichloride by adults and infants; these estimates are summarized in Table 7-6.
From available monitoring data, Letkiewicz et al. (1982) estimated that low,
intermediate and high exposure levels for EDC in air were 0.25, 2.5 and 25 ug/m ,
respectively. Daily intake of EDC for adults in intermediate exposure levels was
estimated at 0.82 [xg/kg.
Dispersion models have also been used to estimate ambient concentrations.
Based on modeled results for individual plants, maximum concentration levels in
the vicinity of various emission sources have been calculated (SRI
7-18
-------
TABLE 7-6
Estimated Respiratory Intake of Ethylene Dichloride
by Adults and Infants3
Low:
Intermediate:
High:
Exposure
Hg/m3
0.25
2.5
25
Level
(ppb)
(0.062)
(0.62)
(6.20)
Intake
Adult
0.08
0.82
8.21
(ug/kg/day)
Infant
0.06
0.57
5.71
Assumptions: 70 kg=man, 3.5 kg-infant, 20 m of air inhaled/day (man),
0.8 nr of air inhaled/day (infant).
aSource: Letkiewicz et al., 1982
TABLE 7-7
Maximum Concentration Level in the Vicinity of
Various Emission Sources3
Source
Maximum Annual Average EDO
Concentration Level (ppb)
EDC Production Facilities
End Use Production Facilities
Gasoline Service Stations
Automobile Emissions
Automobile Refueling
0.60 - 0.99
0.01 - 0.029
0.01 - 0.029
<0.01
Source: SRI International, 1979
An accurate estimate cannot be determined from this reference, but it is
thought to be in the range of 10-15 ppb.
7-19
-------
International, 1979). Table 7-7 shows a summary of estimated maximum
concentrations that people may be exposed to in the vicinity of specific emission
sources.
7.2.2. Exposure from Water. Letkiewicz et al. (1982) have projected that EDC
levels in all groundwater and surface water systems in the United States fall
below 10 ug/Jl, and that most are below 1.0 \i&/H (see Section 7.1.2). The total
estimated population exposed to EDC from both ground and surface water sources is
shown in Table 7-8. The values in the table were obtained by use of the Federal
Reporting Data System data on populations served by primary water supply systems
and data on the estimated number of these water systems that contains a given
level of EDC (see Section 7.1.2).
Letkiewicz et al. (1982) have also estimated daily intakes of EDC from
drinking water. These estimates are given in Table 7-9.
7.2.3. Exposure from Food. EDC is used as a fumigant for grain, so contamina-
tion of flour and bread is possible. Several studies on this question have
indicated that EDC dissipates when the bagged flour is exposed to air, and that
no detectable EDC remains after baking (U.S. EPA, 198lb). No further information
on the presence of EDC in food was uncovered in the literature search.
7.3- CONCLUSIONS
Most EDC exposures result from the production and use of the chemical. The
highest atmospheric concentrations monitored for EDC have been detected near
production and use facilities. Therefore, it appears that the risk for popula-
tion exposure to EDC is greatest in the vicinity of these sources.
7-20
-------
TABLE 7-8
Total Estimated Population (in Thousands) Exposed to Ethylene
Dichloride in Drinking Water at the Indicated Concentration Ranges'
System type
Groundwater
Surface water
TOTAL
(% of total)
Total
served
in U.S.
(thousands)
69,239
126,356
195,595
(100*)
Population (thousands) exposed
to concentrations (ug/S,) of:
<1.0 to 10
69,239
126,356
195,595
(100*)
>10
0.0
0.0
0.0
(0.0*)
Source: Letkiewicz et al.f 1982
TABLE 7-9
Estimated Drinking Water Intake of Ethvlene
Dichloride by Adults and Infants
Exposure Level
1.0
5
10
100
Intake ( ug/kg/day )
Adult Infant
0.029
o.m
0.29
2.9
0.24
1.2
2.4
24.0
Assumptions: 70 kg-man, 3-5 kg-infant, 2 8, of water/day (man),
0.85 I of water/day (infant).
aSource: Letkiewicz et al., 1982
7-21
-------
8. ECOLOGICAL EFFECTS
A variety of studies have demonstrated that EDC, in the role of a fumigant,
is toxic to insects infesting stored grains (Wadhi and Soares, 1964; Ellis and
Morrison, 1967; Vincent and Lindgren, 1965; Snapp, 1958; Krohne and Lingren,
1958; Finnegan and Stewart, 1962; Lindgren et al., 1954; and Bang and Telford,
1966). Little is known, however, of the effects of EDC to soil micoflora.
EDC has been reported to be non-toxic to many economically important plant
species when directly applied to growing plants (Cast and Early, 1956). LeBlanc
(1980) reported the 24-hour LC5Q for Daphnia magna to be 250 mg/fc (nominal
concentration) and the 48-hour LC Q to be 220 rag/5, (nominal concentration). A no
discernible effect concentration was estimated to be <68 mg/S,.
Toxicity of EDC to barnacles (Barnacle nauplii) and unicellular algae has
been reported (Pearson and McConnell, 1975). The LC_0 for Barnacle nauplii was
reported to be 186 mg/£. The effective concentration needed to reduce the
photosynthetic ability of unicellular algae by 50$ was 340 ppm (340 mg/S,).
A 24-hour median tolerance limit of 320 ppm (320 rag/2,) was reported for
brine shrimp under static test conditions (Price and Conway, 1974).
Static 24-hour and 96-hour LC5_ concentrations of >600 and 430 mg/S,,
respectively, have been determined for the freshwater bluegill (Lepomis
macrochirus) (Buccafusco et al., 1981). Garrett (1957a,b) reported that the
concentration of EDC needed to produce >50$ mortality in the marine pinperch
(Lagodon rhomeboides) under static conditions was 175 mg/i.
An acute LC,-0 of 115 mg/S, was reported for the marine flatfish (Limanda
limanda) (Pearson and McConnell, 1975). In acute tests with another marine
species, static 24-, 48-, 72- and 96-hour LC5_s in the range of 130-230 mg/S,
8-1
-------
were determined for sheepshead minnows (Cyprinodon variegatus) (Heitmuller et
al., 1981); the observed no-effect concentration was reported to be 130 mg/fc.
The estimated acceptable concentration of EDC in a 32-day early life stage
toxicity test with freshwater fathead minnows (Pimephales promelas) lies in the
range of 29-59 mg/Jl (Benoit et al., 1982).
8-2
-------
9. BIOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS
9.1. PHARMACOKINETICS
9.1.1. Absorption and Distribution. Ethylene dichloride (EDC; 1,2-dichloro-
ethane) is a colorless, oily liquid with a sweet taste and with a chloroform-like
odor detectable over a range of 6-40 ppra. It is appreciably soluble in water
(0.869 g/100 mi) (Von Oettingen, 1964), with a vapor pressure of 64 torr at room
temperature (20°C). Consequently, EDC is rapidly and extensively absorbed
through the lungs in its vapor form and from the gastrointestinal tract in
solution. Inhalation is considered the primary route of entrance into man from
occupational exposure and air pollution. Absorption after oral ingestion is of
particular interest for EDC as a contaminating component of drinking water and
foodstuffs. Skin absorption is negligible in most industrial vapor exposure
situations, although absorption may be significant by this route with direct
liquid contact, as evidenced by toxic symptomatology in man (Section 9.2), and
also quantitative animal measurements.
9.1.1.1. DERMAL ABSORPTION — Tsuruta (1975, 1977) studied the percuta-
neous absorption of a series of chlorinated organic solvents (including EDC)
applied to a standard area of shaved abdominal mouse skin for 15 minute periods.
Absorption was quantified by the presence of the compound in total mouse body
plus expired air, as determined by gas chromatography (GC). For all solvents,
percutaneous absorption linearly increased with time and the rate was directly
related to water solubility. For EDC, the absorption rate was 479 nmoles/min/cm2
skin, second highest of 8 solvents measured. Tsuruta concluded that skin absorp-
tion from liquid contact could be a significant route for EDC entry into the
body.
9-1
-------
Jakobson et al. (1983) also carried out dermal absorption studies with
guinea pigs for 10 chlorinated organic solvents including EDC. Liquid contact
o
(skin area, 3.1 cm ) was maintained for up to 12 hours and solvent concentration
monitored in blood during and following dermal application. Dermal absorption,
as reflected by blood concentration profile, was again observed to be related to
the water solubility of the solvent. For solvents like EDC, which are relatively
hydrophilic [300-900 mg/dl], the blood concentration increased steadily during
the entire exposure. EDC reached 20 ug/m& blood in 12 hours. With extended
dermal exposure, the blood concentration eventually became fatal. This pattern
of steady blood accumulation indicates that dermal absorption occurs faster than
body elimination by metabolism or pulmonary excretion. Following dermal expo-
sure, the EDC concentration in blood declined in a manner consistent with a two-
compartment kinetic model, i.e., the sum of two exponential terms.
9.1*1.2. ORAL ABSORPTION — In man, gastrointestinal absorption of EDC has
not been specifically studied, although the absorption rate after oral ingestion
has been determined in animal studies. As expected from the neutral and lipo-
philic properties of EDC (Tables 9-1 and 9-2), transmucosal diffusive passage
occurs readily. There are numerous reports of poisoning in humans as a result of
accidental or suicidal ingestion of EDC and the peroral LD5Q approximates 0.2-1
g/kg (NIOSH, 1976).
In animals, extensive gastrointestinal absorption of EDC has also been
clearly demonstrated by the biological effects produced by peroral administra-
tion of a wide range of dosages and dosing schedules in toxicity studies in rats,
nice, guinea pigs and dogs (Section 9.2) and in metabolism studies in rats (Reitz
et al., 1980, 1982; Spreafico et al., 1978, 1979, 1980). Reitz et al. (I960,
1982) found that C-EDC in corn oil given perorally to rats (150 rag/kg) was
completely absorbed by virtue of a complete recovery of radioactivity in exhaled
9-2
-------
TABLE 9-1
Physical Properties of Ethylene Bichloride
and Other Chloroethanes3
Vapor Pressure
at 25°C, torr.
Ostwald Solubility Coeff.. 37°C
Water/
Air
Blood/
Air
Olive Oil/
Air
1,2-Dichloroethane 80
1,1-Dichloroethane 250
1,1,1-Trichloroethane 125
11.3
2.7
0.93
19.5
3.3
187
356
Source: Sato and Nakajima, 1979
9-3
-------
TABLE 9-2
Partition Coefficients for Ethylene Dichloride
MAN
Blood/air, 37°C
Adipose tissue/blood, 25°C
19.5
56.7
Sato and Nakajima, 1979
Bonitenko et al., 1977
RAT. 250 ppra inhalation exposure
Blood/air, 37°C, 250 ppm
37°C, 150 ppm
Adipose tissue/blood
Lung tissue/blood
Liver tissue/blood
a
30.3
11.9
8.7b
O.U51
0.72*
Spreafico et al., 1980
Reitz et al., 1980
Reitz et al., 1980
Reitz et al., 1980
Reitz et al., 1980
Conversion factors:
At 25°C/760 torr, 1 ppm in air = 1.05 mg/m3
1 mg/liter air = 247 ppm
Dose-dependent
-------
air, urine and carcass (Table 9-3). Spreafico et al. (1978, 1979, 1980) found
that 25, 50 and 1 50 rag/kg administered orally to rats in corn oil was rapidly
absorbed with peak blood levels occurring within 20 minutes. Their data, given
in Figure 9-1, show the time-course of blood, liver, lung and adipose tissue
following oral administration of the three dose levels. The peak blood levels,
which, according to linear kinetics, should be proportional to the amount of EDC
absorbed into the rat body, are summarized in Table 9-4. Although the peak blood
levels in this study do not deviate drastically from linearity, the peak tissue
levels are not linearly proportional to the three oral doses. These results
strongly suggest a passive transport across the gastrointestinal tract of EDC and
the occurrence of nonlinear elimination kinetics for EDC, i.e., a nonlinear dose-
dependency related to saturation of liver metabolism of EDC. An influence of the
dose on kinetic parameters has also been found for halogenated ethylene compounds
(e.g., vinyl chloride, vinylidene chloride) by other investigators (Gehring et
al., 1976, 1977, 1978; Bolt, 1978; McKenna et al., 1978; Reichert and Henschler,
1978; Filser and Bolt, 1979).
Table 9-5 gives the absorption rate constants (k ) and areas under the blood
Si
concentration curves (AUC) found by Spreafico et al. (1973, 1979, 1980) for rats
given a single oral dose of EDC. The rate constant for absorption was dose-
dependent and was probably influenced by liver metabolism. A markedly lower
value was observed for the highest dose (150 mg/kg) at which saturation of liver
metabolism probably occurs. Nonetheless, these data show that EDC is absorbed
from the gastrointestinal tract rapidly, with one-half the dose absorbed within
3.3 minutes for the lowest dose given in corn oil (25 mg/kg) and 6.4 minutes for
the highest dose in corn oil (150 rag/kg). As expected, absorption of EDC
occurred significantly faster with an oral dose in water than in oil [k , 0.299
9-5
-------
TABLE 9-3
11
Fate of 1HC-EDC in Rats 48 Hours After Oral (150 mg/kg) or
Inhalation (150 ppm, 6-hr) Exposure3
Oral
umole/kg
metabolites
Inhalation
iimole/kg
metabolites
Body burden
(total radio-
activity)
Charcoal trap
(B)
1539 ± 391
447 + 60
512 + 135
9.4 ± 0.4
9.4 + 0.4
Total metabolites
(A-B)
Urine
CQy trap
Total carcass
(48 hr after
exposure)
Feces
Cage wash
(1092)
926 + 348
83.1 + 11.9
46.9 ± 14.7
26.3 + 14.7
12.5 ± 6.37
(100)
85.7
7.7
4.3
2.1
1.1
(503)
432 + 121
36.1 + 6.89
22.7 ± 3.38
8.90 + 2.84
3.34 ± 1.34
(100)
84.4
7.0
4.4
1.7
0.7
Values are mean ± S.D., with n = 4 for each route,.of exposure and are jimole equiva-
lents of EDC, based on the specific activity of C-EDC (3.2 mCi/mM).
aSource: Reitz et al., 1980
9-6
-------
Ill
I
5
o
o
0.1
2h
6h
Figure 9-1. EDC levels after single oral administration in rats:
Top panel, 25 mg/ kg; middle, 50 mg/kg; bottom, 150 mg/kg
dose. Adipose tissue ( • !, blood ( o ), liver ( • ), lung ( A }.
Source: Spreafico et al. (1978, 1979, 1980).
9-7
-------
TABLE 9-4
Peak Blood and Tissue Levels After Single Oral Dosage
of EDC in Male Rats3
Dose
ig/kg
25
50
150
Blood
13.29
31. 94
66.78
Adipose tissue
ug/ml or |ig/g
110.67
148.92
259.88
Lung
2.92
7.20
8.31
Liver
30.02
55.00
92.10
aSource: Spreafico et al., 1980
9-8
-------
TABLE 9-5
The Absorption Rate Constants (k ) and Area Under Curve
(AUC) for Rats After Single OraT. Doses of EDC in Oil
and in Water as Vehicle and After Intravenous Administration'
V
Dose a
(mg/kg) min~
Oral 25 0.299 (water)
0.209 (oil)
50 0.185 (oil, male)
0.181 (oil, female)
AUC (blood)
jig x rain x ml"
466
466
1700
1685
150
0.109 (oil)
7297
Intravenous (water)
1
5
25
9
5U
595
*Source: Spreafico, 1978, 1979, 1980
-------
Comparison of the area under the blood concentration curve (AUC) after
intravenous administration with AUC after the same oral dose provides a measure
of the extent or completeness of oral absorption. Table 9-5 shows that the AUC
was 595 >ig x min x mA~1 after a 25 mg/kg intravenous dose and was 166 jig x min x
mi~ after 25 mg/kg oral dose. These results indicate that absorption of the
oral dose was very extensive, averaging 78$ of complete absorption, or 100?
absorption if it is assumed that the first-pass effects in liver (metabolism) and
lung (elimination) after oral absorption decreased EDC appearance in systemic
blood (Reitz, 1980, 1982). It is notable that the AUCs for differing intravenous
doses, where intestinal absorption is not a factor (Table 9-5), are not linearly
proportional, providing further evidence of the occurence of Michaelis-Menten
elimination kinetics for EDC.
Withey et al. (1982) investigated the effect of the dosing vehicle on
intestinal absorption of EDC in fasting rats (100 g) following intragastric
intubation of equivalent doses (100 mg/kg) in =1 mi of water or of corn oil. The
post-absorptive peak blood concentration averaged 5 times higher for water
vehicle than corn oil (81.6 vs 15.9 ug/mH); moreover, the peak blood
concentration was reached 3 times faster for water solution than for oil solution
(3*2 vs. 10.6 minutes). The ratio of the areas under the blood concentration
curves for 5 hours after dosing (AUC, 5 hr) was 3; water:corn oil. These results
are in general agreement with those of Spreafico et al. (1978, 1979, 1980)
(Tables 9-1 and 9-5), who found that intestinal absorption was faster with an
oral dose in water than in oil. The observations of these investigators stress
again the dependence of oral absorptive rate in rats not only on the dose, but
also on the vehicle. While these factors are unlikely to affect the
pharmacokinetics of EDC in man in any practical way, they are of importance to
interpretation of data from long-term carcinogenicity tests of EDC in rodents
9-10
-------
where the modes of intragastric dosing employed may differ by both dose and
vehicle.
9.1.1.3. PULMONARY ABSORPTION — EDC has a moderately high vapor pressure
(80 torr at 25°C; Table 9-1) and, in man, a high blood/air partition coefficient
compared to chloroethanes (19.5 at 27°C; Table 9-2). Hence, its vapor in ambient
air is a primary mode of exposure, and the lungs are a principal route of entry
into the body. The total amount absorbed via the lungs (as for all vapors) can be
expected theoretically to be directly proportional to: (1) the concentration of
the inspired air; (2) the duration of exposure; (3) the blood/air Ostwald solu-
bility coefficient; (4) the solubility in the various body tissues; and (5)
physical activity which increases pulmonary ventilation rate and cardiac output.
Hence, the basic kinetic parameters of pulmonary absorption of EDC and its
equilibrium in the body are as valid for the very low concentrations expected in
urban ambient air as for the higher vapor concentrations found in the industrial
environment and the workplace. However, these parameters of pulmonary absorp-
tion have not been studied in any detail in man, although some information is
available from pharmacokinetic studies in rats.
Urosova (1953) reported that women exposed during a normal day in the work-
place to =15.5 ppm EDC in air accumulated the chemical in breast milk and that
initial concentrations in exhaled air following daily exposure were 14.5 ppm.
These observations indicate that the women absorbed EDC through their lungs and
reached blood and total body equilibrium with inspired air concentration within
the daily work period. The deficiencies in the reporting of this work makes the
results only suggestive.
Spreafico et al. (1978, 1979, 1980) and Reitz et al. (1980, 1982) have
studied the kinetics of pulmonary absorption of EDC in rats. Figure 9-2 shows
the time-course of blood concentration of EDC, observed by Reitz et al. (1980,
9-11
-------
i 1 1 1—i r
i i t i i i
100
200 900 400 600
TIME (mlnutM)
10.0
O
O
O
(A
K
O
2
O
i
40 80
TIME (minutes)
Figure 9-2. Top: Blood levels of EDC observed during and following
a 6-hour inhalation exposure to 150 ppm EDC. Data from four male
Osborne-Mendel rats were fitted to a two-compartment open model
as described by Gehring et at. The computer plot is shown. The
arrow indicates exposure termination. Bottom: Semi-logarithmic
plot of EDC blood levels vs. time after exposure termination.
Source: Reitz et al. (1980. 1982).
-------
19&2) during a 6-hour inhalation exposure to 150 ppm EDC. Blood and body
equilibrium with the blood concentration maintained a plateau level of 9 ug/mil
(blood/air partition coefficient, 14.9). Spreafico et al. (1980) exposed their
rats to 50 and 250 ppm EDC for 6 hours. Their data are given in Tables 9-6 and
9-7. Blood and body equilibrium (liver, lung and adipose tissue) was established
at =2 hours for 50 ppm exposure, and at 3 hours for 250 ppm exposure. During
inhalation of EDC, when equilibrium is achieved, the arterial blood concentra-
tion of EDC should theoretically always be directly proportional to inspired air
concentration. This fixed relationship is defined by the blood/air Ostwald
solubility coefficient for EDC. As calculated from the data for 50 and 250 ppm
exposures of Spreafico and co-workers, the blood/air partition coefficients for
EDC are 6.3 and 30.3, respectively. Including the data of Reitz et al. (1980)
for 150 ppm exposure in rats (Figure 9-2), these results do not demonstrate a
direct proportional relationship between inspired air concentration and blood
concentration of EDC, i.e., a constant blood/air partition coefficient value.
Furthermore, linear kinetics for EDC after inhalation exposure cannot be
presumed, and indeed these data suggest otherwise, and are in accord with the
evidence of nonlinear kinetics after oral and intravenous dosing as noted above
(Table 9-5).
9-1.1.4. TISSUE DISTRIBUTION — After pulmonary or peroral absorption, EDC
is distributed into all body tissues. As expected from its general anesthetic
properties in man and in animals, EDC readily passes the blood-brain barrier.
The compound crosses the placental barrier and has been found in the fetus
(Vozovaya, 1975, 1976, 1977). EDC also distributes into human colostrum ar.d
mature breast milk. Urusova (1953) found EDC concentrated in breast milk
(5.4-6.4 mg/2,) in the workplace. The compound remained in breast milk for 18
hours following workday exposure, even though its concentration in exhaled air
9-13
-------
TABLE 9-6
EDC Tissue Levels After 50 ppm Inhalation Exposure
Time of
Exposure
EDC ug/g or ng/ml + S.E.
Blood
Liver
Lung
Adipose Tissue
30 minutes
1 hour
2 hours
4 hours
6 hours
0.48 + 0.05 0.32+0.02 0.14+0.02
0.92 + 0.09 0.67 + 0.04 0.27 ± 0.03
1.34 ± 0.09 0.84 + 0.09 0.34 + 0.03
1.31+0.11 1.14+0.17 0.42+0.05
2.91 + 0.22
7.19 + 0.60
10.31 + 0.94
11.08 + 0.77
1.37+0.11 1.02+0.10 0.38+0.02 10.19+1.00
Source: Spreafico et al., 1980
-------
TABLE 9-7
EDC Tissue Levels After 250 ppra Inhalation Exposure6
Time of
Exposure
Blood
EDC ug/g or (ig/ml + S.E.
Liver
Lung
Adipose Tissue
30 minutes
1 hour
3 hours
6 hours
6.33 ±1.04 3.82 + 0.78 2.19 + 0.21 26.75 + 3.12
11.65+1.12 7.34+0.7U 6.40+0.20 82.64+2.19
2 hours 23.64+0.91 16.39+1.18 14.07+0.47 151.53+12.17
29.36+1.01 20.83+2.21 14.47+1.12 252.18+14.62
31.29+1.19 22.49+1.12 14.14+0.90 273-32+12.46
Source: Spreafico et al., 1980
9-15
-------
fell to 4 ppm. The lack of detailed reporting of this study precludes adequate
evaluation. Sykes and Klein (1957) have demonstrated the presence of EDC in
cows' milk after oral administration.
There is little information on EDC distribution and concentration in the
various body tissues of man after exposure, and few controlled exposure studies
in animals investigating the distribution of EDC in body tissues and defining
dose-dependent tissue concentrations. Spreafico et al. (1978, 1979, 1980)
determined EDC levels in rat blood, liver, lung and epididymal adipose tissue
after single oral administration of 25, 50 and 150 mg/kg. The time-course of EDC
concentrations are shown in Figure 9-1. This figure shows that tissue accumula-
tion occurs most rapidly in the liver where peak concentrations were reached
within 10 minutes of administration. Following complete absorption and body
equilibration after 2 hours, the decay in tissue concentrations of liver, lung
and adipose tissue parallels the first-order decline in blood concentration.
Table 9-8 summarizes blood and tissue concentrations determined at 2 hours
following administration. Blood levels and tissue levels in liver and adipose
tissues were not found to be linearly proportional to dose, but increased
exponentially, providing evidence of liver metabolism saturation and nonlinear
kinetics after oral administration. At oral dose levels of 25, 50 and 150 mg/kg,
adipose tissue displayed the greatest concentration of EDC with adipose
tissue/blood partition coefficients of 17, 9 and 7, respectively. No other
tissue, including liver, demonstrated a partition coefficient >1.0. The very low
concentration levels of EDC in lung tissue may occur as a result of EDC elimina-
tion by this route.
Table 9-8 also gives blood concentrations after chronic oral administration
to rats (11 daily doses) of 50 mg EDC/kg. Comparison of these tissue levels with
those following a single oral administration of 50 mg/kg EDC provides no evidence
of blood or tissue accumulation with chronic administration.
9-16
-------
TABLE 9-8
Blood and Tissue Levels of EDO in Male Rats 2 Hours
After Single Oral Doses in Corn Oila
Tissue
( |ig/ml , or
Hg/g ± SE)
Blood
Liver
Kidney
Brain
Spleen
Lung
Adipose
Dose,
0.
0.
<0.
7.
25
46 + 0.04
32 + 0.01
05
80 + 0.72
5
3
3
2
1
1
55
.89
.81
.43
.96
.79
.44
.78
50
+
+
-»•
+
^
+
+
mg/kg
0
0
0
0
0
0
6
.41
.45
.26
.36
.35
.10
.88
26.
21.
1.
178.
60
62
62
50
150
•t- 1.
± 3.
-i- 0.
± 24
23
99
25
.66
Blood and Tissue Levels of EDC in Male Rats 2 Hours After the
Last of 11 Daily Oral Doses in Oil (50 mg/kg)
Blood 8.11 ± 0.23
Liver 3.95 + 0.48
Lung 2.24 ± 0.47
Adipose 53-71 + 10.07
a
Source: Spreafico et al., 1978, 1979, 1980
9-1?
-------
Sppeafico and his colleagues (1978, 1979, 1980) also investigated blood and
tissue concentrations in rats after inhalation exposure to 50 and 250 ppm EDC.
The results of these experiments are given in Tables 9-6 and 9-7. Blood and
tissue equilibrium (liver, lung and adipose tissue) occurred at 2 hours and 3
hours, respectively, for the two inspired air concentrations. In general, the
blood and tissue distribution of EDC at body equilibrium is similar to the
results obtained with oral administration given in Table 9-8. A very clear dose
dependence in tissue levels of EDC was again found with the two inhalation
concentrations, with differences in EDC concentrations on the order of 20-30
times when blood, liver, lung and adipose tissue are considered. The highest
absolute levels of EDC were measured again in adipose tissue with concentrations
that were between 8 and 9 times greater than those measured in the blood. As
previously noted for oral administration (Table 9-8), liver and lung concentra-
tions were less than blood concentrations.
A comparison of the absolute blood and tissue concentrations at body equili-
brium for oral and inhalation modes of administration (Tables 9-6, 9-7 and 9-8)
indicates that 50 ppm inhalation roughly equates to 25 mg/kg oral, while 250 ppm
inhalation provides blood, liver and adipose tissue levels slightly greater than
a 150 mg/kg oral dose.
9.1.2. Excretion. Elimination of EDC from the body is perforce the sum of
metabolism and excretion of unchanged EDC via pulmonary and other routes.
Unmetabolized EDC is excreted almost exclusively through the lungs; however,
metabolism of EDC is extensive, with the proportion excreted unchanged dependent
on body doses. While no controlled experimental studies have been made on the
kinetics of excretion of EDC in man, recent studies have been performed on
experimental animals.
9-18
-------
9.1.2.1. PULMONARY EXCRETION — Urusova (1953) reported that women exposed
to =15.5 ppm EDC in ambient air of industrial environs demonstrated initial EDO
concentrations in exhaled air of 11.5 ppm. The breath concentration declined to
-3 ppm after 18 hours. These values lead to an approximation of 9 hours for the
half-time of pulmonary elimination of EDC in man. Although this human data was
difficult to evaluate, similar observations have been made for animals (monkey,
dog, cat, rabbit, rat and guinea pig) in early investigations of the anesthetic
properties and toxicities of EDC (Heppel et al., 1945; Kistler and Luckhardt,
1929; Lehman and Schmidt-Kehl, 1936). In controlled studies in mice, Yllner
(1971a) found that up to H5% of an intraperitoneal injected dose of EDC (170
mg/kg) was recoverable unchanged in exhaled air (Table 9-9). The percentage of
EDC recovered unchanged in exhaled air increased exponentially with the dose,
indicating a limited capacity of biotransformation, i.e., non-linear kinetics.
Thus, in mice, pulmonary excretion of EDC is a major route of elimination,
increasing in importance with higher body doses.
Similar observations have been made for the rat by Reitz et al. (1980,
14
1982). In a balance study utilizing C-EDC given orally (150 mg/kg) and by
inhalation (150 ppm, 6 hours), these investigators found that for the oral dose,
29% was recovered unchanged in exhaled air, and that only 1.8? of the lower
inhalation dose was excreted by the pulmonary route (see Table 9-3). Sopikov and
Gorshunova (1979) observed that after an intraperitoneal 250 mg/kg dose to rats,
30$ of the EDC dose was eliminated in the exhaled air within 5 hours.
9.1.2.2. OTHER ROUTES OF EXCRETION — EDC is not ordinarily eliminated in
significant amounts from the body by any route other than pulmonary. However,
Shchepotin and Bondarenko (1978) identified EDC by gas chromatography (GC)
methods in the urine of persons with severe symptoms of EDC poisoning. Studies
of chlorinated compounds in the urine after EDC inhalation exposure or peroral
9-19
-------
TABLE 9-9
Percent Distribution of Radioactivity Excreted fl(48-hr) by Mice
Receiving 1,2-Dichloroethane- C
Dose g/kg
0.05 0.10 0.11 0.17
14C02 (exhaled air) 13 8 4 5
Dichloroethane (exhaled air) 11 21 U6 15
Urinary metabolites 73 70 18 50
aSource: Yllner, 1971 a
9-20
-------
dosage to experimental animals have failed to detect unchanged EDC (Spreafico et
al., 1980; Yllner, 1971 a). Mutagenic metabolites of EDC have been reported in
rat and mouse bile (Rannug and Beije, 1979; Rannug, 1980a), but EDC itself has
not been found. Diffusion of volatile haloalkane anesthetic agents into bowel
space and through skin is known to occur (Stoelting and Eger, 1969), and these
routes of excretion may also be significant for EDC because of similarity of
structure and comparable high lipid/blood partition coefficients.
9.1.2.3. KINETICS OF EXCRETION — While the kinetic parameters of elimina-
tion of EDC for each of the various routes of excretion, i.e., pulmonary, metabo-
lism, etc., are not well defined for either man or experimental animals, several
investigative groups have determined the kinetic parameters for whole-body and
tissue compartment excretion of EDC in the rat (Retiz et al., 1980; Spreafico et
al., 1978, 1979, 1980; Withey and Collins, 1980).
Withey and Collins (1980) determined the kinetics of distribution and
elimination of EDC from blood of Wistar rats after intravenous administration of
3, 6, 9, 12 or 15 mg/kg of EDC given in 1 mil water intrajugularly. For the two
lower doses (3 and 6 mg/kg), the blood decay curves exhibited two components of
exponential disappearance and best fitted a first-order two compartment model
with kinetic parameters of: ke> 0.24 min~ ; Vd, 43 ml; k12 and k21, 0.02 and 0.04
min~ , respectively. For higher doses (9, 10 and 15 mg/kg), these investigators
found their data best fitted a first-order, three-compartment model; they
suggest that the shift from two to three-compartment kinetics may have occurred
either because of a dose-related alteration in the kinetic mechanisms of uptake,
distribution, metabolism and elimination, or that three-compartment kinetics
were followed at all dose levels but with lower doses; the third exponential
component, representing the third compartment, was obscured by the limit of
analytical sensitivity. Average kinetic values for the three-compartment model
9-21
-------
were: ke, 0.094 min"1; Vd, 67.9 ml; k12, k21, k13, k31; 0.06, 0.08, 0.01 and 0.01
min'1, respectively. Withey and Collins state that they found no evidence in
their kinetic analysis of the disposition of intravenous bolus injections of EDC
into the rat of nonlinear or dose-dependent Michaelis-Menten kinetics. These
workers suggested that a dose of 15 rag/kg EDC in the rat is below hepatic
metabolism saturation.
Spreafico et al. (1978, 1979, 1980) intravenously administered EDC in water
to Sprague-Dawley rats as bolus injections of 1, 5 and 25 mg/kg and determined
the whole blood concentration of EDC as it decayed with time. Figure 9-3 shows
the semilog plot of the results of these experiments. A biphasic decline of EDC
blood concentration was evident for all doses, indicative of a two-compartment
system, with a distributive phase (a) and an excretion phase (B). The data for
intravenous treatment were analyzed by computer fitting to a two-compartment
open model with the results shown in Table 9-10. Of immediate note is the fact
that the kinetic parameters are not independent of the dose as required by linear
kinetics. Thus the half-time (T..,-) of body elimination from central or blood
compartment increases with the dose, while whole-body clearance (Cl) and the
volume of distribution (V.) decrease with an increase of the dose. These results
d
indicate that the rate of whole-body excretion of EDC is largely determined by
the metabolism of EDC, a saturable process and a major route of elimination.
Spreafico and his co-workers (1978, 1979, 1980) have also investigated the
whole-body kinetics of oral administration of EDC in rats. Figure 9-1 shows
semilog plots of blood and tissue concentrations during and after absorption
(excretory phase) following single oral doses of 25, 50 and 150 mg/kg given in
corn oil. In general, the slopes of the disappearance curves (excretion compo-
nent) for adipose, liver and lung tissues roughly parallel and reflect the blood
decay curve. The data for blood were computer-fitted to the kinetic equations
9-22
-------
I I I I I I I I I I I I I
001
0.5 h
VBh
Figure 9-3. Blood levels of EDC m rats after i.v. administration. ( • ) 25 mg/kg i.v.;
( O ) 5mg/kg i.v.; ( A) 1 mg/kg i.v.
Source: Spreafico et al. (1978. 1979, 1980).
-------
TABLE 9-10
Pharmacokinetic Parameters of EDC Administered as Single Bolus
Intravenous Injections in Saline to Sprague-Dawley Male Rat|.
Parameters Calculated from a Two-Compartment Open Model.
Parameter
Co, ng x ml'1
Kel> min'1
K-pi min~
K, 1 , min~
AUC, fig x min x
ml"1
T 1/2 B min
Clearance
ml x m~
Dose , mg/kg
1 5 25
1.50 8.00 38.12
0.277 0.142 0.078
0.121 0.063 0.040
0.158 0.140 0.118
9 54 595
7.30 9.49 14.07
21.98 17.46 7.98
Vol. distr., ml 231 239 162
«
K12
•^^^ ^
*21
el (inhalation)
or
V / (Kffl + C) (oral)
Source: Spreafico et al., 1979, 1980
9-24
-------
for a two-compartment open model for oral absorption; the results are given in
Table 9-11. Like the kinetics observed for intravenous dosing, the values for
the parameters, T^gi Cl and V., are dose dependent. Furthermore, the oral
dosing vehicle also affects the parameters. Comparison of the absorption rate
constants, K half-times of elimination, T.,~ an<* volumes of distribution, Vrf,
for 15 mg/kg EDC in oil and 25 mg/kg EDC in water indicates a faster absorption of
EDC in water, a shorter half-time of body elimination and a small volume of
distribution. Nonetheless, the AUCs indicative of the total absorbed dose were
similar for oil and water vehicles, as were the body clearance rates of EDC. On
the other hand, no significant differences in the kinetic parameters were seen
for male and female rats given the same dose, nor were significant differences
observed between a single dose and 11 daily administrations of the same dose (50
mg/kg) of EDC.
Spreafico et al. (1980) included in their comprehensive studies on the
pharmacokinetics of EDC in the rat, experiments to determine the kinetics of EDC
after inhalation dosing. Rats were exposed to 50 and 250 ppm EDC for 5 hours, a
period sufficiently long to establish and maintain whole body steady-state
conditions with these inspired air concentrations (Tables 9-6 and 9-7). The
exposure was terminated, and blood and other tissues were sampled to determine
the decay of EDC concentrations in these tissues with time. The results of these
experiments are presented as semilog plots of tissue concentrations versus time
in Figure 9-1. As for oral dosing, the slopes of the tissue decay curves
parallel and reflect generally the blood concentration curves. In contrast to
intravenous and oral dosing, which have several components to their curves
representing absorption and/or distribution, the curves after steady-state
inhalation are monophasic, representing only excretion. Thus, the
pharmacokinetic parameters calculated from the blood decay curves following the
9-25
-------
TABLE 9-H
Pharmacokinetic Parameters of EDC Administered as Single Oral
Doses in Corn Oil and Water to Sprague-Dawley Male Rats^
Parameters Calculated from a 2-Compartment Open Model.
Parameter
Oil Vehicle
Ka, mln~1
K , min"
AUC, fig x min x ml
T 1/2 B min
Clearance, ml x min"
Vol. Distr., ml.
Aqueous Vehicle
K , min"1
ci
K lf min"
AUC, fig x min x ml"
T 1/2 B min
Clearance, ml x min"
Vol. Distr., ml.
Dose, mg/kg
25 50
0.209 0.185
0.029 0.017
446 1700
24.62 44.07
10.64 5.58
367 328
0.299
0.046
446
14.12
10.20
221
150
0.109
0.010
7297
56.70
3.90
390
Source: Spreafico et al., 1979, 1980
9-26
-------
1000
1
8
2
o
o
100 —
| T
—
I I 1 I 11
i
0.6h
1000
0.1
0.5h
Figure 9-4. Levels of EDC in rats after inhalation exposure
to 50 ppm (top) and 250 ppm (bottom). Levels were mea-
sured at termination of 5-hour exposure period. Adipose
tissue ( • ), blood ( o ), liver ( • ), lung ( A ).
Source: Spreafico et al. (1978, 1979, 1980).
-------
t i-dy-state inhalation conditions are essentially those applicable to a single
c^apartment open model. The kinetic parameters are given in Table 9-12. The
bcdy burden or "dose" of EDO in the animals at termination of inhalation exposure
is not known; however, a comparison of the areas under the blood decay curves, a
reflection of body burden, suggests that the body burden after 250 ppra exposure
was 40 times, not the 5 times expected of the 50 ppm exposure. Similarly, the
half-life of EDC and the zero-time blood concentrations are dose-dependent.
These results from inhalation exposure are in accord with the dose dependent
pharmacokinetics exhibited after intravenous and oral dosing of EDC. They
strongly indicate that the kinetics of absorption, distribution and elimination
of EDC are appropriately described by nonlinear kinetics that take into account
saturable processes, presumably in this case the metabolism of EDC by the
organism.
The pharmacokinetics of EDC after inhalation have also been explored by
Reitz et al. (1980, 1982) in male Osborne-Mendel rats. These investigators
determined the whole-blood levels of EDC during and following a 6-hour inhalation
exposure to 150 ppm EDC. Figure 9-2 shows the data obtained from 4 rats,
computer-fitted to the equations of a two-compartment model and the idealized
computer-fit plotted. Within 2 hours of exposure, the animals approached a
steady-state blood concentration (9 ug/m£) with the inspired air concentration;
but, following termination of exposure at 6 hours, the blood concentration of EDC
rapidly decayed to near zero within 10 hours. Figure 9-2 shows also the semilog
plot of computer-fitted data of EDC blood concentration levels vs. time after
exposure termination. In contrast to the observation of Spreafico et al. (Figure
9-4) that only a raonophasic plot was obtained following inhalation exposure,
Reitz et al. found a biphasic elimination with an initial rapid alpha phase and a
slower beta phase. Table 9-13 gives the pharmacokinetic parameters calculated
from a two-compartment open model. These results may be compared with data
obtained by Spreafico et al. for inhalation exposure (Table 9-12).
9-28
-------
TABLE 9-12
Pharmacokinetic Parameters of EDC Following Termination
of Steady State Inhalation Conditions (5-hr Exposure) of 50 and 250 ppm
to Male Sprague-Dawley Rats.
Parameter
CQ, ug x ml~
i
K ., min
el
T 1/2, min
i
AUC, }ig x min x ml
Exposure
50
1.42
0.0561
12.69
26
Concentration, ppm
250
30.92
0.0313
22.13
1023
aSource: Spreafico et al., 1979, 1980
-------
TABLE 9-13
Pharmacokinetic Parameters of EDC Following Termination
of Steady State Inhalation Conditions; 6-Hr Exposure, 150 ppm to Male
Osborne-Mendel Rats. Parameters Calculated from a Two-Compartment Model
Parameter
CQ, jig x ml~1 9.0
K, min"1 0.092
K21> min"1 0.025"
AUC, |ig x min x ml~1 3018
T1/2B min 35
Vol. Distr, ml. 513
aSource: Reitz et al., 1980
9-30
-------
Reitz et al. (1982) also determined in the rat the pharmacokinetics after
single oral dosing (150 mg/kg in corn oil). In this case, a semilog plot of blood
EDC levels after absorption produced an elimination curve typical of nonlinear
kinetics. These workers therefore computer-fitted the data to a two-compartment
nonlinear model and calculated the rate of elimination from the central compart-
ment (blood) according to Michaelis-Menten kinetics. Table 9-13 gives the
phartnacokinetic parameters calculated from the model.
Several differences exist between the observed data and calculated
parameters of Reitz et al. for inhalation vs. oral exposure (Table 9-13) as well
as those of Spreafico et al. (Table 9-12). The EDC inhalation concentration used
by Reitz et al. (150 ppm) was intermediate to those of Spreafico et al. (50 and
250 ppm). Plateau blood concentrations in the three inhalation studies (1.1 at
50; 8.3 at 150; and 30.9 ug/m£ at 250 ppo), clearly demonstrate saturation of an
elimination process, presumably metabolism. Reitz et al. found peak blood levels
following oral dosing (30-44 ug/m£ at 150 mg/kg) to be similar to those found by
Spreafico et al. after a 250 ppra inhalation exposure. Reitz et al. also found a
biphasic elimination after both oral and inhalation exposure with the second or
3 phase essentially equal (T1/2 = 28 rain), and similar to the monophasic
elimination calculated by Spreafico et al. for the 250 ppm exposure (T1/2 = 22
min). The relatively slow alpha elimination phase (T1 ,~, 90 min) following oral
dosing of 150 mg/kg suggested to Reitz et al. a saturable elimination process.
The data of Spreafico and co-workers are generally in agreement with this
treatment; however, several kinetic parameters are not in good agreement between
the studies. Of particular note are the biphasic and monophasic elimination
curves and the large discrepancy of AUG. The explanation for these differences
may lie in the different kinetic models, species differences and inability of
linear kinetics to accurately describe the kinetics of EDC body elimination.
9-31
-------
9.1.2.4. BIOACCUMULATION — There is no definitive experimental evidence
in the literature concerning bioaccumulation in man after chronic or repeated
-:uj.ly exposure to EDC. While EDC has a moderately high fat tissue/blood parti-
_on coefficient (Table 9-2), its half-time of elimination in man appears to be
=6-8 hours (Urusova, 1953). In the rat, Spreafico et al. (1978, 1979, 1980) and
Reitz et al. (1980, 1982) have provided estimates of half-time of whole-body
elimination of 25-57 minutes for acute oral dosing, and 13-35 minutes after 5-6
hour inhalation exposures. The. half-time of elimination of EDC from the
epididymal adipose tissue was essentially the same as whole-body elimination.
These relatively short half-times of elimination indicate that the risk of
significant bioaccumulation of EDC is small. Based on the pharmacokinetic data
from rats, these investigators predict essentially complete elimination of EDC
from the body in 24 hours following acute oral exposure or five 6-hour exposure
periods via inhalation. Furthermore, Spreafico et al. (1979, 1980) found no
significant differences in the kinetic parameters of EDC elimination between a
single dose and multiple daily oral administration of EDC.
9.1.3. Metabolism
9.1.3.1. KNOWN METABOLITES — Metabolism of EDC in man during or after
exposure has not been studied. However, one of the earliest suggestive reports
of an active mammalian biotransformation of EDC is related to human exposure. In
1945, Bryzkin reported that EDC underwent rapid transformation to an "organic
chloride" in patients who died after ingesting 150-200 mi; EDC itself was not
detectable in tissues at autopsy. Although Heppel and Porterfield demonstrated
as early as 1948 that both ethylene dichloride and dibromide were dehalogenated
by rat liver enzyme preparations, current knowledge of mammalian metabolism of
EDC derives primarily from studies performed within the last 10 years. Metabo-
lites that have been identified either in vivo in mice and rats or in liver and
9-32
-------
kidney tissue crude enzyme systems are listed in Table 9-11. In addition to the
very active metabolite, 2-haloacetaldehyde, other possible reactive interme-
diates formed during the metabolism of 1,2-dihaloethylenes by glutathione-depen-
dent reactions, such as an episulfonium ion, may be involved in the covalent
binding to tissue macromolecules leading to tissue damage (Rannug and Beije,
1979; Hill et al., 1978; McCann et al., 1975; Guengerich et al., 1980; Anders and
Livesey, 1980).
9.1.3.2. MAGNITUDE OF EDC METABOLISM -- The capacity of mammalian
organisms to metabolize EDC has been studied only in the mouse and rat. In these
two species, the extent of biotransformation of single doses of EDC has been
11
estimated by balance studies utilizing C-EDC.
Yllner (1971a) administered intraperitoneally doses of 50-170 mg/kg of
11
C-EDC to mice in metabolism cages and collected the expired CO. and volatile
organic metabolites using the appropriate solvent traps. Yllner recovered
97-100$ of radioactivity in exhaled air and urine within 21 hours. The results
of his balance studies are summarized in Table 9-9* For the low dose of 50 mg/kg,
Yllner found that EDC was metabolized to CO. (13$) and water and to other
metabolites appearing in the urine (73$). Unchanged EDC did not appear in the
urine, but 11$ of the dose was eliminated unchanged in exhaled air. An increase
of the intraperitoneal dose resulted in a smaller percentage of the d se being
metabolized. For the largest dose, 170 mg/kg, 55$ was metabolized to C02 (5$)
and urine metabolites (50$). These results indicate that extensive biotransfor-
mation (55-86$) occurs in the mouse. Furthermore, the extent of metabolism is
dose dependent, suggesting a limited capacity for biotransformation with satura-
tion of the metabolizing system(s). Saturation of metabolism to C0p and urinary
metabolites occurred at a dose level of between 100-110 mg/kg and 50-100 mg/kg,
respectively.
9-33
-------
TABLE 9-11
Identified Metabolites of Ethylene Dichloride and Ethylene Bromide.
Metabolites
System
Reference
Inorganic halide
co2
2-Chloroethanol
2-Chloroacetic acid
Bromoace taldehyde
Thiodiglycolic acid
Ethylene
S-(2-hydroxyethyl)-cysteine
N-acetyl-S-(2-hydroxyethyl)-
cysteine
N-acetyl-S-(2-hydroxyethyl)-
oxide
S-carboxymethyl cysteine
S-(2-hydroxyethyl)-glutathione
S-(2-hydroxyethyl)-S-oxide
rat liver
cytosol
mouse, rat
exhaled air
mouse, rat
urine, blood
mouse urine
rat liver
microsomes
mouse, rat
urine
rat liver
cytosol
rat liver
cytosol,
rat urine
rat urine
blood
mouse urine
rat liver
cytosol,
tissue
S,S'-ethylene-bis-glutathione rat liver
Heppel and Porterfield, 1918
Nachtorai, 1970
Yllner, 1971b
Reitz et al., I960
Yllner, 1971b
Kokarovtseva and
Kiseleva, 1978
Yllner, 1971b
Hill et al., 1978
Yllner, 1971b
Spreafico et al., 1979
Livesey and Anders, 1979
Nachtorai et al., 1966
Edwards et al., 1970
Edwards et al., 1970
Yllner, 1971b
Nachtorai, 1970
Nachtomi, 1970
9-31
-------
Reitz et al. (1980, 1982) have found that EDC is also extensively metabo-
14
lized in the rat. These investigators conducted balance studies with C-EDC in
Osborne-Mendel rats after oral (150 rag/kg) and inhalation (150 ppm for 6 hours)
exposure. Their results are summarized in Table 9-3- In the oral balance study,
14
150 mg/kg of C-EDC (or, 1520 ^moles/kg) were administered. The sum of radio-
activity recovered in exhaled air, urine and feces, and that remaining in the
carcass was 101$, or complete recovery. Some 29% of the dose was excreted
unchanged in exhaled air; 5% was metabolized completely to COp and 60% of the
14
dose was metabolized to C-metabolites appearing in the urine. Less than 3%
radioactivity remained in the body 48 hours after dosing (probably as covalently
bound metabolites). These results indicate that the extent of total metabolism
of the oral dose was =70% in these rats. For the inhalation exposure study, the
absolute dose administered by a 150 ppm, 6-hr exposure was unknown. From the
recovery after termination of exposure of C-radioactivity in exhaled air,
urine and feces, and radioactivity remaining in the carcass 48 hours after
exposure, it was estimated that the inhalation dose was 50.5 mg/kg (512
^.moles/kg) or one-third of the oral dose. Of this dose, 2% was excreted
unchanged in exhaled air, 7% was metabolized completely to CO-, and 84$ was
14
metabolized to C-metabolites appearing in the urine. Less than 5% of the
radioactivity was left in the body 48 hours after inhalation exposure. Ignoring
the pharmacokinetic differences between acute oral and inhalation dosing, the
results of Reitz et al. (1980) demonstrate extensive biotransformation in the rat
(70-91$) and also suggest a dose dependency with greater metabolism for lower
doses, as Yllner (1971a) observed in the mouse.
The balance studies of both Yllner (oral dosing) and Reitz et al. (oral and
inhalation dosing) demonstrate that the primary route of elimination of EDC is by
metabolism. Renal excretion of nonvolatile metabolites predominates; however,
9-35
-------
5-10? is metabolized completely to C02 and water. It is of interest to note that
a small amount of EDC or its metabolites appear in the feces (<2%), even from
inhalation exposure. Yllner (1971a) identified the metabolites appearing in the
urine of mice after dosing with 14C-EDC (170 mg/kg) and found six l4C-metabo-
lites; the three principal metabolites were S-carboxymethylcysteine (455&),
thiodiacetic acid (33*) and chloroacetic acid (15*) (Table 9-15). However,
Ml
Spreafico et al. (1979) dosed rats with 50 and 150 mg/kg C-EDC and found the
principal metabolite to be thiodiacetic acid; chloroacetic acid was not detected
and S-carboxymethylcysteine was found only in trace amounts.
9.1.3.3. PATHWAYS OF METABOLISM — A number of studies have been carried
out describing the metabolism of 1,2-dihaloalkanes. Two principal pathways,
involving microsomal and cystosolic enzymes, respectively, have been proposed.
These pathways account for the formation of the metabolites identified in both iji
vivo and in vitro studies listed in Table 9-14 and also suggest the nature of the
reactive intermediates involved in the covalent binding to cellular
macromolecules (protein and DNA) which has been demonstrated in both in vivo and
in vitro studies (Section 9.1.3-5).
9.1.3.3.1. Microsomal Reactions — Yllner (1971a) originally proposed
that the degradation of 1,2-dichloroethane to 2-chloroacetic acid involved a
primary reaction in which chlorine was removed from one of the carbon atoms
(hydrolytic dehalogenation) to yield 2-chloroethanol. As evidence for this
reaction, he found chloroethanol to be a minor metabolite in the urine of mice
injected with EDC (Table 9-15). Following 2-chloroethanol formation, Yllner
proposed that alcohol was enzymatically converted to 2-chloroacetic acid (a
major urinary metabolite) via 2-chloroacetaldehyde. Kokarovtseva and Kiseleva
(1978) have also identified chloroethanol in the blood of rats (T1/2 ca. 9
hours), and in rat liver tissue within 1 hour and for 24-48 hours after oral
9-36
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TABLE 9-15
Percent Distribution of Radioactivity Excreted (18-hr) as Urinary
Metabolites by Mice Receiving 1,2-Dichloroethane- C or
2-Chloroacetic Acid- C.
After After
dichloroethane chloroacetate
Metabolite (0.17 g/kg) (0.10 g/kg)
Chloroacetic acid 16 13
2-chloroethanol 0.3
S-carboxymethylcysteine 45 39
Conjugated S-carboxymethyl- 3 3
cysteine
Thiodiacetic acid 33 37
S,S-ethylene-bis-cysteine 0.9
Glycolic acid — 4
Oxalic acid -- 0.2
aSource: Yllner, 1971a,b
9-37
-------
administration of EDC (750 rag/kg). Van Dyke and Wineman (1971), and subsequently
Salmon et al. (1978, 1981), found that rat microsomal fractions, in the presence
of Qy and NADPH, dechlorinated a series of haloalkanes including EDC. The
reaction for EDC had a Vfflax of 0.24 nraol/min/mg protein with a Km of 0.11 mM
(Salmon et al., 1981). Thus, EDC and other haloalkanes interact with cytochrome
P-450 to give a Type I difference spectra associated with the metabolism of these
substrates by direct C-hydroxylation (Ivanetick et al., 1978). Besides EDC,
Ivanetick et al. (1978) observed that chloroacetaldehyde and chloroethanol also
interacted with cytochrome P450 with K values of 10.3, 30 and =15, respectively.
s
Hill et al. (1978) reported that 1,2-dibromoethane was activated to an
irreversibly bound species by microsomal mixed-function oxidases, and also
provided evidence that 2-bromo-acetaldehyde was formed in such reactions. More
recently, Guengerich et al. (1980) showed that 2-chloroethanol was a product of
rat microsomal mixed-function oxidation of EDC, requiring 02 and NADPH. The
reaction was blocked by classic P450 inhibitors and increased by pre-treatment
with phenobarbital. Guengerich et al. (1980) proposed that part of the
microsomal mixed-function oxidative metabolism of EDC proceeded from oxygen
insertion into a C-H bond to form an unstable chlorohydrin which spontaneously
dehydrohalogenates to form 2-chloroacetaldehyde. Figure 9-5 illustrates the
proposed reaction and pathway. Chloroacetaldehyde is then reduced to
chloroethanol by alcohol dehydrogenase in the presence of NADH, or oxidized to 2-
chloroacetic acid by aldehyde dehydrogenase. Johnson (1967) previously had
observed that chloroethanol was readily dehydrogenated to chloroacetaldehyde by
alcohol dehydrogenases from yeast or horse liver. The reaction, therefore,
proceeds in either direction depending on enzyme, substrate and cofactor
concentrations. Williams (1959) also had suggested that chloracetic acid
appeared in vivo via chloroacetaldehyde.
9-38
-------
P450.0, r ii0-H i
-+• X-6-C-X
NADPH L M J
-HX
Alco. D
-X-C-(
BINDING —I H'
PROTEIN. DNA 2 haloacat. I
Hi
aldehyde * Aid. D
2 haloathanol
2-haloacatic acid
I'i1 P450.0, , F 5'1?' 4 "1
-OH
2 haloethanol
2 haloacataldahyda
T
Aid O
H. ^)
CI-C— C-OH
2 haloaeatle acid
Figure 9-5 Postulated microsomal oxidative metabolism of 1 ,2-dihaloethanes.
Source: Adapted from Guengench et al. (1980) and Anders and Livesey (1980).
9-39
-------
Further evidence for this reaction sequence has been provided by Guengerich
et al. (1980). These workers observed that covalent binding to microsomal
protein and DNA was inhibited 30-40? by inclusion of alcohol or aldehyde dehydro-
genases in the reaction mixture, strongly indicating that chloroacetaldehyde was
the reactive intermediate responsible for this portion of the total irreversible
binding of EDO. Chloroethanol itself, when added to the microsomal system, gave
only a low level of irreversible binding. In order to account for the remainder
of the microsomal mixed-function oxidative irreversible binding of EDC,
Guengerich et al. (1980) proposed the oxidative formation of the reactive meta-
bolite 1-chloroso-2-chloroethane, which spontaneously rearranges to 1-chloro-
acetaldehyde via a hypochlorite. The reaction is illustrated in Figure 9-5.
These workers noted that a reactive chloroso compound could react directly with
raacromolecules, or hydrolyze (with release of hypochlorite ion) to form
2-chloroethanol.
As a consequence of microsomal P-450-mediated oxidation of EDC as
illustrated in Figure 9-5, further metabolism of the oxidative metabolites,
2-chloroacetaldehyde and 1-chloroso-2-chloroethane, leads either to a
"detoxification" or to a further formation of reactive intermediates,
respectively, as illustrated in Figure 9-6.
Regarding detoxification reactions of chloroacetaldehyde with glutathione,
Johnson (1955, 1966a,b, 196?) found that Chloroethanol, administered orally to
the rat, caused rapid depletion of liver glutathione (GSH) with a concomitant
formation of S-carboxymethylglutathione. In vitro, the reaction with a rat liver
cytosol fraction required stoichiometric amounts of GSH (1 mole) and NAD
(2 moles). Since pyruvate was also required for reaction, Johnson (1967)
postulated that Chloroethanol was converted by alcohol dehydrogenase to
chloroacetaldehyde, which then conjugated with GSH to give
9-40
-------
"OH T>H
2-cMoroacataldahyda 2-chioroacatata
,-cr
GSH
GT
GSH 1 7 -Ci-
ty J
GS-C-C-H
S-formylmathylglutathlona
H. *,
DH
i
cs-c-
-------
S-forraylmethylglutathione, and thence by an NAD requiring dehydrogenation to S-
carboxymethylglutathione. Johnson (1966b) reported that chloroacetaldehyde also
rapidly conjugates with GSH in vitro by a non-enzymatic reaction at pH 7.0. He
also has shown that S-carboxymethylglutathione is rapidly degraded by rat kidney
homogenate to yield glycine, glutamic acid and S-carboxymethylcysteine, part of
which is further metabolized to thiodiglycolic acid. Yllner (1971a) found that
these latter two metabolites were the two major urinary compounds after
administering EDC to mice (Table 9-15). Since these two compounds were also the
major urinary metabolites after administering 2-chloroacetate to mice (Table
9-15), Yllner (1971b) proposed that 2-chloroacetic acid could also conjugate
with GSH with chloride excision forming S-carboxymethylcysteine and thereby
enter the pathway (Figure 9-6). Spreafico et al. (1979) found that after rats
were given oral doses of 50 and 150 mg/kg EDC, 2-chloroacetic acid did not appear
in the urine, but thiodiglycolic acid did appear as the major urinary product.
The difference between the urinary metabolites found in mice by Yllner and those
observed in rats by Spreafico et al. may be explained by the availability of GSH
in the livers of the animals in the two studies.
Reactions of putative 1-chloroso-2-chloroethane: Guengerich et al. (1980)
noted that the chloroso compound that they proposed was formed by microsomal PU50
oxidative metabolism of EDC, would be extremely reactive and could be expected to
either rearrange to 2-chloroacetaldehyde, hydrolyze to 2-chloroethanol, or react
directly with microsomal protein (Figure 9-5). In addition, these workers
postulated that the chloroso compound may react with GSH to form S-(2-
chloroethyDglutathione, a half sulfur mustard, which could react via an
episulfonium ion intermediate with macromolecules (Figure 9-6). However, in an
Ames test with S. typhimurium TA1535, EDC activated with rat liver microsomes,
NADPH, 0 and added GSH, did not enhance mutagenic activity. In contrast, the
9-12
-------
use of 100,000 g liver supernatant containing GSH and GSH transferases markedly
increased the number of revertants per 1,2-dichloroethane plate. These results
suggested that either the putative chloroso compound is not formed in the micro-
somal system, or that differences in the subcellular systems contribute to
differences in covalent binding and rautagenicity.
9.1.3.3.2. Cytosolic reactions — Heppel and Porterfield (1948)
obtained an enzyme preparation from rat liver capable of hydrolyzing the carbon-
halogen bonds of chloro derivatives of methane and ethane, including EDC. Bray
et al. (1952) also studied the dehalogenation of dichloroethane and other
halogenated hydrocarbons by rabbit liver extracts and nonenzymatic
dechlorination by direct interaction with sulfhydryl groups of GSH and cysteine.
Naehtomi et al. (1966, 1970), found that an enzyme system from the soluble
supernatant fraction of rat liver catalyzed a reaction between EDC and GSH. The
formation of inorganic halide in these studies could occur as a consequence of
attack by GSH resulting in the excision of halide, and the formation of S-(2-
haloethyl)-GSH (Figure 9-7). This metabolite is a reactive intermediate, a half
sulfur mustard, which, via its episulfonium ion and further reaction with a
second GSH or with water, may be expected to form S,S-ethylene-bis-GSH or S-(2-
hydroxyethyl)-GSH. The S,S'-ethylene-bis-GSH may be presumed to be subject to
degradation to S,S-ethylene-bis-cysteine by glutathione in liver and kidney.
Yllner (1971a,b) found small amounts of S,S'-ethylene-bis-cysteine in the urine
of mice injected with EDC (Table 9-15). Nachtorai et al. (1970) found that the
products of the soluble fraction of rat liver reaction with EDC were S,S'-
ethylene-bis-glutathione and S-(1-hydroxyethyl)glutathione. Nachtorai et al.
(1970) identified the same two compounds plus the sulfoxides of the former in
urine of EDC- and dibroraoethane-treated rats. Naehtomi et al. (1966) have also
identified N-acetyl-S-(2-hydroxyethyl)cysteine in the urine of EDC- and
9-13
-------
H,H,
ci-6-c-ci
1
CT
GSH
GS-CH,-CH,-CI
S-C2-chloro«thylVGSH
HOH
Ethylana
-------
1,2-dibromoethane-treated rats. Jones and Edwards (1968) and Edwards et al.
(1970) confirmed the work of Nachtorai with dibromoethane, and moreover, isolated
the sulfoxides of both S-(2-hydroxyethyl)cysteine and its mercapturic acid from
the urine of dibromoethane-treated rats.
The half sulfur mustard, S-(2-chloroethyl)-GSH, and its episulfonium ion
formed by cytosolic GSH-dependent transferase metabolism (Figure 9-7) has been
suggested by Rannug et al. (1978, 1979, 1980a) to be responsible for the
mutagenic action of EDC in S. typhimurium TA1535. These workers found that
activation of EDC occurred only with the soluble fraction of rat liver
homogenates or with purified GSH transferase enzyme in the presence of GSH.
Their findings have been confirmed by Guengerich et al. (1980). In addition,
Rannug and co-workers demonstrated that S-(1-chloroethyl)cysteine and N-acetyl-
S-(2-chloroethyl)-cysteine produced direct mutagenic effects when tested on
Salmonella, whereas S-(2-hydroxethyl)cysteine showed no mutagenic effect. These
results showed that the enzymatic degradation of the GSH moiety does not abolish
the mutagenic properties of the conjugate, whereas a substitution of the chlorine
with a hydroxyl group does.
9.1.3.3.3. Formation of ethylene — Livesey and Anders (1979) have
identified ethylene in rat liver and kidney cytosol incubated with EDC. Ethylene
was produced in only small amounts which were, however, linearly independent on
the cytosolic protein concentration. The enzymatic conversion was glutathione
dependent and specific for this thiol. Studies of substrate specificity with
1,2-dihaloethanes showed that reactivity following the halide order with C-Br
bond cleavage and elimination occurring at a faster rate than C-C1 bond breakage.
Whereas EDC conversion was primarily an enzymatic reaction, ethylene formation
occurred equally well from 1,2-dibromoethane by a nonenzymatic reaction with
GSH. The microsomal P-450 inhibitor, SKF 525-A, had no effect on EDC metabolism,
9.115
-------
but the reaction was inhibited by p-chloromercuribenzoic acid, methyl iodide and
diethylmaleate. Livesey and Anders proposed that the intermediate in the conver-
sion of EDC to ethylene was S-(2-chloroethyl)glutathione (Figure 9-7). An analog
of this intermediate, S-(2-chloroethyl) cysteine, was nonenzymatically converted
to ethylene in the presence of glutathione and other thiols.
9.1.3.3.4. Oxidation of EDC to CO- — Following oral administration of
14C-EDC to mice, Yllner (1971a), and rat, Reitz et al. (1982), found 5-10$ of the
dose administered was metabolized to CO.. Yllner (1971b) also observed that
after 2-chloroacetate administration, small amounts of glycolic and oxalic acids
appeared in urine (Table 9-15). Since these acids are known to be metabolized to
CO-, Yllner proposed that 2-chloroacetate, arising from EDC metabolism (Figure
9-5) is enzymatically hydrolyzed to glycolate by dehydrohalogenation, a portion
of which is further oxidized to oxalic acid.
9.1.3.3.5. Comparison of the Metabolism of EDC to Vinyl Chloride —
Rannug and Beije (1979) have drawn attention to the similarities in the
biotransformation pathways of EDC and vinyl chloride. For both metabolisms, S-
(2-chloroethyDcysteine, thiodiglycolic acid, S-(2-hydroxyethyl)cysteine and
its mercapturic are involved as end products. Guengerich et al. (1980)
considered the possibility that reductive dechlorination of EDC would yield
chloride ion plus a chloroethyl radical, which could either react with
macromolecular targets, or lose a hydrogen atom to form vinyl chloride.
Alternatively, some other dehydrohalogenation mechanism (-HC1) could give rise
to vinyl chloride. Vinyl chloride could then be microsomally oxidized to 1-
chloroethylene oxide, which can either react with macromolecules, or be
rearranged to 2-chloroacetaldehyde and proceed through the pathway of Figure
9-6. However, Guengerich et al. found that reductive dechlorination of EDC to a
chloroethyl radical was an unlikely reaction because metabolism and binding of
EDC was dependent upon the presence of 0_ in microsomal incubations containing
9-46
-------
NADPH. Guengerich et al. calculated that the rate of EDC conversion to either a
total nonvolatile or to an irreversibly bound metabolite was =25-50 times too
high to support an obligatory role for vinyl chloride as an intermediate.
Moreover, they found that irreversible binding of label from vinyl chloride to
microsomal protein was inhibited 95$ by either alcohol or aldehyde dehydrogenase
in microsomal incubations while these conditions produced only 30-10$ inhibition
of binding for EDC. These investigators concluded that the metabolism of EDC
probably does not involve vinyl chloride as an intermediate.
9.1.3.1. METABOLISM, TOXICITY AND MODIFIERS — Dichloroethane metabolites,
chloroacetaldehyde, chloroethanol (oral LD Q for rats, 96 mg/kg), and chloro-
acetic acid (oral LD,.Q for rats, 76 mg/kg) are several times more toxic than EDC
itself (oral LD5Q for rats, 770 mg/kg) (Heppel et al., 1945, 1946; Woodward et
al., 1941; Ambrose, 1950; Hayes et al., 1973). Since the symptoms of poisoning
from both accidental and occupational exposures of humans and experimental
exposures of animals to these metabolites are very similar to those resulting
from EDC, it can be assumed that the toxicity of EDC for both man and animals is
in large part the result of biotransformation to these metabolites. Johnson
(1967) was the first to suggest that chloroacetaldehyde may be the premier toxic
metabolite, since this very reactive compound is capable of both enzymatic and
non-enzymatic interaction with cellular sulfhydryl groups (Figure 9-6).
However, Yllner (1971a,b) found that chloroacetic acid also reacted extensively
with sulfhydryl compounds in vivo.
Heppel et al. (1945, 1946, 1947) found a high mortality (35$) in rats given
1.3 g/kg EDC orally. Mortality was reduced by pre- or post-administration of
methionine, cysteine and other sulfhydryl compounds. Sulfur-containing amino
acids, cysteine and methionine, also protected young rats from inhalation
9-47
-------
exposure. This protective effect of sulfhydryl compounds is clearly related to
the marked depletion of glutathione levels that occurs in the livers of rats
given EDC, chloroethanol or chloroacetaldehyde (Johnson, 1965, 1966, 1967) and
to the enzyme pathways of metabolism of EDC (Figures 9-5 to 9-7). Johnson (1965,
1967) has noted that the morbidity and mortality of young rats given
chloroethanol orally was reduced by concomitant administration of ethanol. He
postulated that the protective effect of ethanol was due to simple substrate
competition for alcohol dehydrogenase which catalyzes the conversion of
chloroethanol to chloroacetaldehyde (Figure 9-5). Ethanol also inhibited early
effects of chloroethanol on liver glutathione depletion in these animals. This
author also suggested that the minimal toxicity observed with chronic low
inhalation doses of EDC in different animal species may be simply explained by
the rapid replenishment of tissue glutathione.
Similar observations on the relation of the level of glutathione in liver
and other tissues, and toxicity of the chlorocarbons, have been made by Jaeger
and his co-workers (1974, 1979). Like EDC, vinylidene chloride is detoxified by
glutathione-dependent pathways. Jaeger et al. (1974) found in rats that an 18-
hour overnight fast decreased the LC,.- from a 4-hour inhalation concentration of
vinylidene chloride from 15,000 ppm (fed) to 600 ppm (fasted) and decreased the
concentration of vinylidene chloride that produced a significant plasma eleva-
tion of a-ketoglutarate transaminase, evidence of liver cytoxicity (2000 ppm,
fed; 150 ppm, fasted). Increased susceptibility to hepatotoxicity was shown to
be related to a decreased hepatic glutathione concentration associated with the
fasting. Jaeger (1979) also demonstrated in rats a circadian rhythm for tissue
glutathione concentration of liver, blood, lung and kidney. For rats maintained
on a 12-hour light-dark cycle (6 pm to 6 am, dark), hepatic glutathione content
was lowest at 6-10 pm and highest at 4-12 am; these periods correspond to the
9-48
-------
greater and lesser hepatotoxicity respectively associated with vinylidene
chloride exposure during these two periods.
Nakajima and Sato (1979) studied the metabolism of the chlorocarbons
in vitro with microsomes from livers of fed and fasted male rats and found that
the metabolism of EDC increased 1.5-fold as the result of a 24-hour fast,
although fasting produced no significant increase in the microsomal protein and
cytochrome P-450 liver contents. Microsomes from livers of fasted female rats
displayed even more active metabolism of EDC (3.6-fold over fed rats). These
observations suggest that the increased toxicity of EDC that occurs with food
deprivation may be due not only to decreased liver glutathione content, but also
to a greater production of toxic metabolites: chloroacetaldehyde, chloroethanol
and chloroacetic acid.
Sato et al. (1980) have studied the effect of chronic ethanol consumption on
hepatic metabolism of chlorinated hydrocarbons in rats to provide information on
the effect of ethanol consumption on the toxic effects of chlorinated hydro-
carbons in the work environment. Male rats were maintained on a daily intake of
ethanol amounting to 30% of their total energy intake for 3 weeks. The hepatic
microsomal metabolism of EDC was increased 5.5-fold over microsomes from livers
of ethanol-free rats, although ethanol feeding produced only a slight increase in
the microsomal P-450 content. The increase in enzyme metabolism of EDC occurred
only with microsomal fractions and not with cytosolic fractions, and one-day
withdrawal of ethanol feeding almost completely abolished the effect of chronic
alcohol consumption.
Sato et al. (1981) also studied the effects of an acute single dose of
ethanol (given as an aqueous solution by gastric intubation) on the metabolism of
chlorinated hydrocarbons in rats. Hepatic metabolism of EDC (by a 10,000 g liver
fraction) added in vitro was accelerated (up to 2.5-fold) by doses of ethanol up
9-49
-------
to 14 mg/kg without causing any increase in P-U50 content. The stimulation was
not seen and even suppression occurred with higher doses of ethanol. Increased
metabolism was most marked 16-18 hours after ethanol administration. Ethanol
added directly to the incubation mixture, however, depressed EDC metabolism.
These investigators suggest that ethanol is capable of exerting a dual effect on
EDC-metabolizing enzymes, i.e., inhibition and stimulation, depending on ethanol
concentration.
9.1.3.5. METABOLISM AND COVALENT BINDING — The metabolism of EDC results
in the production of reactive metabolites of an electrophilic nature such as
those proposed to be formed in the metabolic schemes of Figures 9-5 to 9-7. The
occurrence of these metabolites, chloroacetaldehyde, the putative 1-chloroso-2-
chloroethane, the half-mustard S-(2-chloroethyl)glutathione and its episulfonium
ion, provides a theoretical basis for the covalent binding observed with EDC to
cellular macromolecules such as protein and DNA with consequent possibility of
damage to cellular integrity and genetic apparatus. Such covalent binding reac-
tions are known to be related to teratogenesis, mutagenesis and carcinogenesis,
although chemical reactivity alone is not evidence of genetic damage (Lutz,
1979). The amount of covalent binding, and hence damage potential, is related to
the pharmacokinetics of EDC exposure. At least in the rat, there is ample
evidence from pharmacokinetic studies of EDC that the metabolic capacity for EDC
metabolism is saturable (Sections 9.1.2 and 9.1-3). It may therefore be specu-
lated that the organism when confronted with low quantities of EDC utilizes
glutathione conjugation detoxification reactions (Figure 9-6) and
nonelectrophilic metabolites are primarily produced, but when confronted with
higher EDC doses, increasing amounts of electrophilic metabolites are produced
to a saturating and limiting rate for their enzymatic pathways (Figures 9-6 and
9-7). Reitz et al. (1980, 1982) determined total covalent binding and binding
9-50
-------
to DNA in rats exposed to 1l|C-EDC by the oral (150 mg/kg) and inhalation (150
ppm, 6 hour) routes. The results of their study with these presumably saturating
doses are given in Table 9-16. These results show no striking difference between
the two routes of exposure in the distribution of covalent binding and DNA
binding among the various organ tissues, although the tissue levels of covalent
binding are generally higher after inhalation exposure for total binding and
after gavage exposure for DNA binding. The exposure doses used in these studies
of Reitz et al. are comparable to the oral doses of EDC use in the lifetime NCI
carcinogenicity study (NCI, 1978) and to the inhalation doses of the
carcinogenicity studies in rats of Maltoni et al. (1980). No excess tumors were
reported by Maltoni et al., while liver and forestomach were sites of malignant
tumors in the NCI study. Recently, Storer et al. (1982) determined DNA damage
produced in vivo in the livers of male B6C3F1 mice following single oral doses of
EDC (100 mg/kg). DNA damage was evaluated by sedimentation of liver nuclei in
alkaline sucrose density centrifugation, and by DNA recovery, which measure
single strand breaks in DNA or alkaline label sites. DNA from EDC-treated mice
isolated U hours after dosing sedimented more slowly and the total DNA recovery
was decreased 16£ from untreated mice. However, dimethyl nitrosamine, used as a
positive control, decreased recovery of DNA 61$.
In contrast to the low covalent binding potential of EDC in. vivo, inhalation
exposure of rats to only 20 ppm, 1,2-dibromoethane (EDB; 7 hours/day, 5 days/week
for 18 months) produced tumors in the liver and kidney, sites of covalent binding
(Hill et al., 1978). Furthermore, Plotnick et al. (1980) found that the carcino-
genicity of EDB was enhanced by the dietary addition of disulfiram (0.055S by
weight; an inhibitor of aldehyde dehydrogenase) which blocks the further oxida-
tion of broraoacetaldehyde formed in the metabolism of EDB (Figure 9-5) and which
presumably results in increased tissue levels of bromoacetaldehyde with an
9-51
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TABLE 9-16
Total Macromolecular Binding and DNA Binding in Selected Tissues
of Rats After Exposure to C-EDC by Oral or Inhalation Routes
Nanomole equivalents EDC/g tissue
oral inhalation
(150 mg/kg) (150 ppm 6 hr)
Total Binding (n=4)
Liverb 175 + 24 268 + 45
Kidney 183 + 25 263 + 48
Spleen 65+21 130 » 22
Lung 106+34 147+16
ForestomaGh 160+19 71 ± 19
Stomach 90+2 156+29
DNA Binding (n=3)
Experiment 1
Liver"
Spleen
Kidney
Stomach
Experiment 2
Liver
Spleen
Kidney
Stomach
21.3 + 7.4
5.8 + 0.7
17-4 + 2.3
14.9
13.9 + 2.1
2.5 + 0.3
14.5 + 6.2
6.7
8.2 + 3-3
1.8 + 0.3
5.2 + 3.7
2.8
3.3 + 1.2
1.8 + 0.5
2.0 + 0.3
1.9
Results are reported as nanomole equivalents of EDC/g tissue + S.D.
Animals were sacrificed U hr after oral dosing or* immediately following a 6-hr
inhalation exposure.
Source: Reitz et al., 1982
Site where malignant tumors were observed in the NCI study (1978) after EDC was
given by gavage, also forestomach. No excess tumors were reported in the inhala-
tion carcinogenicity bioassay (Maltoni et al., 1980).
9-52
-------
increase of covalent binding. Plotnick et al. (1980) showed that dietary disul-
14
firam markedly increased covalent binding of C-EDB in the nuclei of liver cells
in rats dosed with EDB (Table 9-17).
Covalent binding of metabolites from both oxidative rnicrosomal and cyto-
solic metabolism of EDC has been demonstrated in vitro by several investigative
groups. Van Duuren and his colleagues (Banerjee and Van Duuren, 1979; Banerjee
and Van Duuren, 1978, Banerjee et al., 1979, 1980) observed microsomal-activated
covalent binding of both EDC and EDB to native DMA from salmon sperm, and to
liver and lung microsomal protein from rats and mice. Binding to DMA did not
occur in the absence of raicrosomes or in the presence of denatured microsomes.
Cytosolic metabolism produced insignificant metabolic activation and binding.
The microsomal-activated binding was enhanced by phenobarbital and 3-
methylcholanthrene pretreatment, whereas the addition of glutathione to the
microsomal reaction reduced the binding. Similar results were obtained by Sipes
and Gandolfi (1980). Van Duuren and his co-workers suggested that EDC and EDB
were biotransformed by microsomal oxidative reactions to the reactive transient
metabolites, 2-haloacetaldehyde, 2-haloethanol or haloethylene oxide, all of
which are electrophilic in nature. Banerjee et al. (1979) demonstrated that both
2-bromoacetaldehyde and 2-bromoethanol bind covalently to protein and DNA
14
without metabolic activation. Guengerich et al. (1980) have found that C-EDC
is metabolized by both microsomes and cytosolic systems to metabolites that
covalently bind to protein and calf thymus DNA. Cytosolic metabolism depended
upon the presence of glutathione and involved glutathione transferases (Figure
9-7), although glutathione inhibited microsomal-activated binding to protein but
stimulated binding to DNA. These investigators also produced evidence that 2-
chloroacetaldehyde, S-(2-chloroethyl)glutathione, and their putative 1-
chloroso-2-chloroethane are the metabolites involved in the covalent binding.
9-53
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TABLE 9-17
14
Effect of Dietary Disulfiram Upon the C Content of Liver
Nuclei Isolated 24 or 48 Hours After Administration of a
Single Oral Dose of 15 rag/kg [U-1 C]EDBa
Time Interval Control Disulfiram
24 hours 687 ± 82b 1773 ± 314
48 hours 460 + 42 1534 + 197
aSource: Plotnick et al., 1980
bResults are expressed as dpra/pellet (mean + S.E.M.) of duplicate determinations
on 6 animals per group at 24-hr and 5 animals per group at 48 hr.
9-54
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9.1.1. Summary and Conclusions. At ambient temperatures, EDC is a volatile
liquid with appreciable solubility in water, and hence the principal routes of
entry to the body are by pulmonary and oral absorption. The pharmacokinetics of
absorption and excretion of EDC have been studied extensively in the mouse and
rat. Absorption from the gastrointestinal tract is rapid and complete, occurring
by first order passive processes with a half-time of <6 rain. A dose-dependent
first-pass effect with pulmonary elimination of unchanged EDC occurs with oral
ingestion, thus decreasing the amount reaching the systemic circulation. At the
low concentration of EDC existent in food and water, nearly complete extraction
by the liver is expected. With inhalation exposure, a blood/air partition
coefficient of =20 at 37°C has been observed. Tissue distribution of EDC is
consistent with its lipophilic nature. The chemical crosses the blood brain and
placental barriers and distributes into breast milk. The adipose tissue/blood
partition coefficient varies from 7-57 depending on dose.
Excretion of unmetabolized EDC is almost exclusively via the lungs;
however, metabolism and excretion of the metabolites by other routes is extensive
and dose related. In the mouse and rat, after both oral and inhalation exposure,
the half-life of EDC in blood increases with dose, although it is <60 rain at high
doses. The parameters of total body elimination are compatible with a 2-corapart-
ment system and Michaelis-Menten kinetics. The short half-life of EDC suggests
that the risk of bioaccumulation from intermittent multiple oral and inhalation
exposures is small. Daily oral dosing of rats does not result in significant
bioaccumulation in blood or other tissues.
Pharmacokinetic parameters on multiple doses of EDC are very limited.
Ill
Treatment of animals with EDC prior to C-EDC may alter the detoxification
mechanisms, thus leading to enhanced toxicity and carcinogenicity.
Biotrans format ion of EDC has been shown to occur by multiple pathways, in both
9-55
-------
liver microsomal and cytosol fractions. In the mouse and rat, up to 90% of low
oral or inhalation doses (<50 mg/kg) are metabolized, with a decreasing
percentage of the dose metabolized as the dose approaches and exceeds saturation
of metabolic capacity. Metabolism produced 2-chloroacetaldehyde, S-(2-chloro-
ethyDglutathione (a half sulfur mustard) and other putative reactive metabo-
lites capable of covalent binding to cellular macromolecules, as well as nonreac-
tive glutathione conjugates from "detoxification reactions." The intensity of
covalent binding of reaction metabolites to proteins, lipids and DNA, however, is
considerably less than that observed with the carcinogenic bromine analog and
other known carcinogens.
Exposure to EDC decreases liver levels of reduced glutathione in a dose-
dependent manner. Glutathione plays an important role through glutathione
conjugation detoxification mechanisms in protecting against binding and cellular
toxicity. Covalent binding, hepatotoxicity and mortality of experimental
animals are all reduced by administration of sulfhydryl amino acids or gluta-
thione. Conversely, toxicity is enhanced by low liver and tissue concentrations
of glutathione. Phenobarbital and other inducers of P-450 metabolism increase
the metabolism of EDC, the toxicity and covalent binding. Fasting reduces gluta-
thione tissue content and increases both the metabolism and toxicity of EDC.
Ethanol, acutely or chronically administered, may either enhance or inhibit EDC
metabolism and toxicity depending on the tissue alcohol concentration.
9-56
-------
9.2. ACUTE, SUBCHRONIC AND CHRONIC TOXICITY
9.2.1. Effects in Humans. The preponderance of reports on the toxicity of EDC
to humans was published in the foreign literature (primarily German, Russian, and
Polish). Many of these reports involved exposure to high levels of compound,
involved single cases or small numbers of individuals, were anecdotal in nature
and discussed observations that related to the overall toxicity of this chemical
in man, and/or provided little detailed correlation between toxic effects and the
amount or duration of exposure. Since detailed reviews of the foreign studies
were available from NIOSH (1976) and the U.S. EPA (1979), these sources were used
in part as a basis for the following discussion of human health effects. Trans-
lations were obtained and evaluated independently for all foreign studies that
provided dose-response data of relevance to human risk assessment. Reports in
which exposures were not primarily to EDC (i.e., most reports of mixed solvent
exposures) were not discussed.
9.2.1.1. ACUTE EXPOSURES
9.2.1.1.1. Case Reports and Surveys
9.2.1.1.1.1. Oral Exposure. Information on the effects of ingested EDC
is available solely from clinical case reports and surveys involving accidental
and intentional exposures. Many of the cases involved fatalities and described
symptoms and signs of poisoning that preceded death. The progression of
signs/symptoms and the outcome/findings of cases in which the quantity of EDC
ingested leading to death was reported are summarized in Table 9-18. Ingestion
of quantities of EDC estimated to range from 8 to 200 mH ( = 143-3571 mg/kg,
assuming 70 kg body weight) were reported as lethal to adult males. Following
ingestion there was often a latent period, generally 30 minutes to 3 hours, prior
to the onset of symptoms. Symptoms were indicative of CNS effects and gastro-
9-57
-------
4 males/20 to 29 years
3 males/19 to 27 years
vC
00
a
Male/NS
Hale/63 years
TABLE 9-18
Effects Associated with Acute Lethal Oral Doses of Ethylene Dlchlorlde In Humans
Patient8
Sex/Age
Amount
Ingested
Symptoms and Signs Outcome and Findings
Reference
150 to 200 mi
(»188 to 250.6 g)
70, 80 and 100 ml
(=67.7, 100.2 and
125.3 g)
=82 ml ("102.7 g)
mixed with coffee
and beer
2 oz. (»75.2 g)
mixed with orange
Juice and ginger ale
Symptoms were not specified,
but appeared after 3 to 1 hours.
Onset of symptoms within a
few minutes: vomiting;
weakness; dizziness; lost
consciousness
Intoxication; vomiting;
diarrhea; unconsciousness;
dypsnea
Onset of symptoms after 2 hours:
nausea; faintness; vomiting.
Subsequently, cyanosis; dilated
pupils; coarse rales; weak
and rapid pulse; diarrhea;
increased cyanosis; absence
of pulse and heart sounds;
dypsnea
Deaths after 10, 15, 33 and Bryzhin, 1915
35 hours. Punctuate
hemorrhaging In the epi-
cardlum, pleura, and mucous
membranes of the stomach and
duodenum; varying degrees of
liver damage with focal
hemorrhaging in one case; yellow-
white flbrinous bundles of blood
In the heart cavities and lesser
circulatory vessels; hemolytic
jaundice of the endocardium,
aortal Intima, and dura mater;
evidence of decomposition of
circulating erythrocytes
Deaths after 5 to 8 hours Kalra, 1966
Hyperemla and hemorrhagic
lesions (see text);
evidence of overt bleeding
into the visceral organs
Death after 6 hours
Hyperemla and hemorrhagic
lesions (see text)
Death after 22 hours
attributed to circulatory
failure. Extensive
hemorrhagic colitis; nephrosls
with calcifications of the
tubular epithelium and tubular
and vascular elastic membranes;
fatty degeneration of the liver
spleen and lungs; multiple
perivascular hemorrhages of
the brain
Noetzel, 1944
Hueper and Smith,
1935
-------
TABLE 9-18 (cont.)
Patient8
Sex/Age
Amount
Ingested
Symptoms and Signs
Outcome and Findings
Reference
Male/16 years
Male/80 years
Male/18 years
Male/57 years
50 ml
(=62.7 g)
50 mi
(=62.7 g)
50 ml
(=62.7 g)
10 mSL
(= 50.1 g)
Vomiting; epigastric pain;
muscle spasms, hiccups, rapid
pulse and lack of eyelid
response to light on the 4th
day
Elevated serum enzymes: LDH;
SCOT; SGPT; alkaline phosphatase
glutamlc dehydrogenase; RNAase.
Onset of symptoms after 1 hour:
somnolence and cyanosis.
Diarrhea after 4 hours.
Impaired blood coagulation
after 5.5 hours: Increased
prothrombln time; decrease In
clotting factors II and V;
thrombocytopenla; no Increase
In fibrlnolysls
Somnolence; vomiting; sinus
tachycardia; ventricular
extrasystoles; regained
consciousness after 14 hours;
dypsnea; loss of blood
pressure; cardiac arrest.
Impaired blood coagulation
observed after 24 hours;
prolonged bleeding from
venlpunctures; reduction in
activity of clotting factors
II, V, VII and VIII; complete
defibrinatlon; thrombo-
cy topenla; Increased thrombin
time
Death after 91 hours
Death within a few hours
Death after 17 hours
attributed to circulatory
shock. Intravascular
thrombosis were not found.
Roubal, 1947
Secchl et al., 1968°
Schonborn et al.,
1970°
Death after 24 hours.
Thrombi In the pulmonary
arterloles and capillaries;
hemorrhages into the mucosa
of the esophagus, stump of the
stomach, rectum and in the
subeplcardial, subendocardlal
and myocardial tissues. The
coagulation disorder with
thrombocytopenla attributed
to disseminated intravascular
coagulopathy and hyper-
flbrlnolysis
Martin et al., 1969
-------
TABLE 9-18 (cont.)
Patient3
Sex/Age
Hale/30 years
Amount
Ingested
40 ml
(» 50. < g)
Symptoms and Signs
Slight cough; reddened
conjunctlvae; shock; weak,
Outcome and Findings
Death after 28 hours
Hyperemia and hemorrhagic
Reference
Garrison and
Leadingham, 1954°
Hale/50 years
Hale/55 years
(asthmatic)
Hale/NS
Hale/NS
=•30 ml
(=37.6 g)
20 ml
(=25.1 g)
=20 ml
(=25.1 g)
920 ml
(=25.1 g)
rapid pulse. Regained
consciousness after
3 hours; hyperactlvlty
alternated with
semlconatose condition
Onset of symptoms after
30 minutes: unconsciousness;
vomiting; cyanosis;
dilated and fixed pupils;
pulmonary edema; extreme
dypsnea
Epigastric pain; extreme
dizziness; sleeplessness;
vomiting; slow pulse
Onset of symptoms after 1 hour:
repeated vomiting. Cyanosis
and dypsnea after 12 hours
Symptoms not reported
lesions (see text); evidence
of overt bleeding into the
visceral organs
Death after 10 hours
Diffuse hemorrhagic
gastritis; bilateral
pulmonary congestion;
acute necrotizlng
bronohlolltls and
bronchitis; acute toxic
nephrosis; diffuse hepatic
necrosis; hyperemla of the
brain with scattered perl-
vascular hemorrhages In
the pons
Death after 24 hours
Death after 13 hours
Death within 12 hours
Lochhead and Close,
1951
Roubal, 1947b
Flowtow, 1952"
Flowtow, 1952
-------
TABLE 9-18 (cont.)
Patient"
Sex/Age
Amount
Ingested
Symptoms and Signs
Outcome and Findings
Reference
Male/11 years
15 mH
(» 18.8 g)
vO
Male/32 years
8 ml
(=00 g)
Male/32 years
glass
Onset of symptoms after 2 hours:
Progressive appearance of
severe headache; staggering;
lethargy; periodic vomiting;
decreased blood pressure;
ollgurla; dypsnea; somnolence;
Increased dypanea; and ollgurla,
hemorrhaglc nasogastrlc aspirate;
ecohymoses; sinus tachycardia;
cardiac arrest, pulmonary
edema; refractory hypotension.
Hypoglycemla on day 2 and
hypercalcemla on day 4.
Progressive decrease In blood
coagulation ability: increased
prothrombln time; all clotting
factors except VIII were
markedly decreased on day 4.
Death after 6 days
Extensive mid-zonal liver
necrosis; renal tubular
necrosis; focal adrenal
degeneration and necrosis
Yodaiken and
Babcock, 1973
Burning sensation in mouth,
throat and stomach; drank milk
and vomited; weakness; speech
retardation; lethargy; asthenia;
cold sweat; muffled heart sound;
weak and rapid pulse. 22 hours
after ingestion: excitation;
restlessness; delirium; flushed
face; systolic murmur; respiratory
depression; circulatory weakness;
anuria
Vomiting, weakness, stomach
pains. On 3rd day: rest-
lessness, coated and dry
tongue; bloody diarrhea;
abdomen painful on palpitation;
enlarged liver; dry, moist rales;
rapid, weak pulse; anuria;
increasing cyanosis; un-
consciousness
Death after 56 hours
Bogoyavlenski et al.,
19686
Death on the 3rd day
Hyperemia and hemorrhagio
lesions (see text)
Agranovlch, 19*18
-------
TABLE 9-18 (cont.)
Patient3
Sex/Age
Amount
Ingested
Symptoms and Signs
Outcome and Findings
Reference
Hale/27 years
half glass
Hale/43 years
(alcoholic)
4 drinks diluted
with orange Juice0
o
i
ro
Hale/43 years
(alcoholic)
Male/NS
4 drinks diluted
with orange juice0
several mouthfuls
Onset of symptoms after
2.5 hours: Unconsciousness
that was regained after
12 hours; vomiting
(dark vomitus); burning
sensation in the digestive
tract; dypanea; nausea;
cyanosis; depressed
respiratory rate; moist
rales; muffled heart sounds;
rapid pulse; extrasystoles;
anurla
Unconsciousness
Death after 19 hours
Hyperemia and henorrhagic
lesions (see text); evidence
of overt bleeding into the
visceral organs
Confusion; deep sleepiness;
unconsciousness; vomiting
with blood
Onset of symptoms after
30 min: vertigo; nausea;
vomiting; slightly muffled
heart sound; tachycardia.
After 17.5 hours: headache;
substernal pain; cyanosis;
rapid, weak pulse; dry rales;
decreased blood pressure;
vomiting with bile; diarrhea;
subconjunctliral hemorrhage;
ollgurla; paranephric hemorrhage,
5th day pulmonary edema and
unconsciousness
Bogoyavlenskl et al.,
1968
Death after 8 hours
Hyperemia and hemorrhagic
lesions (see text);
evidence of overt bleeding
into the visceral organs
and lungs. Death after
24 hours. Hyperemia and
hemorrhagic lesions (see
text); evidence of overt
bleeding into the visceral
organs and lungs
Death after 24 hours
Hyperemia and hemorrhagic
lesions (see text); evidence
of overt bleeding into the
visceral organs and lungs
Death on the 5th day
Hulst et al., 1946
Hulst et al., 1946
Horozov, 195B
-------
7ABLB 9-18 (cont.)
Patient8
Sex/Age
Amount
Ingested
Syoptons and Signs
Outcome and Findings
Reference
Hale/63 years
1 or 2 sips
Male/1 1/2 years
Hale/79 years
Hale/2 years
Hale/23 years
1 alp
1 sip
1 alp
1 alp
Unconsciousness shortly after
Ingestion, but soon
regained; strong vomiting;
period of improvement;
unconsciousness again
after 10.5 hours; falling
blood pressure
Extreme weakness; comatose;
vomiting
Vomiting; weakness; pale;
cyanosis; scarcely conscious;
vagueness; rapid, regular
pulse; blood pressure not
measurable
Vomiting; diarrhea; tonic
spasms; increasing loss of
consciousness; dypsnea;
impaired circulation
Onset of symptoms after 1 hour:
dizziness; nausea; unconscious-
ness; vomiting; cyanosis; no
pupil reaction; no corneal
reflex; dypsnea; strong motor
unrest
Death after 14 hours
attributed to circulatory
failure. Hyperenia and
hemorrhagic lesions (see t«xt)
Death the next day
Hyperemia and heoorrhaglc
lesions (see text)
Death after 10 hours
attributed to heart and
circulatory failure.
Hyperenlc and hemorrhagic
lesions (see text)
Death after 20 hours
Hyperemia and hemorrhagic
lesions (see text)
Death after 8 hours
attributed to respiratory
and circulatory failure
Hyperemia and heoorrhaglc
lesions (see text); evidence
of overt bleeding Into the
visceral organs
Freundt et al.. 1963
Keyzer,
Weisa, 1957
Relnfried, 1958°
Relnfrled, 1954
"NS s Hot stated
bData compiled from NIOSH, 1976
cPresumably EDC was mixed instead of alcohol
-------
intestinal disturbances, and frequently included dizziness, nausea, headache,
periodic vomiting, diarrhea, epigastric pain or tenderness, dilated pupils and
lack of corneal reflex, rapid and weak pulse rates, progressive cyanosis, dyspnea
and unconsciousness. These symptoms of toxicity are characteristic of those
produced by many of the chlorinated aliphatic hydrocarbons. Somnolence,
oliguria and anuria, muffled heart sounds and motor unrest have also been
described in a number of cases.
The results of several health surveys of large numbers of orally exposed
individuals have recently been reported by Russian investigators, and summarized
by PEDCo Environmental, Inc. (1979) (Akimov et al., 1976, 1978; Shchepotin and
Bondarenko, 1978; Bonitenko etal., 1974, 1977; Luzhnikov et al., 197**;
Luzhnikov and Savina, 1976) and Chemical Abstracts (Andriukin, 1979).
Information regarding the design of these surveys is not available, and dosage
information is either not available or not adequately reported (i.e., not
correlated with effects) (Table 9-19). The clinical symptoms and signs of
exposure recorded in the surveys are consistent with those described in single
case reports (see Table 9-18) (i.e., neurological effects and evidence of liver
and kidney dysfunction), but there appears to be a more frequent indication of
cardiovascular insufficiency. The range of doses that elicited effects in the
surveys (=12.5 to 250.6 g) also appear to be consistent with the single case
reports (see Table 9-18), but incidences of fatalities were not presented in the
available summaries. Although none of the effects reported in the surveys were
correlated with specific doses, several of the studies indicated that the
severity of poisoning was related to blood levels of EDC (Bonitenko, 197U, 1977;
Luzhnikov et al., 1971*, 1976).
The clinical syndrome of oral EDC poisoning in children is similar to that
seen in adults, but the lethal dose range is somewhat lower (0.3 to 0.9 g/kg)
(Hinkel, 1965). This range of doses did not always cause death, and is derived
9-61
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TABLE 9-19
Effects of Acute Oral Ingestlon of 1,2-Dichloroethane (Survey Results)
Route No. of Subjects
Dose
Principal Findings
Reference
Oral
121
20 to 200 ml
(=25 to 250.6 g)
Oral
Inhalation
211
37
NR
NR x 20 to 30 minutes
a\
MRa
110
NR
Mild to severe poisoning characterized by
pronounced DCE odor on breath (911), dry akin
(759), hypotension (71J), extreme pupil dilation
(72*), mucosal cyanosis (67*), tachycardia (62*),
respiratory difficulty (99%)i muscular
hypotonla (46*), decrease in tendon reflexes (46»,
loss of consciousness (42*). Neurological syn-
dromes noted in 118 aubjecta: comatose (42J),
atactic (42*), asthenlo with autononio vascular
insufficiency (27*), extrapyranidal (23*),
convulsive (7.If), psychotic (51). Lethality
not reported.
Aggregate findings for 218 total patients reported.
Neurological disorders (incl. unconsciousness,
respiratory depression) 000*), cardiovascular
insufficiency (incl., arrhythmias, hypotension,
reduced cardiac output, decreased peripheral
resistance) (60», liver dysfunction (inol.,
enlargement, hyperbilirubinemia, increased
serum albumin and asparagine transaminase (35*).
Nephropathology (ollgurla, proteinurla, azotemia,
acute renal failure with disturbed acid/base
balance) particularly associated with inhalation
exposures. Gastroenteritis milder in subjects
exposed via inhalation. No correlation between
severity of poisoning and the blood or urine
concentration or the amount inhaled.
Acute gastritis with vomiting (77», neurological
disorders including coma (81*), acute cardio-
vascular insufficiency (57$), hepatitis (56<)
with liver enlargement and dysfunction
(abnormal bromoaulfaleln, plasma billrubln,
plasma glutamlne - aparagine transaminase).
Clinical symptoms of poisoning were observed
at blood concentrations of 0.5 mg J, and coma
developed at 5 to 7 mg J.
Aklmov et al.,
1976, 19T8
Schepotln and
Bondarenko, 1978
Luzhnikov et al.,
1976, 1978
-------
TABLE 9-19 (cont.)
Route No. of Subjects
Dose
Principal Findings
Reference
NSa
160B
NA
o
i
Oral
Inhalation
2H
3
10 to 100 ai (=<12.5 to 125 g)
NR
Oral
32
NR
Hemodynamlc shock due to myocardlal dysfunction.
Characterized by compensatory (increased
peripheral resistance, normal or slightly
Increased arterial blood pressure, decreased
cardiac output and blood volume, decreased
cardiac isometric contraction, increased
ventricular expulsion time, asynchronous
contractions) and deoompensatory (pronounced
and progressive hypotension, decreased cardiac
output, normal or slightly decreased peripheral
resistance, decreased myocardlal contractile
force during ventricular systole, prolonged
periods of asynchronous contractions) phases.
EKG changes including arrhythmias observed in
both compensated and decompensated shock.
Pathologic changes in the myocardium charac-
terized by capillary endothellal cell edema,
capillary lumen stenosis, edema of the
myooardlal Interstices with leukocyte
accumulation and miorofocal hemorrhages,
decreased glycogen, degenerative changes, and
decreased mitoohondrlal enzyme activities.
17 patients exhibited manifestations of
light/mild poisoning: headache, vertigo,
abdominal pain, nausea, vomiting, and signs of
cardiac Insufficiency (increased contraction
rate, decreased minute volume, decreased
circulating blood volume, decreased cardiac Index);
5 of 17 had hepatomegaly, Icteric skin, solera,
and Increased bilirubin levels. The other 10
patients were comatose and had more pronounced
symptoms of cardiac Insufficiency.
Gastroenteritis and protelnurla. Elevated
leukocyte count and elevated alanine/aapartate
amlnotransferase activities correlated with
severity of poisoning. Coma was associated with
blood concentrations of 15 to 30 mg %, and
consciousness returned at levels below 8 to
10 mg ). Adipose tissue and blood levels of
68 and 1.2 mg t, respectively, at autopsy.
Luzhnlkov et al.,
1576, 1978
Andrlukin, 1979
Bonitenko et al.,
1971, 1977
-------
TABLE 9-19 (cent.)
Route
No. of Subjects
Dose
Principal Findings
Reference
Oral0
7 children
(1 to 6 years)
Clinical syndrome similar to that In adults:
severe and persistent vomiting (within 1 hour),
and subsequent (Immediate to 10 to 12 hours)
narcotic effects (e.g., somnolence, motor unrest,
reflex Increases, convulsions or coma). Less
frequent Indications of circulatory failure,
kidney and liver functional disturbances, and
tachycardia. No remarkable hematologic changes.
Typical pathological anatomical findings.
U>20 reportedly ranged from 0.3 to 0.9 8/kg.
Hlnkel, 1965
aRoute is probably oral, but not specifically stated In the EPA (1979) summary of this study.
bThe EPA summary of this study stated that "at least" 160 patients were studied.
°Exposure to a "nerve balsam" medicine that was 75% 1,2-dlchlorethane (other components not stated in EPA summary).
vO
en
-------
from a review of seven cases of accidental poisoning in children that ranged in
age from 1 to 6 years.
Foreign reports of individual non-fatal cases of EDC ingestion (lenistea
and Mezincesco, 1943; Bloch, 1916; Stuhlert, 1949; Flowtow, 1952; Kaira, 1966;
Rohmann et al., 1969; Gikalov et al., 1969; Pavlova et al., 1965; Agranovich,
1948) were in most instances not detailed in the NIOSH (1976) or U.S. EPA (1979)
reviews, presumably because the effects were similar to those that preceded death
with lethal exposures, and because the quantities ingested were not known. Bloch
(1946) did note bloody diarrhea and evidence of adverse liver (enlargement,
slightly increased serum bilirubin, urobilinogen, abnormal galactose and alcohol
load test results) and kidney (oliguria, albumin and casts in the urine,
temporary retention of nitrogenous substances) effects in one man who claimed to
have swallowed "only a small amount" of EDC.
Death was usually ascribed to circulatory and respiratory failure, and
autopsies revealed tissue congestion, cellular degeneration, necrosis and
hemorrhagic lesions of most organs, including the stomach, intestines, liver,
kidneys, spleen, heart, lungs, respiratory tract and brain (see Table 9-18).
There was occasionally evidence of gross bleeding into the visceral organs or
lungs. Several of the more recent reports (Martin et al., 1969; Schonborn
et al., 1970; Yodaiken and Babcock, 1973) indicated that the hemorrhagic effects
may have been exacerbated by a condition known as disseminated intravascular
coagulation (DIG). DIG is characterized by a depletion (consumption) of clotting
factors, hypofibrinogenemia and thrombocytopenia, and the resultant effect is a
severe bleeding tendency.
The results of the studies by Luzhnikov and coworkers (1976, 1978) suggest
that EDC can exert a direct toxic effect on the myocardium (see Table 9-19).
Pathologic examinations of EDC-exposed individuals (amount ingested and number
9-68
-------
examined not available) revealed significant edema in the capillary endothelial
cells and stenosis of the capillary lumens, pronounced edema of the myocardial
interstices with accumulation of polymorphonuclear leukocytes, microfocal hemor-
rhages, diminished glycogen content, and degenerative changes. Mitochondrial
damage was indicated by a decrease in enzyme activities. Myocardial functional
changes (e.g., decreased contractile force, asynchronous contractions) were also
observed prior to death. The cardiotoxic effect of EDC apparently precipitated a
clinical state of shock; hemodynamic disorders commonly observed in at least
160 patients included increased peripheral resistance, decreased cardiac output,
hypotension and EKG changes including arrhythmias.
9.2.1.1.1.2. Inhalation Exposure. Reports of numerous cases of acute
occupational exposure to EDC vapor have been published (NIOSH, 1976). These
acute exposures have involved both fatal (27 cases) (Wendel, 1948; Brass, 1949;
Hadengue and Martin, 1953; Ollivier et al., 1954; Domenici, 1955; Salvini and
Mazzuchelli, 1958; Guarino and Lioia, 1958; Troisi and Cavallazzi, 1961) and non-
fatal (57 cases) outcomes (Wirtschafter and Schwartz, 1939; Jordi, 1944;
Agronovich, 1918; Baader, 1950; Paparopoli and Cali, 1956; Menschick, 1957;
Smirnova and Granik, 1970), but none of the reports provided exposure concentra-
tions. Furthermore, although EDC was usually reported to be the primary vapor to
which the workers were exposed, the preponderance of exposures were poorly
characterized mixtures of EDC and other solvents, or EDC of unknown purity. The
occupational exposures were generally associated with maintenance and cleaning
operations or the use of EDC as a paint thinner, but many (39 cases) involved
exposure to Granosan, a fumigant composed of 30% carbon tetrachloride and 70% EDC
(Domenici, 1955? Salvini and Mazzucchelli, 1958; Guarino and Lioia, 1958;
Paparopoli and Cali, 1956).
9-69
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The effects associated with the above cited fatal and non-fatal acute
inhalation exposures were very similar to those found after ingestion (NIOSH,
1976). In general, the initial symptoms appeared to be consistent with both CNS
effects (headache, dizziness, lethargy, feelings of drunkenness, unconscious-
ness) and gastrointestional disturbances (nausea, vomiting, diarrhea), which are
characteristic of chlorinated aliphatic hydrocarbon toxicity. In some cases
workers were asymptomatic and were not overcome during exposure, but later became
unconscious. Other signs and symptoms of inhalation exposure commonly included
cyanosis, epigastric tenderness and/or pain, hepatomegaly, and jaundice.
Laboratory studies were consistent with these observations and indicative of
hepatic dysfunction (increased serum bilirubin and urobilinogen, hypoglycemia,
abnormal function tests) and renal disturbances (oliguria, anuria, abnormal
function tests). In addition, conjunctivitis, respiratory tract irritation and
inflammation, rales and leukocytosis were occasionally associated with the acute
vapor exposures.
Death from acute inhalation exposure was generally attributed to respira-
tory and circulatory failure (NIOSH, 1976), although it is not certain if EDC
exerted a direct toxic effect on the cardiopulmonary system or precipitated a
shock-like state that could have elicited many of the changes observed at
autopsy. Autopsies frequently revealed pulmonary edema, congestion of internal
organs (liver, kidneys, spleen, lungs, brain) and cellular degeneration and
necrosis (liver and kidneys). Hyperemia and hemorrhagic lesions were also
observed in the kidneys, brain, respiratory tract, lungs, and heart.
9.2.1.1.1.3. Dermal Exposure. Several reports described instances (a
total of 6 cases) in which skin contact with EDC occurred concurrently with
inhalation exposures (Wirtschafter and Schwartz, 1939; Anonymous, 1916;
Rosenbaum, 1917; Hadengue and Martin, 1953)* Severe dermatitis was a commonly
9-70
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reported result of the dermal exposures, as is the case with many chlorinated
hydrocarbons which produce defatting of the skin. Several of these reports also
suggest that dermal absorption may significantly contribute to total exposure
(characteristic symptoms of intoxication were noted in workers who were wearing
EDC-soaked clothing).
9.2.1.1.2. Experimental Investigations.
9.2.1.1.2.1. Physiologic Effects. Borisova (1957) examined eye sensi-
tivity to light and plethysmographic and spirographic responses in a small number
of subjects (3 to 4) who were experimentally exposed to concentrations of EDC
that ranged from =1 to 12.4 ppm (4 to 50 mg/m3). This range of concentrations
represented subthreshold, threshold and above threshold odor perception concen-
trations (Section 7.1.2.2). The concentration of EDC in air was determined
nephelometrically; the method is sensitive enough (0.001 mg/sample reported) to
monitor EDC at levels lower than the lowest concentration tested (4 mg/nr).
In the light sensitivity experiment, baseline perception thresholds were
determined prior to the exposures for each of 3 subjects (17 to 24 years old),
and the subjects inhaled EDC vapor for 15 minutes prior to testing (Borisova,
1957). When tested after 40 minutes of dark adaptation, the threshold (inten-
sity) at which light was perceived was found to be higher during exposure to EDC,
and the threshold appeared to increase with increasing concentrations of EDC
(Table 9-20). No change in the light sensitivity of eyes was observed in any of
the subjects at 1 ppm, and the lowering of eye sensitivity to light was most
marked at 12.4 ppm.
The effect of low concentrations of EDC on blood volume (vascular con-
striction) and pulse fluctuations was studied with a finger plethysmograph
(Borisova, 1957). Four subjects were exposed to vapor concentrations of 1, 1.5,
3, 5.7 and 12.4 ppm for 30 seconds or 15 minutes. A 30-second exposure at
9-71
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TABLE 9-20
Effect of Ethylene Dichloride Exposure on Eye Sensitivity to Light6
Concentration
ppm (mg/nr)
1 (4)
1.5 (6)
2.2 (9)
3 (12)
4.3 (17.5)
5.7 (23.2)
6.2 (25)
7.4 (30)
12.4 (50)
Subject 1
no effect
-0.2
-0.25
-0.25
-0.3
-0.45
-0.45
-0.5
-1.2
Perception Threshold
(Units of Optical Density)
Subject 2
no effect
<0.05
-0.3
-0.25
-0.35
-0.4
-0.5
-0.5
-1.2
Subject 3
no effect
no effect
+0.15
no effect
-0.45
-0.25
-0.7
-0.75
-0.6
a
Source: Borisova, 1957
bEye sensitivity to light was determined after 40 minutes of adaptation in the
dark at different EDC concentrations.
9-72
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1.5 ppm resulted in a temporary vasoconstriction in all 4 subjects. Complete
data were not reported, but vasoconstriction appeared to be more pronounced at
3 ppm and higher concentrations. Results of tests in which EDC exposure was
extended to 15 minutes reportedly showed that the above concentrations caused
"characteristic changes" in the plethysmograms of all subjects that included
increased pulse beat, pulse waves, and pulse amplitude. The observations at the
higher concentrations in both the 30-second and 15-rainute exposure experiments
also suggested that the degree of response was proportional to the exposure
concentration. Exposure to concentrations of 1.5, 3, 5.7, and 12.4 ppm for
1 minute produced changes in depth of breathing in 4 subjects (as indicated by
increased amplitude of respiratory waves on spirograms), but concentrations of
1 ppm reportedly had no effect on respiration (Borisova, 1957).
9.2.1.1.2.2. Odor Detection and Recognition Thresholds. Hellman and
Small (1973, 1974} determined an absolute odor threshold of 6.0 ppm and an odor
recognition threshold of 40 ppm for EDC. A trained odor panel was used to
characterize the odor properties of this compound and 101 other petrochemicals,
but the number of panelists was not stated. The absolute odor threshold is the
concentration at which 50$ of the odor panel observed an odor, and the recogni-
tion threshold is the concentration at which 100$ of the panel defined the odor
as representative of EDC. Borisova (1957) determined the odor threshold of EDC
with 20 subjects and a total of 1256 tests. Thirteen subjects perceived EDC
vapor at a concentration of approximately 6 ppm (23.2 to 24.9 mg/m3), 6 subjects
perceived EDC at 4.5 ppm (17.5 mg/m3), and 1 subject detected the compound at
3 ppm (12.2 mg/m3).
When diluted in water,the odor recognition threshold of EDC was determined
to be 29 ppm (v/v water) (Amoore and Venstrom, 1966). An odor panel of 13 men
and 16 women was used for the determination, and the threshold defined by the
9-73
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panelists was the concentration of EDC that yielded 70$ correct response (signi-
ficant at the "\% level) in a paired comparison against pure water as a control.
9.2.1.2. REPEATED AND CHRONIC EXPOSURES.
9*2.1.2.1. Case Reports — Repeated exposure to EDC vapor in the work-
place has resulted in effects that are consistent with those resulting from acute
exposures. Symptoms and signs including anorexia, nausea, vomiting, weakness
and fatigue, nervousness, epigastric pain/discomfort and irritation of the
respiratory tract and eyes were described in numerous case reports of industrial
exposures (McNally and Fostvedt, 194"i Siegel, 1947; Rosenbaum, 1939; tfatrous,
1917; Rejsek and Rejskova, 1947; Delplace et al., 1962; Suveev and Babichenko,
1969). In one study (Suveev and Babichenko, 1969), examination of 12 symptomatic
workers who were brought to a clinic revealed paleness and cold sweat (12 of 12),
bradycardia (9 of 12), systolic murmur (5 of 12), diarrhea (5 of 12; 3 of 12 with
blood) and enlarged livers that were soft and tender to palpitation (9 of 12);
muffled heart sounds, increased rate of respiration, rales, coated and dry
tongues were also observed but incidences of occurrence were not stated. Signs
indicative of nervous system dysfunction have also been reported in cases of
chronically exposed workers; these include nystagmus, fine tongue tremors and
sluggish patellar reflex (MoNally and Fostvedt, 1941), encephalic disorders
(Delplace et al., 1962), and decreased muscle tone, loss of reflexes, a positive
Romberg's sign, and deafness (Suveev and Babichenko, 1969). Complaints of hand
and arm eczema that appeared within the first year of exposure were recorded in
11 of 16 cases by Delplace et al. (1962). It should be noted that exposure
concentrations and information on the types of exposures were not provided in any
of the above cited reports.
Rosenbaum (1947) discussed experiences with cases of EDC intoxication in
Russian industries from 1931 to 1945. He observed over a period of 10 years that
9-74
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characteristic symptoms of acute poisoning could develop with repeated exposure
to concentrations of 75 to 125 ppm in air. Fatalities have resulted when workers
experienced these symptoms 2 or more times in a period of 2 to 3 weeks. There was
no mention, however, of the number of people affected or durations of exposure.
Byers (1943) noted that a "number of persons" exposed to EDC complained of
delayed toxic effects. The workers stated that the worst effects, which varied
from lassitude and malaise to nausea, vomiting and abdominal pain, occurred after
the evening meal. These symptoms were associated with exposure to concentrations
"only slightly in excess of 100 ppm for 7.5 hours daily", and were not completely
alleviated when ventilation procedures reduced the EDC concentration to 70 ppm.
Exposure conditions were described in partial detail for 2 workers who were
chronically exposed to EDC for 7 and 9 months during the manufacture of hexa-
chlorophene (Guerdjikoff, 1955). CDC was used as a catalyst in this process.
During one of the operations, which was repeated for 2 to 3 minutes several times
a day for a total of about 30 minutes, exposures were associated with adding EDC,
trichlorophenol and sulfuric acid to the reaction vat. EDC exposure concentra-
tions were not measured during the filling operations, but the workers wore an
air-supplied respirator and were probably only exposed to EDC occasionally due to
improper fit. During another operation which lasted 10 minutes and was repeated
3 to 4 times a day, the exposure concentration of EDC was about 120 ppra. During a
final operation, the workers were exposed once a day for =10 to 15 minutes when
an EDC pipe was cleaned; these exposure concentrations were not measured but were
considered by Guerdjikoff (1955) to be more than 120 ppm. Both workers exhibited
similar symptoms, typical of EDC exposures, of anorexia, epigastric pain,
fatigue, irritability, and nervousness after about 3 weeks of exposure. Neuro-
logical effects such as hand tremors, hyperhidrosis and difficulty in talking
were eventually experienced.
9-75
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9.2.1.2.2. Health Surveys — Kozik (1957) reported the results of a
health and morbidity survey of Russian aircraft industry workers. All of the
workers in the study group (size not stated) were employed in a shop where glue
that contained EDC as a solvent was used to bond rubber sheets to metal forms in a
soft tank fabrication process. Most of these workers were gluers, but a small
number worked inside the completed tanks to disassemble the forms. EDC was
emitted to the air during application and glue drying.
About 500 atmospheric measurements of EDC were reported to have been taken
(Kozik, 1957). Although the sampling and analytical methods were not mentioned,
NIOSH (1976) felt that the data were presented in sufficient detail to permit
estimations of TWA exposures. NIOSH estimated that M to 16$ of the total
exposure occurred during the gluing operations, when the TWA concentrations were
s28 ppm during application and =1 6 ppm when the glue was drying. When other
operations were performed in the same shop (during the second half of the work-
shift), the EDC TWA concentrations were ^11 ppm. The TWA for the total shift was
estimated to be 15 ppm. Concentrations ranged from approximately U to 50 ppm;
concentrations in excess of 20 ppm were associated only with the gluing and
drying operations and occurred about 15$ of the time. NIOSH (1976) has noted,
however, that the aforementioned TWA concentrations may be an overestimate of
most of the workers' exposures for several reasons. First, the tabulation of
measurements in the glue application category also contained high values (45 to
52 ppm) that were experienced only by an insignificant number of workers who
disassembled the molds within the finished tanks. Second, the measurements were
apparently not breathing zone measurements and third, the ventilation system was
designed with the exhaust ducts on the floor. In light of these considerations,
NIOSH concluded that a more realistic appraisal of the TWA exposure of the
majority of the workers is 10 to 15 ppm.
9-76
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Workers (total number not stated) who were engaged in the production of soft
tanks during the years 195" to 1955 experienced increased morbidity and los1:
workdays when compared *rith workers in the entire factory (Table o_2i). Disease
categories examined included acute gastrointestinal disorders, neuritis and
radiculitis, and other diseases. An in-depth analysis of the morbidity rate with
temporary disability for 195^ to 1955 reportedly showed high rates for gastro-
intestinal diseases, liver and gall bladder diseases, and diseases of the muscle,
tendons and ganglion (Table 9-21). The liver and gall bladder diseases were
considered by Kozik (1957) to be related to a specific toxic effect of EDC (the
dyspeptic symptoms it causes reportedly are often diagnosed as gastritis), but
the diseases of the muscle, tendon and ganglia were associated with the numerous
repetitive motions the workers had to make when applying the glue. Farther
examination of 83 of the gluers revealed diseases of the liver and bile ducts (19
of 83), neurotic conditions (13 of 83), autonomic. dystonia (n of 83), asthenia
conditions (5 of 83), and goiter and hyperthyroidism (10 of 33).
Visual-motor reactions were studied at the beginning and end of 11 workdays
in 1 7 of the gluers and 10 control machinists (Kozik, 1957). It was stated that a
device wag used to determine simple and complex reaction (color differentiation)
times, as well as reaction times in a modification of the complex reaction task;
a total of 3700 reaction tests were conducted, but details of the tests were not
given. A comparison of the mean rates for all three reactions show no signifi-
cant differences in the two groups either before or after work. Nervous system
dysfunction was suggested, however, by the inadequately reported results of the
complicated reaction tests. "Most" of the gluers made errors in the complex
reaction task, while the machinists did not make any errors (additional data are
not available). In the modifier! complex reaction test, errors were committed
9-77
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TABLE 9-21
o
i
Morbidity and Lost Workdays of Aircraft Industry Gluers Exposed to Ethylene Diohloride
(Rates/100 Workers)
Year
1951
1952
1953
1951
1955
Total
Morbidity
Cases
Days
Cases
Days
Cases
Days
Cases
Days
Cases
Days
Plant
120.2
995.8
121.0
960.9
135.6
101(0.8
150.7
"75.9
127.6
978.1
Shop
159.8
1115.5
137.6
996.0
163.9
1236.5
191.8
1563.2
176.6
1162.1
Acute Gastro-
intestinal
Disorders
Plant
5.
19.
1.
15.
11.
15.
5.
19.
1
3
2
1
4
6
3
3
3.6
12.1
Shop
11.6
13.5
5.7
23.1
6.2
19.1
9.6
31.8
5.0
15.3
Neuritis
and
Radiculltis
Plant
5.2
59.9
5.0
11.8
7.5
67.3
7.9
73.8
5.9
51.1
Shop
13.0
127.0
9.7
91.5
16.5
116.0
16.7
182.8
10.3
90.2
Diseases of
the Muscles,
Other Liver and Call Acute Chronic Tendons,
Diseases Bladder Diseases Gastritis Gastritis and Ganglia
Plant
31.1
351.2
31.0
335.2
35.3
338.3
10.8
386.4
37.9
315.7
Shop
13.2
511.8
10.8
378.7
53.5
521.0
63.8 21
596.2 251.5
63.3 21
640.5 290
.
.
.
13 6 US
64.5 45 170
8 3 8
27 14 50
^Source: Kozlk, 1957
The Incidences are reportedly elevated, but reference values were not given.
-------
both before and after work by 15 of 17 gluers; 4 of 10 machinists made errors, but
only at the end of the workday.
Cetnarowicz (1959) investigated the possibility of EDC poisoning in Polish
oil refinery workers. The workers were employed in a mineral oil purification
(dewaxing) process that involved mixing oils with a solvent that contained 80$
EDC and 20% benzene at 40°C; the mixture was subsequently cooled to -24°C, and
precipitated paraffin was separated by centrifugation. Durations of exposure
were not stated, but concentrations of CDC ranged from =10 to 200 ppm (0.04 to
0.8 mg/Jt), with the highest excursions in the centrifuge room (Table 9-22).
Benzene concentrations ranged from =3.1 to 7.8 ppm (0.01 to 0.025 mg/O, but were
not specifically reported for the different work areas detailed in Table 9-22.
Nineteen workers (18 to 48 years old; 18 men, 1 woman) who had been employed for 2
to 8 months were included in the initial study group; 2 men and the woman were not
examined due to medical history complications. The results of these examinations
are summarized in Table 9-23 and in the text below.
Ten of the 16 workers were employed in the centrifuge room, where EDC
concentrations ranged from 62 to 200 ppm. All 10 of these workers complained of
a burning sensation in the eyes and lacrimation, and 6 of the 10 workers stated
that they had experienced dryness of the mouth, an unpleasant sweet aftertaste,
dizziness, fatigue, drowsiness, nausea, occasional vomiting, constipation, and
loss of appetite. Three of these workers also complained of pain in the epigas-
trum. All of the above-mentioned subjective complaints disappeared when the
workers changed workplaces, but reappeared upon reexposure. Of the 6 workers
employed in the pump and recrystallization rooms (10 to 37 ppm), only 1
complained of similar symptoms. As detailed in Table 9-23, clinical evaluation
of the 16 workers indicated the likelihood of liver effects (tenderness to
palpitation with minimal enlargement, epigastric pain, elevated urobilinogen
9-79
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TABLE 9-22
Concentrations of Ethylene Dichloride in Oil Refinery
Mineral Oil Purification Process Air*
Location
Centrifuge room
Pump room 1
Pump room 2
Pump room 2
Crystallization room
(repeated
6U
16
25
30
30
ppm
measurements)
62
10
13
37
37
200
17
-
-
-
•Source: Derived from Cetnarowicz, 1959 by NIOSH, 1976.
9-80
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TABLE 9-23
Effects Observed in Polish Oil Refinery Workers*
Concentration of 1,2-DCE
Effect
62, 61 and 200 ppm
f 10 to 37 ppm
Co
10 to 200 ppm
Dryness of the mouth; unpleasant sweet aftertaste;
dizziness; lassitude; sleepiness; nausea;
vomiting; poor appetite
Burning sensation of the eyes that disappeared with
adaptation to the atmosphere
Epigastrium pain
Insignificant tenderness of the epigastrium
Livers tender to palpitation with minimal enlargement
Normal arterial blood pressure
Relaxed pulse (60 to 65/minutes)
Intensified reflexes and automic neuroses
Complaints similar to those associated with
exposure to 62, 61 and 200 ppra
Sweet aftertaste; dizziness; nausea; vomiting; lack
of appetite
Livers tender to palpitation with insignificant
enlargement
Epigastrium pain
Emaciation (2 to 10 kg below expected weight)
Unremarkable opthalmologic examination
Unremarkable examination of upper respiratory tract,
lungs and heart
Augmented reflexes and vegetative neurosis
Incidence
6/104
10/10
3/10b
7/10°
4/10°.
10/10
6/10°
3/10b
1/6°
3/42
16/16
16/16
16/16
3/16
-------
TABLE 9-23 (cont.)
Concentration of l,2-DCEa
10 to 200 ppm (cont.)
vO
Effect
Incidence
Icteric skin coloration 1/16
Elevated urobilinogen levels "majority"
of 16
X-ray observable chronic catarrh of the stomach with 6/16
atrophy of the mucous membrane
Periodic spasm of the pylorus 3/16
Moderate hyperchromic anemia 1 /1 3e
(3,430,000 erythrocytes/cu mm and 60H hemoglogin)
Slight reticulocytosis (0.1 to 0.3*) 1/136
Diminished osmotic fragility of erythrocytes in NaCl 6/13
Slight leukocytes (11,200/cu ram) 1/136
Low platelet count (10,000 to 55,000 cu mm) 2/1 3e
Decreased number of neutrophils (10 to 5058) 2/1 3e
Slightly increased number of neutrophils (70 to 78$) 6/13e
with decreased number of lymphocytes (15 to 25%)
"Abnormal" distribution of white blood cells 1/1 3e
Increased number of erythrocytes, increased percentage 5/1 3e
of polymorphonuclear neutrophils, mild stimulation
of erythropoiesis with a less significant increase
of leukopoiesis in the bone marrow
Elevated serum bilirubin (2.3 »g %) 1/16
-------
TABLE 9-23 (cont.)
U)
Concentration of 1,2-DCEE
10 to 200 ppm (cont.)
Effect
Elevated blood nonprotein nitrogen (55 mg %)
Normal overall quantity of serum protein
Diminished serum albumin
Elevated serum globulin levels
Diminished blood fibrin content
Positive Takata-Ara liver function test
Borderline Takata-Ara liver function test
Positive Cadmium turbidity teat
Borderline Cadmium turbidity test
Negative glucose tolerance test
Decreased secretion of gastric hydrochloric acid
X-ray observable catarrhal changes of gastric mucosa
Periodic pyloric spasms
Incidence
1/16
16/16
6/16
8/16
3/16
1/16
5/16
6/16
5/16
8/16
12/16
6/16
3/6f
Source: Cetnarowicz, 1959 (derived from NIOSH, 1976)
Concurrent exposure to =3 to 8$ benzene (see text)
Workers employed in the centrifuge room (see Table 9-22)
°Workers employed in the pump and crystallization rooms (see Table 9-22)
12 workers initially examined; effects on these workers prompted further examinations of 16 workers from
one shift (see below).
Heraatological examinations were performed on 13 workers.
f3 of the 6 with catarrh
-------
levels, positive Takata-Ara liver function tests, negative glucose tolerance
tests), and changes in the gastrointestinal tract (X-ray observable chronic
gastritis with mucosal inflammation, pyloric spasms, impaired hydrochloric acid
secretion). The distribution of these findings by room (i.e., high or low
exposure) was not, however, given. Other abnormal findings included blood and
bone marrow changes that may be attributable to the concurrent exposure to
benzene (Table 9-23).
Rozenbaum 09t7) reported the results of studies of approximately 100
Russian workers from different industries (not specified) who had experienced
exposure to EDC at levels below 25 ppm (<0.1 ing/1) in air for 6 months to
5 years. "Many" of these workers exhibited non-specific functional nervous
system disorders (e.g., >*ed dermographisra, muscular torus, bradycardia,
increased perspiration), and "some" of the subjects complained of fatigue,
irritability, headaches and insomnia. Hematologic or functional organ changes
(not elaborated) were not, however, noted. The incidence of the observed
effects, average exposure concentrations, the range of exposure concentrations
to which the workers were exposed and information on experimental design were not
reported.
Brzozowski et al. O951*) considered that absorption of EDC through the skin
was largely responsible for adverse health effects among certain agricultural
workers in Poland. EDC was used as a fumigant and work practices involved
transport of the liquid to fields in barrels, pouring the EDC by hand into
buckets, carrying the open buckets to the site of application, and pouring the
EDC into a series of holes in the ground. The workers were exposed to particu-
larly high concentrations of EDC vapor during pouring. Significant dermal
exposure is indicted by the frequent spilling of quantities of EDC on clothes and
sleeves during the transport of the open buckets to the places of application
9-84
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(resulting in soaked clothing that was not changed), and by the fact that the
workers used EDC to wash their skin. A single atmospheric sample, comprising 10
subsaraples collected from locations representative of the working zone (the
collection apparatus was moved from place to place with the workers), showed a
concentration of 4 ppm of EDC. Because of the practical difficulties of sample
collection (the workers did not stay at one location sufficiently long to collect
an entire sample), conditions were simulated in a laboratory to estimate
potential exposure concentrations better. Analysis of laboratory samples showed
concentration of =14.5 to 15 ppra; a sample taken during the pouring of EDC into
buckets, considered to be the maximum exposure of a worker, contained 60 ppm.
Medical examinations were performed on 118 of the agricultural workers
(Brzozowski et al., 1954). Ninety had positive findings that included conjunc-
tival congestion (82/118), weakness (54/118), reddening of the pharynx (50/118),
bronchial symptoms (43/118), metallic taste in the mouth (40/118), headache
(39/118), dermatographism (37/118), nausea (31/118), cough (30/118), liver pain
(29/118), conjunctival burning sensation (24/118), tachycardia (21/118), and
dyspnea after effort (21/118). The Quick Test for hippuric acid was used to
evaluate liver function and was reported to be positive in 40 of 56 investiga-
tions. Skin sensitization tests with pieces of gauze soaked with 0.1% EDC in
alcohol or 50% EDC in soybean oil were reportedly negative when scored after
40 hours of contact, but the number of workers tested was not stated.
9-85
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9.2.2. Effects in Animals
9.2.2.1. ACUTE EXPOSURE
9.2.2.1.1. Exposure — The observed toxic effects of ingested EDC in
laboratory animals summarized in Table 9-21 tend to confirm the findings from
cases involving human oral exposures. The LD for a single oral exposure was
reported to be 680 mg/kg in a study that employed 80 rats and 4 dosage levels
(McCollister et al., 1956) and 770 mg/kg in a range-finding study of rats (Smyth
et al., 1969). These data suggest that EDC is moderately hazardous by the oral
route, and indicate that the compound is more acutely toxic than 1,1-dichloro-
ethane, 1,1,1-trichloroethane, pentachloroethane, and hexaehloroethane (Patty,
1981). According to a World Health Organization report (1970; cited by Torkelson
and Rowe, 1981) rabbits and mice are as susceptible as rats to EDC. Heppel et al.
(1945) observed 60J mortality (6 of 10 treated) in mice 10 days after an acute
oral dose of 700 mg EDC/kg body weight, which was administered as a 5% solution
in olive oil. Similar treatment with 900 mg/kg EDC in 5% olive oil produced
mortality in 10/10 mice, but 6/6 survived a dose of 500 mg/kg given as a
10 percent solution in olive oil. Munson et al. (1982) recently determined that
the single oral dose LD_ 's of EDC for male and female CD-1 mice were 189 and 413
3U
mg/kg, respectively. In these studies, EDC was apparently administered in water
solution, and all mice died over a 46-hour period. Gross pathologic examination
indicated that the lungs and liver appeared to be the target organs. Dogs and
humans may tolerate larger doses. Since EDC causes vomiting, the higher
tolerance in dogs and humans is likely attributed to the ability to vomit in
these two species. Ristler and Luckhardt (1929) found that oral doses >500 mg/kg
tended to be vomited by dogs.
In 2 unanesthetized dogs (weighing 9 and 12.5 kg) administration of 0.63
g/kg EDC by gavage produced initial excitement (8 minutes after treatment)
9-86
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TABLE 9-21
Effects of Acute Exposure to Ethylene Dlchloride
Route
Oral
Oral
Oral
Oral
Oral
Species
rata
rats
alee
alee
(CD-I, male)
nice
(CD-I, female)
Number
BO
6/group
10
10
6
MS
NS
Doae
0.68 g/kg
0.77 g/kg
0.90 g/kg (in olive oil)
0.70 g/kg (In olive oil)
0.50 g/kg
0.489 g/kg (In water)
0.413 g/kg
Effect
LD50
"So
10/10 dead
6/10 dead
no deaths
LD50
LD50
Reference
McCol lister et
Smyth et al.,
Heppel et al.,
Nunson et al.,
Hunson et al . ,
al., 1956
1969
19»5
1982
1982
oo
Inhalation
Inhalation
rata
rats
(female)
6 male or female 1000 ppm for 4 h
Sherman Strain
(100-150 weight)
4-6/group 12,000 ppm for 0.1 h
0.2 h
3000 ppm for 0.3 h
0.5 h
1000 ppm for 1.5 h
3.0 h
300 ppm for 3*0 h
5.5 h
200 ppa for 7.0 h
2/6, 3/6 or 4/6 dead In
11 day observation period.
Ho adverae effecta
Adverse effect
Mo adverae effect
Adverae effect
No adverse effect
Adverse effect
No adverse effect
Adverse effect
No adverae effect
Carpenter et al., 1919
Spencer et al., 1951
-------
TABLE 9-24 (cent.)
i
0.1
co
Route Spec lea Number
Inhalation rats 20
22
40
DO
51
44
32
44
22
10
30
10
41
24
10
32
31
32
30
20
33
20
20
Inhalation rats 20
15
15
12
13
mice 22
20
23
guinea pigs 14
12
rabbits 16
cats 3
hogs 2
raccoons 2
Dose
3000 ppm for 6 h
3000 ppm for 5 h
3000 ppm for 4 h
3000 ppn for 3 h
3000 ppn for 2 h
3000 ppm for 1.6 h
3000 ppn for 1 .0 h
3000 ppn for 0.7 h
3000 ppm Tor 0.5 h
1 500 ppm for 8 h
1 500 ppm for 7 h
i 500 ppm for 6 h
1 500 ppm for 4 h
i 500 ppm for 3 h
1 500 ppm for 2 h
1000 ppm for 8 h
l 000 ppn for 7 h
1000 ppn for 6 h
800 ppn for 7 h
600 ppn for 8 h
600 ppn for 7 h
600 ppm for 5 h
300 ppn for 7 h
3000 ppm for 7 h
3000 ppn for 3>5 h
3000 ppn for 1.5 h
1 500 ppn for 7 h
1 500 ppm for 4 h
3000 ppn for 7 h
i 500 ppm for 7 h
1500 ppm for 2 h
3000 ppn for 7 h
i 500 ppn for 7 h
3000 ppm for 7 h
3000 ppn for 7 h
3000 ppm for 7 h
3000 ppn for 7 h
Effect Reference
20/20 dead Spencer et al., 1951
22/22 dead
38/40 dead
24/40 dead
6/51 dead
1/44 dead
1/32 dead
1/44 dead
0/22 dead
7/10 dead
24/30 dead
7/10 dead
2/41 dead
1/24 dead
0/10 dead
20/32 dead
17/31 dead
5/32 dead
10/30 dead
4/20 dead
3/33 dead
0/20 dead
0/20 dead
20/20 dead Heppel et al., 1945
1 5/1 5 dead
0/1 5 dead
4/20 dead
0/13 dead
22/22 dead
20/20 dead
1/23 dead
14/14 dead
6/12 dead
12/16 dead
0/3 dead
2/2 dead
0/2 dead
-------
TABLE 9-2* (Cont.)
Route
Species
Number
Intraperitloneal guinea pig I/group
(male)
Subcutaneous
dogs 6
oats NS
rabbits NS
albino rats NS
vo
A,
VO
Subcutaneous
Subcutaneous
rats
mice
10/group
4/group at
the lowest dose
10/group
Dose
150, 300 and 600 ng/kg
1 dose
0.94 or 1.26 g/kg body weight
0.94 or 1.26 g/kg body weight
0.9» or i .26 g/kg body weight
0.91 or 1.26 g/kg body weight
0.31, 0.5, 0.75, 1.0,
1.25 g/kg (In olive oil)
0.25, 0.3U, 0.75 g/kg
(In olive oil)
Effect
Reference
No effect on liver histology
2*t hours after Injection.
No effect on serum ornlthlne
carbamyl transferase (OCT)
Activity 24 hours later at
1 50 or- 300 rag/kg.
Increased OCT activity at
GOO rag/kg; death of 1 animal.
Death within 21 hours at
higher dose; histology: mild
perllobular fatty degeneration
in liver, swelling of tubular
cells and mild hemorrhage In
the kidney, lung edema hls-
tological changes more marked
in dogs.
In dogs only: corneal opacity
evident by 1 0 hours; necrosis
of corneal endothelluo; corneas
became clear by fifth day.
0.75/kg: 50J mortality;
0.38 g/kkg: no deaths
Divlncenzo and Krasavage,
Kuewabara et al., 1968
0.38 g/kg:
0.25 g/kg:
8Of mortality
no deaths
Heppel et al., 1945
Heppel et al., 1945
h = hours
-------
followed by decreased excitement. Progressive incoordination and salivation was
evident. In 24 minutes, sleep (from which the dogs could be aroused) supervened
for another 20 minutes, at which time depression was relieved. Vomiting occurred
subsequently, after which signs of recovery were evident (Kistler and Luckhardt,
1929). Larger doses of 1.26 g/kg in 2 dogs, 1.89 g/kg in one dog, and 2.51 g/kg
in- two dogs produced similar symptoms of greater severity. Vomiting occurred
earlier after administration of the compound; little food or water was ingested
and vomiting persisted in one dog that received 1.26 g/kg EDO. The corneas of
the eyes in all five animals were opaque within 24 hours.
9.2.2.1.2. Dermal Exposure — Skin absorption LD Q values of 4.9 g/kg
(Torkelson and Rowe, 1981) and 2.8 g/kg (Torkelson and Rowe, 1981) have been
determined with rabbits. Strong erythema, edema, or slight necrosis was produced
upon application of 0.01 mil of undiluted EDO to the uncovered rabbit belly (Smyth
et al., 1969).
9.2.2.1.3* Inhalation Exposure — Sayers et al. (1930, cited in
Browning, 1965 and NIOSH, 1976) reported that death occurred in guinea pigs
within a few minutes of exposure to 100,000 ppm EDC, and on the day following
exposure to 10,000 ppm EDC for 25 minutes. Progressive signs of intoxication
were observed in groups of 3 or 6 guinea pigs that were exposed to EDC at
concentrations ranging from 600 to 60,000 ppm for durations up to 8 hours. One-
third of each group was sacrificed immediately after exposure, another one-third
sacrificed after 4 days of exposure and the final one-third was sacrificed at the
end of the eighth post-exposure day unless death supervened. The signs of
intoxication (squinting and lacrimation of the eyes and rubbing of the nose,
vertigo, static and motor ataxia, retching movements, unconsciousness, incoordi-
nation of extremities, and marked changes in respiration) appeared in less than
10 minutes, and death occurred in 30 minutes, after exposure to 60,000 ppm EDC.
9-90
-------
Exposure to a lower concentration (10,000 ppm) for 15 to 20 minutes produced
these symptoms in 25 minutes and delayed death a day or more after exposure. No
mortality or signs of intoxication occurred in guinea pigs exposed to 1200 ppra
for 8 hours.
Congestion and edema of the lungs and generalized passive congestion of the
visceral organs were observed in animals that died during exposure (Sayers et
al., 1930, cited in NIOSH, 1976). In animals sacrificed immediately after
exposure, congestion of the liver, spleen, lungs, and kidneys was observed.
Pulmonary congestion, pulmonary edema and renal hyperemia were seen in those
animals that died 1 to 8 days after exposure, and the renal and lung effects were
more pronounced in the animals sacrificed 3 to 4 days post-exposure than in those
sacrificed immediately after exposure. Partial resolution of visceral effects
was observed by 8 days after exposure. In these studies, the severity of
pathological changes was dependent on duration and concentation of exposure.
Heppel et al. (1945) reported that a single exposure to 3000 ppm EDO (12.4
mg/i) for 7 hours produced death in all of 14 treated guinea pigs within 3 days of
exposure. The guinea pigs were inactive and breathing was labored after expo-
sure. Microscopic examination of 11 guinea pigs revealed congestion in the
liver, lung and adrenal glands. Focal necrosis of the adrenal cortex in 5 of the
guinea pigs, and slight to moderate fatty degeneration of renal tubular epithe-
lium was noted in 8 of the animals. In another group of 8 guinea pigs similarly
exposed to 3000 ppm EDC, fatty infiltration of the myocardium was observed in all
of 7 animals that were examined histologically. Changes in the lung, kidney and
adrenal cortex, similar to those observed in the first group of animals were also
found.
Keppel et al. (1945) also found that inhalation exposure to 3000 ppm EDC
(12.4 mg/£) for 7 hours was fatal to rats, mice and rabbits within 3 days of
9-91
-------
exposure. Varying degrees of narcosis were observed in the animals during
exposure, and dyspnea and increasing weakness preceded death. Pulmonary conges-
tion, mild to moderate degeneration of renal tubular epithelium and liver, and
occasional necrosis of the adrenal cortex were observed at autopsy; congestion of
the spleen was additionally found in rats. Inhalation of 3000 ppm EDC for 7
hours was fatal for 2 hogs but was not fatal for 2 raccoons or 3 cats. Reduction
of dose by exposure to one-half the concentration of EDC (1500 ppm) for a similar
duration (7 hours) was lethal to 6 of 12 guinea pigs and to less than one-fifth of
the rats (4 of 20). This lower dose was fatal, however, to all of a group of 20
mice. Reduction of the duration of exposure in rats to 4 hours and mice to
2 hours decreased mortality (Heppel et al., 19*14).
Spencer et al. (1951) subjected groups of 4 to 6 female rats to varying
concentrations of EDC (200, 300, 1000, 3000 or 12,000 ppm) for varying durations
to determine the doses and durations of single exposures that produced and did
not produce adverse effects. No-adverse effect exposures ranged from 200 ppm
(0.8 mg/i) for 7 hours to 12,000 ppra (48.6 rag/i) for 0.1 hours. Doubling the
duration of exposure at 12,000 ppm produced adverse effects. Exposure to 3000
ppm EDC for 0.3 hours was without effect, but increasing the duration of exposure
to 0.5 hours caused adverse effects.
Spencer et al. (1951) also sacrificed groups of 10-54 rats after inhalation
exposure to EDC at concentrations ranging from 300 to 20,000 ppra for durations of
8.0 to 0.1 hours. A decrease in body weight, increase in liver and kidney
weight, increase of blood urine nitrogen, increase of plasma prothrorabin
clotting time, decrease in serum phosphatase, increase in liver lipids and histo-
logical changes in the kidneys, liver and adrenal glands were observed. The
kidney changes consisted of tubular damage that ranged from slight parenchyma-
tous degeneration of the epithelium to complete necrosis accompanied by
9-92
-------
interstitial edema, congestion and hemorrhage. Changes ranging from slight
congestion and slight parenchymatous degeneration to marked hemorrhagic necrosis
were observed in the liver. Some parenchymatous degeneration of the adrenal
cortex was found in the most severely affected animals. Pulmonary congestion and
edema was observed at concentrations above 3000 ppm. Carpenter et al. (1949)
reported an inhalation LC5_ in rats to be 1000 ppm in air for a U-hour exposure.
9.2.2.1.3.1 Central Nervous System and Cardiovascular System. Inhala-
tion of 3000 ppm EDC (12.4 mg/Jl) for 7 hours in five species (rabbits, guinea
pigs, hogs, rats and mice) produced varying degrees of narcosis (Heppel et al.,
1945). Spencer et al. (1951) partially attributed the inactivity or stupor and a
slower response to handling in rats, at vapor concentrations of 3000 ppm and
lower, to toxic injury to organs other than depression of the central nervous
system. At concentrations of 12,000 ppm and lower, varying degrees of "drunken-
ness" were attributed to depression of the central nervous system. Considerable
depression of the central nervous system was produced at 22,000 ppm causing death
in rats within O.U hours. Animals that died from exposure to 20,000 ppm EDC were
in a state of deep anesthesia during the period of exposure. At all vapor
concentrations a large proportion of the rats died suddenly after leaving the
exposure chamber. Marked cyanosis, reduced body temperature, stupor or coma, and
failing respiration were observed. Spencer et al. (1951) hypothesized that this
response is suggestive of cardiovascular collapse. Other deaths, which occurred
over a period of two to seven days, were accompanied by loss in body weight and
other signs of toxicity; the authors attribute these deaths to renal injury.
Inhalation of EDC (0.1 to 5 cc, introduced by volatilization into a Jackson
Carbon Dioxide Absorption Apparatus) produced a drop in blood pressure, depres-
sion of the knee jerk reflex and stimulation followed by a short cessation of
respiration in sodium barbital anesthetized dogs (number unspecified) (Kistler
9-93
-------
and Luckhardt, 1929). These dogs had been subjected to spinal cord transection
prior to EDC treatment. Similar effects on blood pressure and the knee jerk were
observed in sodium barbital anesthetized dogs (11-20 kg) after intravenous
injection of amounts as small as O.i cc, while 0.3 cc caused a cessation of
respiration for a duration of up to 40 seconds.
9.2.2.1.3.2. Eyes. Inhalation of EDC was observed to produce a clouding
of the cornea in dogs and foxes, but not in rabbits, guinea pigs, mice, rats,
hogs, raccoons, chickens or cats (Heppel et al., 1944). A single exposure to a
vapor concentration of 1000 ppm for 7 hours produced turbidity in the eyes of
dogs (Table 9-25). A higher concentration of 3000 ppm caused turbidity in the
cornea of the red fox after a single exposure of 7 hours duration. Repeated
exposure interspersed with two days of no exposure at a lower concentration of
400 ppm produced clouding in the cornea of six dogs, which cleared during the
days free of exposure. Development of resistance to toxic effects in the cornea
after repeated exposure was observed.
Corneal opacity was also observed within 10 hours in 6 dogs that received a
subcutaneous injection of 0.94 g/kg EDC (Kuwabara et al., 1968) (Table 9-25).
Marked swelling of most of the endothelial cells of the cornea was seen at 12
hours, and the reparative process was found to occur by 24 hours. Similar
corneal changes were produced in dogs, rabbits and cats when EDC (a 1$ suspension
dissolved in mineral oil and homogenized in 0.556 gelatin solution) was instilled
directly into the anterior chamber of the eye. The authors suggested that the
apparent vulnerability of the dog eye to EDC by routes other than direct local
application was due to "a greater amount of dichloroethane coming in contact with
the dog endothelium rather than an unusual susceptibility of the eye itself."
9-94
-------
Inhalation
Inhalation
TABLE 9-25
Effects of Ethylene Dlchlorlde on the Cornea
vO
1
\o
-------
TABLE 9-25 (Cont.)
Route
Species
Number of
Animals
Dose
Effect
Reference
Inhalation
Inhalation
Inhalation
dog
guinea pigs
rata
rabbi ta
dog
10
US
NS
NS
3
vD
vO
Inhalation
dog
10
Inhalation
eats NS
monkeys
chickens
rodents (species, NS)
1000 ppm for 7 h,
i exposure
1500 ppm for 7 h,
repeated exposure
1500 ppm for 7 h, repeated
exposure
1000 ppm for 7 h, 5 d/uk,
repeated exposure,
length NS
1000 ppn for 7 h, 5 d/ufc,
repeated exposure, length NS
Symmetric turbidity in the
corneas of 8; up to 3 weeks
required for partial
regression.
Death in aost animals
after 1 exposures;
no adverse effect on eyes.
Intense bilateral corneal
opacity in both eyes 48 h
after first exposure; death
after 5, 6 or 30 exposures.
Turbidity In eyes retained
throughout.
Increasing turbidity of
cornea during 5 exposure days,
clearing during 2 non-exposure
days; increased tolerance with
successive bouts of exposure;
complete resistance of cornea
finally.
No adverse effects in eyes
Heppel et al., 1901
Heppel et al., 1944
Heppel et al. 1944
Heppel et al., 1944
Heppel et al., 1944
-------
TABLE 9-25 (Cent.)
Route
Subcutaneous
Inhalation
Species
dogs
(puppies and
adults)
cats
rabbits
albino rats
dogs
Number of
Animals
6
NS
NS
NS
6
0.91
0.94
0.94
0.94
400
Dose
g/kg
g/kg
g/kg
g/kg
ppm for 7 h, 5 d/wk,
Effect
Corneal opacity evidence within
10 hours; necrosis of corneal
endotnelium; clearing by
fifth day.
No effect on cornea
No effect on cornea
No effect on cornea
Hild clouding during first
Reference
Kuuabara et al
Kuwabara et al
Kuwabara et al
Kuwabara et al
Heppel et al . ,
. . 1968
., 1968
. . 1968
.. 1966
1944
for 10 weeks
week, cleared during 2 non-
exposure days; 5th week of
exposure; faint opacity of
cornea 10th week of exposure:
no trace of turbidity.
vo
h = hour; d = day; wk = week
-------
9.2.2.2. SUBCHRONIC AND CHRONIC EXPOSURE
9.2.2.2.1. Oral Exposure — EDC has been tested for carcinogenicity in
an NCI bioassay with Osborne-Mendel rats and B6C3F1 mice (NCI, 1978). The
compound was administered at time-weighted average doses of 95 and 47 mg/kg (rats
of both sexes), 195 and 97 mg/kg (male mice) and 299 and 119 rag/kg (female mice)
for a period of 78 weeks, according to the regimen described in Section 9.5.
Fifty animals of each sex were treated/dose level, and the animals were observed
for an additional 28 weeks (rats) or 12-13 weeks (mice) after pretreatment.
As detailed in Section 9.5, no distinct dose-related mean body weight
depression was apparent in either male or female rats relative to vehicle
controls. Mortality was, however, early and severe in many of the dosed rats,
particularly those given the highest dose. Mean survival was =55 weeks for the
high-dose males and females. Toxic rather than carcinogenic effects of EDC
appeared to be responsible for the deaths. No distinct, dose-related mean body
weight depression was observed in male mice or in low-dose female mice, but mean
body weight depression for high-dose female mice was apparent as early as the
1 5th week of treatment. A significant positive association between increased
dosage and elevated mortality was found for female mice; 72$ (36/50) of the
females died between weeks 60 and 80. The presence of one or more tumors in these
mice suggests that these deaths may have been tumor related. There was no
statistically significant association between dosage and mortality for male
mice.
Munson et al. (1982) evaluated the effect of subchronic 1 M-day and 90-day
oral EDC exposures on the immune response of male CD-1 mice. EDC was adminis-
tered daily by gavage in the 11-day study at levels of U.9 and 19 mg/kg, which
represent 0.01 and O.i times the single dose LD_0 determined in a preliminary
9-98
-------
study (Section 9-2.2.1.1). For the 90-day study, EDC was administered in the
drinking water at levels intended to be equivalent to those administered by
gavage; in addition, a 10-fold higher dose was added because the results of the
11-day exposure indicated that doses >19 mg/kg could be tolerated. The calcu-
lated time-weighted average doses of EDC delivered in the 90-day study based on
actual fluid consumed were reported to be 3, 21 and 189 mg/kg. Results of
standard toxicological analyses showed that body weight was unaltered during the
14-day study, but that EDC did elicit a dose-dependent decrease in growth rate
and fluid consumption when administered over 90 days. Thirty-two mice per dose
were weighed in the 90-day study but the numbers weighed in the 11-day study were
not stated. EDC did not alter the weights of selected organs (liver, spleen,
lungs, thyraus, kidneys, brain) in either of the studies. It was further found
that exposure to 49 mg/kg/day EDC caused a 30% decrease in leukocyte number in
the 11-day study, although the number of leukocytes was normal after 90 days of
exposure. Other hematologic parameters (hematocrit and hemoglobin evaluated
after 11 or 90 days, erythrocytes and platelets evaluated after 90 days), coagu-
lation values (fibrinogen and prothrombin time evaluated after 11 days), and
clinical chemistry parameters (lactic dehydrogenase, serum glutamic-pyruvate
transaminase and blood urea nitrogen evaluated after 11 days) were unaltered by
EDC exposure. The hematological/coagulation/clinical chemistry parameters were
assessed in 10-12 and 16 mice/dose in the 11-day and 90-day studies, respec-
tively.
The status of the humoral immune system was determined by measuring the
number of IgM spleen antibody-forming cells (AFC) to sheep erythrocytes (sRBC)
after 11 and 90 days, the serum antibody level to sRBC after 90 days, and the
lymphocyte response to the B-cell mitogen LPS (lipopolysaccharide from
Salmonella typhosa 0901) after 90 days (Munson et al., 1982). Results of assays
9-99
-------
with 10-12 mice (14 days) and 16 mice/dose showed that EDC produced a significant
(P<0.05) reduction in AFCs at 4.9 and 49 mg/kg in the 14-day study (25 and 40$
suppression, respectively), but that EDC exposure for 90 days caused no signifi-
cant (P<0.05) change in the number of AFC spleen cells (although there was an
indication of suppression at 189 mg/kg/day). There also appeared to be an EDC
dose-dependent reduction in hemagglutination titer after 90 days, although this
response was not significant.at the p<0.05 level. EDC did not alter the spleen
cell response to three concentrations of LPS. Cell-mediated immunity was
assessed by measuring the delayed hypersensitivity response (DTH) to sRBC and the
response to the T-lymphocyte mitogen, concanavalin A. EDC produced a slight but
significant (P<0.05) non-dose-dependent inhibition at both 4.9 and 49 mg/kg in
the 14-day study, but 90-day exposure did not alter DTH response or spleen
lymphocyte response to concanavalin A. EDC did not alter the functional activity
of the reticuloendothelial system, as measured by the vascular clearance rate and
tissue (i.e., liver, sple.en, lungs, thymus or kidneys) uptake of 51Cr after 90
days. Dexamethasone was used as a positive control in the above studies:
Results of similar tests with female mice were not presented, but reportedly were
not remarkably different from the males.
The results of the above experiments (Munson et al., 1982) indicate that EDC
produced a suppression of both humoral and cell-mediated immunity when adminis-
tered daily for 14 days by stomach tube, but not when consumed in the drinking
water for 90 days (although there were trends toward suppression at the high
exposure level). Two explanations were offered for these results. First, the
effective dose at the immunocompetent cell may be higher with the bolus presenta-
tion than with the semi-continuous self-administration in the drinking water.
Second, EDC may induce its own metabolism over the longer exposure period,
thereby effectively reducing the amount of chemical reaching the immune cells.
9-100
-------
9.2.2.2.2. Inhalation Exposure — Four animal species (15 rats of both
sexes, 8 guinea pigs of both sexes, 2 male and 1 female rabbits, and 2 male
monkeys) were exposed to EDC vapor at levels of 100 pptn (1.62 rag/1) and 100 ppm
(0.105 rng/5.) for 7 hours daily, 5 days/week for 6 months (Spencer et al., 1951).
In addition, 1 5 rats of both sexes and 8 guinea pigs of both sexes were similarly
exposed to 200 ppm EDC (0.8l mg/R.) for 151 and 180 7-hour periods, respectively.
Repeated exposure at 100 ppm did not produce adverse effects in any of the four
species as determined by general appearance and behavior, mortality, growth,
body and organ weights, and gross and microscopic examination of tissues (Table
9-26). No adverse effects were indicated by periodic hematological examination
of the treated rats, guinea pigs, or rabbits. Exposure to 200 ppm EDC did not
produce any adverse effects in the rats, but did elicit slight parenchymatous
degeneration of the liver with a few diffusely distributed fat vacuoles in half
of the guinea pigs. A slight increase of total lipid, phospholipid, neutral fat
and free and esterified chloresterol was also found in the guinea pigs exposed to
200 ppm EDC, and the final body weights of the treated male guinea pigs were
significantly different from control animals. Severe toxic effects were found in
the rats, guinea pigs, and monkeys that were exposed to 400 ppm EDC (1 .62 mg/£)
(see Table 9-26). The three rabbits that were exposed to 400 ppm EDC were not
adversely affected, although Heppel et al. (1946) reported a high mortality in
rabbits exposed to this concentration of EDC.
Heppel et al. (1945) exposed rats, mice, rabbits, guinea pigs, hogs and dogs
to 1500 ppm EDC (6.4 mg/S,). Almost all of these animals died before 6 daily
exposures of 7 hours each were completed. Hemorrhaging in the lungs, gastro-
intestinal tract and adrenals; fatty degeneration of the myocardium; degenera-
tion within the renal tubules; and congestion of the liver and intestines were
found at autopsy.
9-101
-------
TABLE 9-26
Effeota of Subohronlo Exposure to Ethylene Dlchlorlde
Route
Speolea
Number
Dose
Effect
Deference
Inhalation
rata
26 1000 ppm (3.0 mg/1) 7 h/d x 5 d/wk 20 dead within 15 exposures; Heppel et al., 1946
(average weight: progressive weakness culmln-
19B g) atlng In Inability to stand.
Histology of 4 rata:
degeneration and proliferation
changes in renal tubular
eplthellun; chronic splenltls
In 20 rats; pulmonary congestion
In 2 rats.
Inhalation
Inhalation
o
K)
rabbits
guinea pig
guinea pig
guinea pig
6 1000 ppn (3.9 mg/l) 7 h/d x 5 d/wk 5 dead within 43 exposures
(average weight: survivor exposed 64 days
2480 g>
Heppel et al., 1946
15 1000 ppn (3.9 mg/l) 7 h/d x 5 d/wk 10 dead within 2 exposures Heppel et al., 1946
(average weight:
161 g)
10 1000 ppm (3.9 mg/l) 7 h/d x 5 d/wk 10 dead within 2 exposures Heppel et al., 1916
(average weight:
558 g)
16 1000 ppm (3.9 mg/l) 7 h/d x 5 d/wk 16 dead within 4 exposures Heppel et al., 1946
(average weight: lacrimatlon and marked
900 g) inactivity during exposure;
congestion of lung, liver,
heart, kidney, adrenal gland
and spleens.
Inhalation
dogs
6 (7000-8300 g) 1000 ppm (3.9 mg/l 7 h/d x 5 d/wk
I dead after 30 exposures
1 dead after 43 exposures
Heppel et al., 1946
-------
TABLE 9-26 (Cent.)
Route
Species
Number
Doae
Effect
Reference
Inhalation
cats
6 (2660 g 1000 ppa (3.9 ng/l) 7 h/d x 5 d/wk 2 dead after «3 exposures
average weight) congestion and fatty meta-
morphosis In liver of all.
Heppel et al., 1916
Inhalation monkeys 2 (1880 g 1000 ppa (3.9 ag/l) 7 h/d x 5 d/wk 1 dead after 2 exposures Heppel «t al., I9t6
average weight) 1 dead after 43 exposures;
fatty degeneration of liver
and slight fatty changes In
kidney; focal myocarditis In
1 Donkey.
Inhalation
cats
500 ppa 6 h/d x 5 d/wk for 6 wks
No deaths; dilated hearts; Hoffmann et al., 1971
blood urea nitrogen Increased
to 11H og/100 mi.
o
10
Inhalation
Inhalation
rabbita
guinea pigs 10
500 ppa 6 h/d x 5 d/wk for 6 wks
500 ppa 6 h/d x 5 d/wfc for 6 wks
3/4 dead after 10-17 exposures; Hoffman et al., 1971
dilated hearts.
9/10 dead after 4-11 exposures; Hoffman et al., 1971
apathy and weight loss, fatty
degeneration and necrosis-of
myocardium, liver, kidney and
adrenals.
Inhalation
rata
10
500 ppm 6 h/d x 5 d/wk for 6 wks
All dead after 1-5 exposures;
hypereuia of lungs; fatty
degeneration and necrosis of
myocardium, liver, kidney,
and adrenals.
Hoffman et al., 1971
-------
TABLE 9-26 (Cont.)
Route
Speoiaa
Number
Dose
Effect
Reference
Inhalation
rata
Inhalation
guinea pigs
Inhalation
Inhalation
rabbits
dogs
15 male
1 female
(average weight:
167 g)
23 controls
IB male
2 female
(average weight:
303-737)
30 controls
2 male
3 female
(average weight:
3110 g)
4 controls
3 male puppies
(average weight:
1330 g)
6 female adult
(average weight:
9100 g)
4 controls
MOO ppm (1.54 mg/l) for
7 h/d s 5 d/wk for
60 exposures
400 ppm (1.54 mg/l) for
7 h/d x 5 d/wk
400 ppm (1.54 ag/l} for
7 h/d x 5 d/wk
400 ppm (1.54 mg/l) for
7 h/d x 5 d/wk for
167-177 exposures
Total dead: 9
(6 dead after 4 exposures)
Histology In 6 survivors:
no changes in 5; diffuse
myocarditis and fatty
degeneration of liver,
kidney and heart in 1.
1 dead.
Histology: no change.
13 dead after 45 exposures
1 dead after 65 exposures
Histology in 10: fatty
degeneration of liver and
kidney In 4 dead; fatty
degeneration of heart in
2 dead; no histologlcal
change in 6 survivors of
exposure.
3 deaths.
All dead after 97 exposures.
No hematological changes.
No deaths.
No deaths; 6 adults: no
effect on mean arterial
pressure, Bromsulphaleln
excretion rate, prothrombin
time, plasma total protein,
albumin, globulin, non-
protein nitrogen, Icterus
index, urine; slight fatty
change In livers of 5 and
kidney of 1.
No deaths.
Heppel et al., 1946
Heppel et al., 1946
Heppel et al., 1946
Heppel et al., 1946
-------
TABLE 9-26 (Cont.)
Route
Specie?
Number
Doae
Effect
Reference
Inhalation
Inhalation
rata
rata
15 male,
15 female
20 male,
20 female
400 ppo (1.62 mg/l) 7 h/d,
5 d/wk
400 ppm (1.62 mg/l) 7 h/d,
5 d/wk
i
o
Inhalation
Inhalation
guinea pigs 8 male,
8 female
guinea pigs 2 male
400 ppo (1.62 ng/l) 7 h/d,
5 d/ wk
400 ppffl (1.62 mg/l) 7 h/d,
5 d/wk
Females died within 10
exposures.
Kales died within 40
exposures.
60ft mortality after 2 or 3
exposures; rapid loss of
body weight; slight Increase
of liver and kidney weight;
slight cloudy swelling of
liver with a few large fat
vacuoles.
No significant differences In
blood urea nitrogen, non-
protein nitrogen, serum
phosphatase and plasma
prothrombln olotting time.
Hales died within 10
exposures.
Females died within 24
exposures.
Spencer et al., 1951
Spenoer et al., 1951
Sacrificed after 1, 3, 4, and Spencer et al., 1951
10 exposures; rapid loss of
body weight; Increased liver
and kidney weight; slight to
moderate central fatty
degeneration of liver; slight
to moderate cloudy swelling of
renal tubular epithelium; no
alteration in lungs, heart,
spleen and teates; increased
BUN and Increased blood non-
protein nitrogen levels; no
change In serum phosphatase
or plasma prothrombln clotting
time.
-------
TABLE 9-26 (Cont.)
Route
Inhalation
Species
rabbits
Number
2 male,
i female
Dose
400 ppn (1 .62 ng/fc) 7 h/d,
5 d/wk for 165 exposures
Effect
No effect on general
appearance, behavior;
Reference
Spencer et al., 1951
mortality, body weight,
histology of liver, kidney,
lungs, heart, spleen or teatea;
organ weights, blood non-
protein nitrogen. BUN,
serjo phosphatase, plasma
prothrombln clotting time.
Inhalation
monkeys
2 males
100 ppm (1.62 mg/l) 7 h/d,
5 d/wk
I
.-»
o
One sacrificed In moribund
condition after 8 exposures:
enlarged liver with In-
creased neutral fat and
esterlfled cholesterol
content, marked degeneration
and vacuolation of hepatic
cells; moderate degeneration
of renal tubular epithelium
with cast formation; In-
creased plasma prothrombin
clotting time.
Second sacrificed after 12
exposures: similar, but
milder, changes.
No significant hematologlcal
change.
Spencer et al., 1951
Inhalation
rats
12 male
Osborne-Mendel
(average weight:
72 g)
12 controls
200 ppm (0.73 mg/t,) 7 h/d x 5 d/wk 8 dead after 6 exposures
No deaths.
Heppel et al., 1916
-------
TABLE 9-26 (Cont.)
Route
Species
Number
Dose
Effect
Reference
Inhalation
rata
1 male Wlatar
11 female Ulstar
(average weight:
249 g)
200 ppa (0.73 og/1) 7 h/d x 5 d/wk
1>I controls
7 dead; 1 after 73 exposures,
6 after 14 exposures.
Symptoms: weight loss;
listless; crusting of eyes.
Histology of 5 rats killed
after 86 exposures: fatty
degeneration of kidney In
1 rat; no other changes
noted in liver, heart, lungs,
kidney, adrenals and spleen;
no effect on red and white
blood cell count, hemoglobin
and differential counts
(7 treated compared with 6
control rats).
1 dead.
Heppel et al., 1916
^
o
Inhalation
Inhalation
mice
guinea pigs
20
12 male,
2 female
(average weight:
376 g)
18 controls
200 ppm (0.73 mg/l) 7 h/d x 5 d/uk 18 dead after 7 exposures. Heppel et al., 1916
200 ppm (0.73 Bg/O 7 h/d v 5 d/wk
4 dead after 88 exposures;
1 dead after 115 exposures.
Histology of 9 guinea pigs
killed after 124 exposures:
pulmonary congestion in 4,
liver necrosis in 1,
necrosis of adrenal cortex
in 1.
1 dead.
Histology of 5 guinea pigs:
fatty liver and myocardium
of 2; no other changes noted
in liver, heart, lung,
kidneys, adrenals and spleen.
Heppel et al., 1946
-------
TABLE 9-26 (Cont.)
Route
Species
Number
Dose
Effect
Reference
Inhalation
rabbits
(average weight:
2720)
1 controls
200 ppn (0.73 ng/A) 7 h/d x 5 d/wk
No deaths.
No hlatologlcal changes
found; no effect on red
and white blood cell
count, hemoglobin, and
differential counts
compared with controls.
No deaths.
Heppel et al., 1916
Inhalation
monkeys
2 males
(average weight:
8740 g)
no controls
200 ppm (0.73 mg/l) 7 h/d x S d/wk
for 125 exposures
No deaths.
Histology of both: focal
calcification in adrenal
medulla of 1; fatty
droplets In liver and
myocardium of both.
Heppel et al., 1946
Inhalation
rats
o
oc
15 male,
15 female
Inhalation
guinea pigs 6 male,
8 female
200 ppn (0.81 mg/4) 7h/d,
5 d/wk for 151 exposures
200 ppm (0.61 ug/l) 7 h/d,
5 d/ wk for 180 exposures
No adverse effects on
general appearance, behavior,
growth, mortality, final
body and organ weights,
hematology, gross and
microscopic histology.
Spencer et al., 1951
Final body weight
significantly different
from controls in males, and
growth less than controls,
but not siglflcant In females.
Significant increase in male
liver weights only; slight
parenchyoatous hepatic
degeneration in half of
guinea pigs of both sexes;
no effect on hematology; slight
increase in trial lipid,
phospholipid, neutral fat,
free and eaterified cholesterol.
Spencer et al., 1951
-------
TABLE 9-26 (Cont.)
Route
Species
Number
Dose
Effect
Reference
Inhalation
rats
15 male, 100 ppa (0.105 mg/i) 7 h/d,
15 female 5 d/wk
males: 151 exposures
females: 112 exposures
No adverse effect on general
appearance, behavior,
mortality, growth, final body
and organ weights, hematology,
gross and microscopic histology,
BUN, blood non-protein nitrogen,
serum phosphatase, plasma
prothroobin clotting time,
total levels llpld, phosptio-
llplds, neutral fat In liver,
free and esterlfled
cholesterol In liver.
Spencer et al., 1951
Inhalation
guinea pigs
8 male,
8 female
100 ppm (0.105 mg/JO 7 h/d,
5 d/wk
males: 121 exposures
females: 162 exposures
Inhalation
rabbits
Inhalation
monkeys
2 male,
1 female
2 male
100 ppm (0.105 mg/ft) 7 h/d,
5 d/wk for 178 exposures
100 ppm (0.105 me/£) 7 h/d,
5 d/wk for 148 exposures
No adverse effect on
mortality, growth, final
body and organ weights,
BUN, blood non-protein
nitrogen, serum phosphatase,
plasma prothrombln clotting
time, gross and microscopic
histology, total liver llpld
phosphollplds, neutral fat
In liver, free and esterlfied
cholesterol In liver.
No adverse effect on
appearance, behavior,
growth, final body and
organ weights, gross and
microscopic histology,
hematology.
No adverse effect on
appearance, behavior,
growth, final body and
organ weights, gross and
microscopic histology,
hematology.
Spencer et al., 1951
Spencer et al., 1951
Spencer et al., 1951
-------
TABLE 9-26 (Cent.)
Route
Species
Number
Doae
Effect
Reference
Inhalation
rats
16 female
23 male
100 pom (0.12 mg/O 7 h/d x 5 d/wk
for 1 months
Inhalation
1^
o
guinea plga 10 male
6 female
30 control
100 ppm (0.42 mg/l) 7 h/d x 5 d/wk
for 4 months
Inhalation
nice
19 Juveniles
100 ppm (0.42 mg/H) 7 h/d x 5 d/wk
for 19 exposures
No deaths, no effect on rate
of growth in 15 males
compared with 1 5 male
controls.
i5 of 16 females became
pregnant; rat pups were
unaffected by exposure.
No hiatologleal effects in
liver, heart, lungs, kidney,
adrenal glands and spleen of
10 rats examined.
2 dead (disease of neck with
enlarged oaaeous glands)
No histologlcal effects in
liver, heart, lungs, kidney,
adrenal glands and spleen of
10 animals examined.
3 dead (disease of neck with
enlarged easeous glands).
No deaths; no effect on weight
gain.
Heppel et al., 1946
Heppel et al., 1946
Heppel et al., 1946
Inhalation
cats 4
rabbits 4
guinea pigs 10
rats 10
100 ppm 6 h/d x 5 d/wk for 1 7 wks
No clinical symptoms
No effect on serum levels of
creatlnine, urea, SCOT,
SGPT, on body weight.
No changes In liver, kidney,
and other organs (not specified).
Hoffman et al., 1971
h = hour; d = day; wk = week
BUN = Blood urea nitrogen
-------
In another study, Heppel et al. (1946) found that repeated exposure (7
hours/day, 5 days/week) to 1000 ppm EDC elicited death in most of the exposed
rats, rabbits, guinea pigs, dogs, cats and monkeys (see Table 9-26). Rats,
rabbits and guinea pigs appeared to be more susceptible than the other species
tested. Pathological changes, which were varied, included congestion of lung,
liver, heart, kidney, adrenal gland and spleen in guinea pigs; congestion and
fatty metamorphosis in the liver of all exposed cats; renal changes in exposed
monkeys and rats; and pulmonary congestion in a few rats. Chronic splenitis
occurred in all rats exposed to repeated inhalation of 1000 ppm EDC. Focal
myocarditis was found in one monkey. Similar repeated exposures to 400 ppm EDC
produced high mortality in rabbits, rats, and guinea pigs (see Table 9-26), but
mortality was not observed in 9 dogs subjected to from 167 to 1 77 exposures at
this concentration. No changes were observed in a variety of clinical parameters
that were assessed in 6 adult dogs, although slight fatty changes were noted in
the livers of five and the kidney of one of the dogs. Rat and guinea pig
survivors of 400 ppm EDC exposure did not exhibit histological changes with the
exception of 1 of 6 rats that exhibited diffuse myocarditis and fatty degenera-
tion of the liver, kidney and heart. Repeated inhalation exposure to 200 ppm EDC
produced mortality in mice, rats and guinea pigs, but not in rabbits or monkeys
(see Table 9-26). Pathologic effects of exposure to 200 ppm EDC included a few
cases of pulmonary congestion, fatty degeneration in the kidney of one rat,
necrosis of the liver and adrenal cortex in one guinea pig, and fatty droplets in
the liver and myocardium of both monkeys. No clinical or histopathological
effects were observed in 39 rats or 16 guinea pigs exposed to 100 ppm EDC for 4
months or in 19 juvenile mice exposed to this level of EDC for 19 exposures.
Hoffman et al. (1971) exposed groups of 1 cats, 1 rabbits, 10 guinea pigs
and 10 rats to 500 or 100 ppm EDC for 6 hours/day, 5 days/week for 6 weeks.
9-111
-------
Mortality from exposure to 500 ppm EDC was high (see Table 9-26). Variable
changes in the heart, lungs, liver, kidney, and adrenals were found at autopsy.
No toxic effects were observed in the exposed animals at 100 ppm. An elevation
of BUN and creatinine was noted in one fourth of the rabbits, but the
significance of these changes is uncertain. The exposed cats did not grow as
well as the control cats.
The effect of EDC on blood elements and on renal and liver function was
investigated using groups of 10 rabbits that were exposed to 3000 ppm for 4
hours, or to 3000 ppm for 2 hours/day, 5 days/week for 90 days (Lioia,
1959a,b,c,d). The only significant changes observed after the acute 1-hour
exposure were granulations in about 20$ of the granulocytes. Varying degrees of
anemia accompanied by leukopenia and thrombocytopenia and frequent hyperplasia
of granuloblastic and erythroblastic parenchyma in the bone marrow were observed
after subchronic exposure. There was also a reduction in leukolipids, but no
change in polysaccharides, peroxidase, or ribonucleic acid. In tests for liver
function, a decrease in albumin-globulin ratio, a slightly elevated BSP
retention, slightly elevated values in colloidal tests (cadmium and
cholesterol), and normal Van den Berg (indicating an absence of elevated serum
bilirubin) and blood amino acid levels were observed. Measurement of creatinine
clearance, portal blood flow, and glomerular filtration rate indicated altered
renal function. Congestion, vascular degeneration and small necrotic areas were
found in livers and kidneys.
Dimitrieva and Kuleshov (1971) examined the effect of EDC on brain activity
in 18 albino rats exposed to 1235 ppm EDC (5 mg/i) for 3-5 months. The duration,
frequency and number of exposures were not reported. Electroencephalograms were
recorded before exposure and at monthly intervals thereafter from silver and
platinum electrodes implanted in the brains of the test animals. The stimulus
was composed of an arhythmic photic stimulus of constant intensity and pulse
9-112
-------
duration. A maintained frequency of activity was observed in the EEC of the
treated rats. A progressive diminution of amplitude of vacillations occurred,
with the amplitude of delta rhythms reaching 50-70 MV (amplitude of delta rhythm
before exposures was not specified), while the amplitude of beta rhythm decreased
to 10 to 15 MV (amplitudes of pre-exposure beta rhythms were 30 to 80 MV). A loss
of ability to assimilate an imposed rhythm was observed by the authors.
The results of an inhalation bioassay for carcinogenesis with
Sprague-Dawley rats and Swiss mice have been published (Maltoni et al., 1981).
The experimental design and results of these assays are detailed in Section 9>5,
but it should be noted that exposure to 250 to 150 ppm, 50, 10 or 5 ppm EDC for 7
hours/day, 5 days/week for 78 weeks elicited no treatment-related changes in body
weight or survival ability in rats of either sex or male mice. There was,
however, decreased survival ability in female mice exposed to the high level of
EDC. The animals exposed to 250 ppm at the onset of the study exhibited signs of
toxicity (i.e., ruffling of hair, hypomotility and loss of muscle tone),
prompting dose decrease to 150 ppm after several weeks. Systemic pathological
examinations were performed on all the major organs of all animals with or
without pathological changes, but the only non-neoplastic histologic effects
reported were some "regressive" changes in the liver and adrenal glands that were
not dose-related (Cox, 1982).
In a related study with Sprague-Dawley rats, Spreafico et al. (1981) inves-
tigated the effect of inhalation exposure to identical levels of EDC (250 to 150,
50, 10 and 5 ppm) on blood clinical chemistry parameters. Histological examina-
tions were not performed on the rats exposed to EDC in this study. Eight to 10
animals per dose level were sacrificed after 3, 6, 12 or 18 months of 7
hours/day, 5 days/week exposure. Rats used for the 3, 6 and 18 month raeasure-
9-113
-------
ments were started at 3 months of age, and the 12 month determination was made
with animals that were exposed from 14 months of age.
The results suggest that long-term inhalation exposure of one year or longer
to EDC at levels of 150 ppm does not produce marked toxicity in rats exposed from
3 months of age (Spreafico et al., 1981). There were no statistically
significant changes between treated and control animals with respect to
circulating levels of red blood cells, total white blood cell numbers, platelet
numbers, relative percentages of lymphocytes, granulocytes and monocytes, and
circulating protein levels. Percent albumin values were significantly increased
in both sexes at 3 and 18 months but not at 6 months, but no clear dose-response
relationship was apparent. Gamma globulin percentages were significantly lower
at 3 months in the 150 ppm EDC group. Although changes were not apparent at 6
months, at 18 months there was a significant decrease observed only at 5 and 10
ppm.
No treatment related effects were found on levels of y-glutarayl trans-
peptidase (y-GT), serum glutaraic-oxalacetic transaminase (SCOT), serum
glumatic-pyruvic transaminase (SGPT), serum alkaline phosphatase, bilirubin or
cholesterol (Spreafico et al., 1981). A slight, but not significant, increase in
creatine phosphokinase (CPK) level was seen at 18 months in males exposed to 50
and 150 ppm EDC. Lactic acid dehydrogenase (LDH) levels were significantly
elevated after 3 months of exposure in both treated male and female groups, but a
dose-response relationship was apparent only in males. At 6 months, there was an
insignificant increase in LDH levels. At 18 months, significantly higher LDH
levels were observed only in males exposed to 5, 50 and 150 ppm EDC without an
apparent dose-response relationship. No clear changes were observed for BUN
levels, serum glucose levels or uric acid levels (Spreafico et al., 1981).
No consistent and significant differences in a battery of urinary tests (pH,
proteins, bilirubin, glucose, hemoglobin, erythrocytes, leukocytes, epithelial
9-111
-------
cells, casts, cystals, mucus and microorganisms) were observed after 18 months
of inhalation exposure (Spreafico et al., 1981).
Changes in liver and kidney function were indicated, however, in rats
exposed to EDC at 14 months of age. SGPT levels were significantly increased in
rats of both sexes exposed to 50 and 150 ppm. y-GT levels were significantly
elevated in females treated with 50 and 1 50 ppm. SCOT levels were significantly
elevated at the two lower doses (5 and 10 ppm), and significant decreases were
observed at the 2 higher doses (50 and 150 ppm). Cholesterol levels were signi-
ficantly lower at 50 and 150 ppm in both sexes, but there were no significant
changes in bilirubin, CPK, alkaline phosphatase, LDH or glucose levels. Signifi-
cantly higher levels of uric acid were found at 50 and 1 50 ppm and higher blood
glucose levels were observed at 150 ppm. No significant changes were found in
blood elements or in the battery of tests conducted on urine in the rats that
were initially exposed at 1U months of age (Spreafico et al., 1981).
9.2.3. Summary of Acute, Subchronic and Chronic Toxicity.
9.2.3.1. INHALATION EXPOSURE — Information regarding the acute effects of
inhaled EDC in humans is available primarily from cases of occupational exposure
(see Section 9.2.1.1). Although the reported cases had both fatal and non-fatal
outcomes, many of the reports were foreign and none of the reports provided
quantitative exposure data. Further, although EDC was usually reported to be the
primary vapor to which the workers were exposed, the preponderance of exposures
were to poorly characterized mixtures of EDC and other solvents, or to EDC of
unknown purity. Symptoms and signs of acute inhalation exposure were often
indicative of CNS and gastrointestinal disturbances, and clinical evidence of
liver and kidney dysfunction, as well as irritation of the respiratory tract and
eyes, have also been observed. Death was usually ascribed to respiratory and
circulatory failure, and autopsies frequently revealed pulmonary edema and
9-115
-------
congestion, cellular degeneration, necrosis and hemorrhagic lesions of most
internal organs (e.g., liver, kidneys, spleen, lungs and respiratory tract,
brain, stomach and intestines).
The results of acute experimental studies with three or four subjects
suggest that the threshold of light perception (duration of exposure not stated),
depth of breathing (1-minute exposure) and vasoconstriction (30-second and 15-
minute exposures) increased with exposure to EDC (Borisova, 1957). Spirographic
and plethysmographic responses reportedly first differed from baseline values at
1.5 ppo and appeared to be dose-related to 12.4 ppm, the highest level tested;
exposure to 1 ppm (the lowest level tested) reportedly had no effect on the
physiologic end points. Although these Russian experiments appear to indicate a
threshold of toxic action, the reliability of the data is uncertain due to the
small number of subjects tested and an absence of reported baseline measurements
for each subject. Some assurance of data credibility can be gleaned from the
fact that the range of concentrations tested (i.e., =1-12.4 ppm) represented
corroborated sub-threshold, threshold and above threshold odor perception
concentrates. Borisova (1957) determined the odor perception threshold of EDC to
be =6 ppm, and this value is consistent with an absolute odor threshold of 6 ppm
that was determined by Hellman and Small (1973, 1974) in a Union Carbide study.
Further, the sensitivity of the analytic method used by Borisova (nephelometry)
appears to be more than adequate to determine EDC at the reported experimental
levels. Hellman and Small (1973, 1974) also determined an odor recognition
threshold of 40 ppm for EDC.
Acute inhalation studies with a variety of animal species indicate that the
effects of single EDC exposures are similar to those in humans. Immediate
symptoms of toxicity are related to CNS depression (e.g., narcosis) and delayed
histopathologic changes (e.g., congestion and degenerative effects) have been
9-116
-------
observed primarily in the liver, spleen, kidneys, lungs and adrenals. As
detailed in Section 9.2.2.1.3 and Table 9-21, the severity of effects are depen-
dent upon duration and concentration of exposure. For rats exposed for 5-8
hours, it appears that adverse effects are not elicited by exposure at 200 ppm
(Spencer et al., 1951); signs of intoxication first appear at 300 ppm (Spencer et
al., 1951) and mortality at >600 ppra (Heppel et al., 1974; Carpenter, 1949).
Concentrations as high as 12,000 ppm were inhaled by rats for 0.1 hours without
adverse effects (Spencer et al., 1951).
The dose-response relationships for acute inhalation exposure are not as
thoroughly characterized for other species. Corneal opacity was observed in dogs
exposed via inhalation to 1000 ppm for 7 hours (Heppel et al., 1944). Signs of
intoxication were not present in guinea pigs that were exposed to 1200 ppm EDC
for 8 hours (Sayers et al., 1930), but exposure to 1500 ppra for 7 hours produced
mortality in mice, guinea pigs and rabbits (Heppel et al., 1945). Histopatho-
logical effects (e.g., pulmonary congestion/edema, alterations to kidneys,
liver, spleen and adrenals) were generally apparent in rats, mice, guinea pigs
and rabbits at concentrations of >3000 ppm (Spencer et al., 1951 ; Heppel et al.,
1945; Sayers et al., 1930). Spencer et al. (1930) felt that narcosis that was
elicited by concentrations <3000 ppra was attributable to organ injury rather than
depression of the CMS.
Limited quantitative data are available, primarily from the foreign litera-
ture, regarding the effects of repeated EDC exposure on humans. The studies that
are available are deficient because control data are lacking and because dura-
tions of exposure and numbers of subjects were poorly characterized. The avail-
able data suggest that chronic intermittent exposure in the range of 10-37 ppra
may represent a lowest-observed-adverse-effect level (LOAEL) but, as summarized
9-117
-------
subsequently, the data do not provide a basis for identifying a no-observed-
adverse-effect level (NOAEL) or a no-observed-effect-level (NOEL) for humans.
Case reports (Byers, 19**3; Rosenbaum, 19^7; Guerdjikoff, 1955) and a
foreign health survey (Cetnarowicz, 1959) of workers who were exposed repeatedly
to 1 ,2-EDC vapor indicate that typical symptoms and signs of acute poisoning
developed with exposure to concentrations in the range of =60-200 ppm. In the
Cetnarowicz (1959) survey, 16 workers who were exposed to =10-200 ppm for 2-8
months were examined; 10 of 10 who were exposed to 60-200 ppra complained of
adaptable eye irritation and 6 of the 10 experienced typical symptoms of expo-
sure, but only 1 of 6 exposed to 10-37 ppm were symptomatic. Positive symptoms
and signs of exposure, particularly those indicative of mucous membrane irrita-
tion, were also observed in 90 of 118 agricultural workers who were concurrently
exposed to EDC vapor (=15-60 ppm) and liquid (prolonged dermal contact)
(Brzozowski et al., 1954).
The results of two other foreign health surveys of workers exposed to EDC in
the range of the Cetnarowicz (1999) low exposure group (10-37 ppm) support
Cetnarowicz's apparent finding that overt symptoms may not be the predominant
effect of lower exposures; there appears, instead, to be a prevalence of neuro-
logic and clinical effects. Rosenbaum (19**7) examined 100 workers who were
exposed to <25 ppm for 6 months to 5 years and found disturbances that included
"heightened lability" of the autonomic nervous system, increased hidrosis and
frequent complaints of fatigue, irritability and sleeplessness, but no hemato-
logic or functional changes of the internal organs. In the study by Kozik
(1957), which is particularly notable for its reliable exposure data, workers
exposed to time-weighted average concentrations of 10-15 ppm experienced
increased morbidity, particularly from gastrointestinal, liver and bile duct
disorders. The number of workers surveyed in this study was not clearly stated
9-118
-------
in the available translation, but it was >83. The occurrence of overt symptoms
in these workers was not discussed, but impaired performance on control reaction
time tests suggested a possible effect on the nervous system.
Case reports of workers who were repeatedly exposed to unknown concentra-
tions of EDC also suggest that neurologic effects such as nervousness, irrita-
bility, tremors and loss of reflexes (McNally and Fostvedt, 19^1 ; Delplace et
al., 1962; Suveev and Babichenko, 1969) and complaints of skin and mucous
membrane irritation (Suveev and Babichenko, 1969; Rosenbaum, 1947; Guerdjikoff,
1955) are more prevalent in chronically exposed workers than in workers who were
acutely exposed.
Studies with several species of animals provide information regarding the
threshold region of effects for subchronic and chronic inhalation exposure to
EDC. Three studies in which rats, guinea pigs, rabbits, cats and monkeys were
exposed to 100 ppm CDC for 7 hours/day, 5 days/week for 4-6 months have shown no
treatment-related adverse effects on survival, growth, hematology, clinical
chemistry, organ weights or histology (Heppel et al., 1946; Spencer et al., 1951 ;
Hoffman et al., 1971). Adverse effects were also not noted in juvenile mice that
were exposed to 100 ppm EDC for 19 exposures (Heppel et al., 1946). The apparent
NOAEL of 100 ppm is supported by the results of comprehensive chronic studies
with rats in which similar weekly exposures to EDC (7 hours/day, 5 days/week) at
concentrations of 5, 10 or 50 ppra for 18-19 months had no significant treatment-
related adverse effects on histology (Maltoni et al., 1981) or blood or urine
clinical chemistry indices (Spreafico et al., 1981) in rats of either sex;
exposure to 150 ppm elicited decreased survival in female rats, but no clear
effects on clinical chemistry parameters in either sex. There was an indication
of renal and liver damage (increased SGPT, increased serum Y~GT» decreased
cholesterol, increased uric acid, increased blood glucose) in mature (i.e., 14-
9-119
-------
months-old) rats that were similarly exposed to 50 OP 150 ppm EDC for 12 months,
but hematologic effects were not found and histological examinations were not
conducted.
Subchronic exposure to 200 ppm EDC (7 hours/day, 5 days/week) produced toxic
effects in several species, but the severity of effects appeared to vary with
investigation. Heppel et al. 0946) observed mortality among rats, mice and
guinea pigs exposed to 200 ppm EDC, but not among rabbits or monkeys, and histo-
logical lesions were not found in any of these species. Spencer et al. O951 )
did not observe mortality in rats or guinea pigs similarly exposed to 200 ppm
EDC, but other effects were noted in the guinea pigs (decreased body weight gain
and increased liver weight in males, slight hepatic degeneration in males and
females). Rabbits and monkeys were not tested by Spencer et al. (I95t)> An
unequivocal frank-effect level (FED of UOO ppm is representative for EDC as
indicated by production of high mortality and histopathological alterations in
the liver and kidney in rats and guinea pigs after a few exposures (Heppel et
al., 19^6; Spencer et al., 1951). Spencer et al. (1951) reported that exposure
to 100 ppm EDC had no effect on rabbits (as reported at 200 ppm), but Heppel et
al. O9U6) observed some mortality at this level. Exposure to 500 ppm EDC
produced high mortality in rats, guinea pigs and rabbits within a few exposures
(Hoffman et al., 1971).
9.2.3-2. ORAL EXPOSURE — Limited data are available on the effects of
oral exposure to EDC. Human case reports of accidental or intentional ingestion
of EDC indicate that the toxic response to oral exposure is similar to that of
inhalation exposure (Section 9.2.1.1 .1 .1 ). Ingestion of quantities of EDC esti-
mated to have ranged from 8-200 ml have been reported to be lethal (see Table
9-18). This is equivalent to =110-3570 mg/kg if it is assumed that an average
human weighs 70 kg. Median lethal doses in rats (McCollister et al., 1956; Smyth
9-120
-------
et al., 1969) and mice (Heppel et al., 1945) have been reported in the range of
700 mg/kg. Higher oral doses {up to 2.5 g/lcg) were tolerated by dogs without
mortality (Kistler and Luckhardt, 1929). The apparent higher tolerance in dogs
and some humans may be related to the ability or these species to vomit. Dose
information was not reported in human case reports of non-lethal ingestion of
EDC.
Administration of 189 mg/kg/day EDC in the drinking water for 90 days caused
a decrease in growth rate in mice, but no significant effects on organ weights,
hematology indices, clinical chemistry indices, blood coagulation or immune
system function, although a trend towards immunosuppression was noted (Munson et
al., 1982). Similar administration of 24 or 3 mg/kg/day EDC in the drinking
water for 90 days had no effect on mice.
The only dose-response data that are available for chronic oral exposure to
EDC are the results of a 78-week NCI carcinogenesis bioassay with rats and mice
(NCI, 1978). Administration of 95 mg/kg/day EDC via gavage (5 days/week) caused
early and severe mortality in rats of both sexes, and ^47 mg/kg/day caused
decreased survival in male rats {after 90 days) and female rats (throughout the
experiment). Non-carcinogenic toxic effects appeared to be the cause of the
deaths, as indicated by the presence of a variety of lesions including broncho-
pneumonia and endocardial thrombosis. Body weight depression was not found in
any of the treated male or female rats. Treatment-related weight depression and
mortality was found in female mice that were similarly exposed to 299 rag/kg/day,
but not in females exposed to 1^9 mg/kg/day or in males exposed to 195 or 97
mg/kg/day. The presence of tumors in the high dose female mice suggested,
however, that the deaths may have been tumor-related.
9-121
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9.3- REPRODUCTIVE AND TERATOGENIC EFFECTS
Murray et al. (1980, unpublished) performed a subchronic inhalation study
to determine the effects of ethylene dichloride (1,2-dichloroethane, EDO on the
reproductive ability of rats and effects on their offspring. Tne results of this
study were reported at the fifth annual Banbury Conference (Rao et al., 1980).
Twenty to thirty male and female Sprague-Dawley rats were initially exposed to
25, 75, and 150 ppm EDC (lot no. TAC06228, analyzed as 99.98* pure by Dow
Chemical Laboratories) for 60 days (6 hrs/day. 5 days/week for 12 weeks), and
they were successively bred to produce two litters, the F.. and F1g. The
breeding protocol consisted of caging one male and one female rat for 4
consecutive days, and remating any females that had not mated (no sperm in
vaginal smear) with a different male after a 3 day rest period. The results from
remated animals were combined with those of the first breeding. The females were
exposed to EDC throughout breeding, gestation and lactation (6 hrs/day, 7
days/week) except for day 21 of gestation to day U postpartura to allow for
delivery and rearing of the young. A second mating, for the production of the F
generation, took place after the F litter was sacrificed (day 21 of birth).
During the seventh week of this study, both control and treated animals
suffered from symptoms of sialoacryoadenitis (red crusty material around the
eyes and nose and conjunctivitis), which was thought to have resulted from a
viral infection. The authors noted that more males than females contracted the
disease, but suggested that this greater incidence was related to the housing
accommodations (males were caged away from females) and not to any greater
susceptibility of the males for developing infection after EDC exposure. All
9-122
-------
animals (both control and experimental) recovered one to two weeks after the
onset of the disease with no recurrence for the duration of the study.
Murray et al. (1980) reported that the physical well being of the parental
generation was not significantly affected by EDC exposure. However, during the
first and second weeks of exposure, the females consumed significantly less food
than the controls, although for the rest of the study, there were no alterations
in food consumptions. In the males, food consumption was both sporadically
increased and decreased throughout the study with a general trend for increased
food consumption in males exposed to the highest concentration of EDC (150 ppm)
for longer durations of exposure (weeks 15 to 29).
There was a statistical increase in the absolute liver weight at the 150 ppm
level, and a statistical linear trend for increased liver weights in adult male
rats exposed to EDC (Murray et al., 1980). However, this trend was not observed
for liver weights in relation to body weight. All adult males displayed some
degree of chronic renal disease. However, the severity of the disease did not
appear to be related to treatment. Both control and experimental females
displayed signs of renal disease. There was a greater incidence of inflammation
and edema of the salivary glands in males exposed to 150 ppm. Three deaths
occurred during the course of the study, but these deaths did not appear to be
related to EDC exposure.
The fertility and survival index on days 1, 7, 14, or 21 was not
statistically different at any dose level. However, the greatest pup mortality
was observed in groups exposed to 150 ppm between days 14 and 21. The sex ratio,
average number of live pups per litter and neonatal body weight were not
affected. There were sporadic, statistically significant differences in
9-123
-------
reproductive indices of treated versus control groups. However, these did not
appear to be attributable to EDC exposure. A few neonates exhibited external and
internal malformations. However, the incidences of these effects were sporadic
and statistically insignificant, reflecting natural variability and not the
result of EDC exposure (Murray et al., 1980). Therefore, it was concluded that
EDC exposure, under these conditions, did not adversely affect reproduction in
rats or the development of their offspring.
Lane et al. (1982) evaluated the effects of EDC on the reproductive
capability of mice using a multigenerational reproductive protocol modified for
screening teratogenic and dominant lethal effects. Animals were exposed to EDC
for 35 days, then 10 male and 30 female mice (parental generation FO) were mated
to produce the first set offspring (FIA)« After the F.. were weaned, the FO
adults were remated to produce the second set offspring (P-m)* * parental stock
was chosen from the FIQ litter (30 females, 10 males) to produce a second
generation of offspring ^y^' After weaning, the FIQ adults were remated to
produce offspring (F2B^ for use in teratology and dominant lethal screening
tests.
In this study, the EDC (Aldrich Chemical Co., 99$ pure) was dissolved in a
1$ solution of Emulphor EL-620 (GAF Corp., Linden, NJ) and then further diluted
with deionized water to concentrations of 0.03, 0.09, and 0.29 mg/mJl for nominal
doses of approximately 5, 15, and 50 mg/kg/day. The test animals were
continuously maintained on EDC solutions or control solution (deionized water,
or water containing 0.17 mg/mZ p-dioxane dissolved in 1.0$ Emulphor solution).
9-121
-------
Fresh drinking solutions were prepared twice weekly and placed in amber glass
bottles with cork stoppers and stainless steel drinking tubes. The authors
reported no decrease in fluid consumptions in either the EDC or the solvent
control groups.
Lane et al. (1982) reported that EDC produced no treatment-related signs of
toxicity such as lowered body weights or gross pathological changes in major
organs. There were no significant differences in the fertility index or
gestation index observed in the FIA, FIB or F?A generations. There were sporadic
incidences of increased mortality throughout the generations but these were not
dose-related; the reason for the deaths was not apparent at necropsy. There were
no differences in the litter sizes at birth, pup body weight, or survival of pups
at days H and 21 of birth. There was a decrease in the survival indices for the
F2A Seneration as compared to values for the F , and FIQ generation, but these
decreases were not dose related. In the dominant lethal screening tests, there
were no statistically significant dose-related effects. However, both increases
and decreases were observed in the ratios of dead to live fetuses. In the
teratology screening, continuous administration of EDC produced no apparent
adverse reproductive effects or observable abnormalities. It should be noted
that continuous exposure used in this study is not a recommended protocol for
evaluating teratogenic effects. In teratology testing, the duration of
exposures is usually limited to the period of organogenesis. Also in the study,
the F-c skeletal specimens were lost and, therefore, these effects could not be
evaluated.
From this study (Lane et al., 1982), it is not possible to determine
conclusively whether EDC has the potential to cause adverse reproductive or
teratogenic effects because the doses were not high enough to produce any adverse
9-125
-------
effects, including maternally toxic effects. However, under the conditions of
the experiment, it appears that EDC given in drinking water (nominal doses of 5,
15, 50 mg/kg/day) produced no observable adverse reproductive effects in Swiss
ICR mice.
Schlachter et al. (1979) conducted teratology testing in 16 to 30 Sprague-
Dawley rats and 19 to 21 New Zealand White rabbits. The animals were exposed to
inhalation to either 100 or 300 ppm EDC (Lot Nos. TAC201851L and TA022&51L,
analyzed as >99.9$ pure by the Dow Chemical Laboratories). The rats were exposed
7 hrs/day on days 6 through 15 of gestation. Two thirds of the maternal rats died
when exposed to 300 ppm EDC in contrast to no deaths occurring in the control and
100 ppm groups. In the 300 ppm group, only one rat in six survivors had
implantation sites; all of these sites were resorbed. Two additional rats at the
300 ppm level showed early implantation sites when the uterus was stained with
sodium sulfide. In the 100 ppm group, three rats delivered early on day 21;
however, there was no indication of embryo or fetotoxicity in the offspring. In
addition, the rest of the rats at the 100 ppm level delivered normally with no
alterations in litter size, number of resorptions or fetal body measurements
(crown to rump length).
At the 300 ppm level, one of the six surviving rats had a decrease in mean
body weight gain, while in the 100 ppm level, there was an increase in body
weight on days 6, 8, 10, 16, and 21 of gestation. At the 300 ppm level, the
absolute liver weight was decreased, while the relative liver weight was
increased; at the 100 ppm level there was no difference. In the 100 ppm group,
there was an increase in water consumption on days 15-17 and 18-20 of gestation,
9-126
-------
but food consumption was not altered. There was no increase in the occurrence of
malformations with only isolated sporadic incidents observed within the range of
normal variability.
In this teratology study (Schlachter et al.F 1979), four out of twenty-one
rabbits in the 100 ppm group, and three out of nineteen rabbits in the 300 ppm
group died. Three control rabbits delivered early (days 15, 28, and 29 of
gestation) as did one animal at the 300 ppm level (day 28 of gestation). Two
animals that died at the 100 ppm level also aborted on days 26 and 28 of
gestation. There was no effect on mean litter size, incidence of resorptions or
fetal body measurements (crown to rump length) in animals delivering normally.
Staining of the uterus revealed only one additional implantation site in control
animals. The mean body weights of pregnant animals were generally comparable to
those of controls, but there was an increase in mean body weight gains in the 300
ppm group on days 19 through 28 of gestation. A slight, statistically
insignificant increase in both absolute and relative liver weights were observed
in both the 100 and 300 ppm groups. There was no increase in the incidence of
malformations, although sporadic occurrences within the range of normal
variability were reported. It was concluded from this study that EDC exposure
does not produce adverse effects on the developing conceptus.
Several Russian studies by Vozovaya (1971, 1975, 1976, 1977) have reported
that EDC produces a number of adverse reproductive outcomes which include
lengthening the total estrous cycle, changes in the duration of various stages of
the estrous cycle, increases in the number of perinatal embryonic deaths,
decreases in the weight of newborn animals, and decreases in the weight gain of
offspring after birth. In addition, EDC was reported to concentrate in the
9-127
-------
placenta, amniotic fluid and fetal tissue. However, It Is difficult to evaluate
or seriously consider these reports since they are presented with insufficient
detail or critical scientific review. Inadequacies in these reports include:
lack of original data, various techniques and tests mentioned but results not
presented, no information on the source or purity of the chemical, statistical
analysis mentioned, but the type of statistical tests not stated.
The above comments apply similarly to the study by another Russian
scientist, Urusova (1953), who reported that EDC accumulated in the milk of
nursing mothers. This is obviously an important observation. However, it cannot
be accepted as scientific evidence. This report was presented in a subjective,
anecdotal manner with many inadequacies in reporting. These inadequacies
include lack of details concerning the numbers of women involved in the study,
the women's age or physical status, the concentrations and duration of EDC
exposure after dermal exposure, the repeatability of this finding and number of
samples analyzed.
Because of the concern for EDC contamination in mother's milk, Skyes and
Klein (1957) conducted a study to determine whether oral administration of EDC
might pass into the milk of cows. Only five cows were used in this study; two
cows were exposed to 100 ppm EDC for 22 days; two cows were administered 500 ppm
EDC for 10 days followed by 1000 ppm for 12 days; one cow was used as the control.
Two different breeds of cows, Holstein and Jersey, were used, but it was not
stated which cows received which dose. Seven milk samples collected over a three
week period containing no more than 0.4 ppm EDC. However, the authors stated
that this amount was too low for accurate detection by the Volhard titration
method used in this study. The samples collected from cows ingesting EDC had
consistently higher concentrations of EDC than the controls, but because of the
9-128
-------
small sample size and the lack of sophisticated analytical procedures, it is not
possible to make firm conclusions as to the amount of EDC entering the milk
supply. More importantly, this study was not designed to evaluate the possible
health consequences of EDC ingested in the milk of nursing offspring. However,
recent pharmacokinetic studies do indicate that EDC has a tendency to accumulate
in fatty tissues (Spreafico et al., 1980) although the reproductive health
effects of EDC in breast milk have not been evaluated.
9.3.1. Summary and Conclusions. The available scientific information on the
potential of ethylene dichloride (EDC) to affect reproductive or developmental
processes adversely Includes a teratology test conducted in rats and rabbits
(Schlachter et al., 1980), a single-generation reproductive test conducted in
rats (Murray et al., 1980), a multigenerational reproductive test conducted in
mice (Lane et al., 1981), several reports by Russian scientists describing a
number of adverse reproductive effects observed in laboratory animals and man
(Vozovaya 1971, 1975, 1976, and Urusova, 1953), and an investigation measuring
the amount of EDC found in the milk of cows after ingesting EDC (Sykes and Klein,
1953).
The results of reproductive and teratogenicity testing (Murray et al.,
1980, Schlachter et al., 1979; and Lane et al., 1982) indicate that EDC has
little potential for producing adverse reproductive affects or for adversely
affecting the developing conceptus, except when the mother is exposed to doses
high enough to produce maternal toxicity. The Russian studies describing a
variety of adverse reproductive effects associated with EDC exposure do not
provide conclusive scientific evidence because the studies were inadequately
performed or reported. The study of EDC in the milk of cows is inadequate since
only a few animals were used and the biological effects of EDC-contaminated milk
were not investigated.
9-129
-------
In conclusion, the available studies indicate that EDC has little ability to
affect the reproductive or developmental processes adversely in laboratory
animals except at maternally toxic levels. However, it should be noted that
these studies have inadequacies that weaken the strength of this conclusion. The
multigenerational study by Lane et al., 1982 does not include a range of doses
that include maternally toxic doses. The study by Murray et al. (1980) does not
evaluate reproductive effects for more than a single generation. The studies by
Sykes and Klein (1953) on the effects on lactation are inadequate. In addition,
no properly conducted epidemiological study has been done to evaluate the effects
of EDC on human reproduction. Therefore, although the studies to date indicate
that EDC does not pose a specific hazard to reproductive or developmental
systems, this cannot be conclusively established, especially for humans, without
additional studies. The chemical similarity of EDC to ethylene dibromide (EDB)
and 1,2-dibromochloropropane (DBCP) might suggest that additional testing
related to testicular toxicity would be appropriate.
9-130
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9.1. MUTAGENICm
A review of the literature indicates that ethylene dichloride (EDO has been
tested for mutagenic activity in bacteria, plants, Drosophila, mammalian cells
in vitro, and intact rodents. These studies are discussed below and are
summarized in Tables 9-27 to 9-32. The reader may also refer to published
reviews of the mutagenic potential of EDC (e.g., Fishbein, 1976, 1979; Fabricant
and Chalmers, 1980; Rannug, 1980b; Simmon, 1980).
9.4.1. Gene Mutation Studies
9.1.1.1. BACTERIAL TEST SYSTEMS — Many investigators have studied the
ability of EDC to cause gene mutations in bacteria (Table 9-27). Most reported
marginal positive responses, approximately two-fold above background, without
metabolic activation and stronger positive responses with exogenous hepatic
metabolic activation, indicating that EDC is mutagenic to bacteria by itself but
that metabolites, such as S-(2-chloroethyl)-L-cysteine, are more potent
mutagens.
Ethylene dichloride has been reported positive in four Salmonella/microsome
plate incorporation assays (McCann et al., 1975; Rannug 1976; Rannug and Ramel,
1977; Rannug et al., 1978), in assays testing the mutagenicity of bile obtained
from EDC-perfused rat livers or livers from EDC-treated mice (Rannug and Beije,
1979), and in two Salmonella spot tests. In one of the Salmonella spot tests
(Brera et al., 1971), an analysis of duplicate experiments carried out on at least
three different occasions revealed a twofold increase in revertant counts for
strains TA1530 and TA1535 (mean values of 50 and 51 revertants on treated plates
versus 23 and 26 in control plates for TA1530 and TA1535, respectively). No
difference in revertant counts was noted for strain TA1538. This response is
consistent with that expected for an alkylating agent. The authors stated that
9-131
-------
Summary or Kutagenlcity Testing of EDC: Gene Mutations in Baateria
Reference
Bren et al.,
1971
Teat
System
Salmonella
(spot teat)
Strain
TA1530
TA1535
TA153B
Activation
System
Hone
Chemical
Information
10 |imol on filter disk
Source: Not given
Purity: Not given
Results Comments
Weak positive 1. Could not perform
plate incorporation
testa because of
volatility.
Principe et
al., 1981
Salmonella revertants
Salmonella/
mammalian
mlcrosooe
assay (spot
teat)
S.. coellcolor
forward mutation
assay to Str
A. nldulans
forward mutation
to 8-AGr
TA1535
TA1537
TA1538
TA98
TA100
p<0.018
-59
0
39
17
19
82
188
EDC
Water
Chloramphenicol
Dose u£/plate
+S9
100 0
16 33
8 10
12 21
83 77
188 171
Dose iit/plate Survival t
0
2
10
20
100
0
250
500
100
100
100
100
100
100
100
42
TA1530 Ml 535
50 51
.23 26
20 31
100
103*
a
27
81
169
Strr /plate
2.5 + 0.6
0.2 7 0.2
0.7 T O.U
0.7 * 0.1
1.0 * 0.6
8-AG/plate
2.5 i- 0.9
2.0 + 1.1
1.0 7 0.7
TA153B
19
19
14
1. Positive controls
indicated system
working properly.
2. Positive results In
Salmonella in spot
test with TA1535.
3. Mo precautions taken
to prevent excessive
evaportlon of EDC
and ensure adequate
exposure.
1. Toxicity results
indicate exposure
was minimal.
-------
TABLE 9-27 (cent.)
Reference
McCann et al.,
1975
Test
System
Salmonella/
nlcrosome assay
(plate teat)
Activation
Strain System
TA100 PCB-induced
rat liver S9
mix
Chemical
Information
Concentration Tested:
1.3 * 104 Mg/plate
(13 umol)
Source: Aldrlch
ChBDlcal Co.
Purity: Not given,
but stated
to be highest
purity
Results
Negative or at
best only marginal
positive response.
Induced 25
colonies plate
above background.
(0.19 revertants/
linol) In TA100.
Comnents
1. Non-mutagenlc In
this study.
Reproducible dose-
response curves not
obtained.
2. Metabolic activation
did not increase
positive response.
3. Chloroethanol and
Rannug, 1976
Salmonella/
nlcrosome assay
(plate teat)
TA1535 Liver fractions
from Sprague-
Dauley or R
strain Wlstar
rats Induced
with phenobar-
bltal with and
without NADPH-
generatlng syste
and with and
without gluta-
thlone S-trans-
f erases A, B
and C.
Concentration tested:
Up to 60 umol/plate
Source: BDH Chemicals, Ltd.
Purity: Not given but
reported to be
checked by glass
capillary column
chromatography
using a flame
loniiatlon detector
Marginally
positive
without activation
(two-fold In-
creases positive
response with
activation (ten-
fold increases).
Spontaneous back-
ground 8-14
revertants/plate.
chloroacetaldehyde
(two putative
intermediates in the
metabolism of EDC In
mammals) tested
positive (i.e., 0.06
and 716 revertanta/
umol, respectively).
1. EDC activated by the
liver cytosol
fraction; mixed-
function oxygenases
not Involved.
2. NADPH-independent GSH
S-transferase dependent
activation.
3. Strain differences noted
In ability to metabolize
• EDC.
M. Thought that mutagenlclty
of EDC after activation
caused by formation of
highly reactive half
sulfur mustard,
S-(2-chloroethyl)-L-
cystelne.
-------
TABLE 9-27 (oont.)
Reference
Rannug and Ranel,
1977
Teat
System
Salmonella/
Blcroaoue assay
(plate teat)
Activation
Strain System
TA1535 S9 nix from
livers of un-
induced male
R strain
Wlstar rata
plua NADPH-
generatlng
system
Chemical
Information
Concentration tested:
Up to 45 |imol/plate
Source: BDH Chemicals, Ltd.
Purity: Not given
Results
Positive reaponae
(two- fold Increaae
without activation;
nearly ten-fold
Increase with
activation.)
Negative control a
yielded roughly
15 revertanta/
plates.
Comments
1. Compared mutagenlcity
of EDC tar with EDC.
The level of EDC
present at the highest
dose tested for EDC
tar would only exert
a weak mutagenlc
effect, yet a strong
reaponae was
observed .
Rannug et al.,
1978
Salmonella/
mlcrosome assay
(plate teat)
TA1535
ui
Rannug and Belje,
1979
Salmonella/
Isolated
perfused
rat liver
TA1530
TA1535
Rat liver
mlcrosone
system with
and without
NADP
Isolated
perfused liver
from male R
strain Wlstar
rata
Concentration tested:
15 umol/plate
Source: EDC given BDH
Chemicals, Ltd.
Purity: Not given for
EDC, but checked
using glass
capillary chroma-
tography using
a flame ioniza-
zation detector
Concentration tested:
0.1 ml (1.3 mM)
for up to 4 hours.
Marginal positive
response without
activation (two-fold
increase 24.8 +
3.06 at 15 umol
vs. 13.0 + 1.76
control);
positive reaponae
with activation
(ten-fold Increase)
Independent of
presence of NADP.
Positive. Highest 1.
response 15-60
minutes after
addition of EDC
(45-60 revertanta
compared to 7-10
in controls).
Activation of EDC
tar dependent on
NADPH. EDC activa-
tion Independent
of NADPH.
EDC major component
of EDC tar.
Hutageniclcity of
tar Increased with
metabolic activation
only with addition
of NADP.
Mutagenlcity of EDC
tar not solely due
to EDC.
Positive
responses consis-
tent with conju-
gation of EDC
with glutathlone.
Rannug and Beije,
1979
Salmonella/
(plate test)
TA1535
Bile from male
CBA mice
80 mg/kg EDC l.p.;
removal of liver and
collection of bile 30
and 60 minutes later.
Positive. Greater
than two-fold
increases with bile
from liver removed
30 minutes after
addition of EDC
(28.8 + 27 rever-
tanta compared to
11.3 + l.D.
-------
TABLE 9-27 (cent.)
Reference
King et al.,
1979
Test
System
Salmonella/
nlcroaooe assay
(plate test)
E. coll K 12/
3"»3/ll3
(suspension test
and intrasangulneous
host-mediated assay)
Strain
TA1535
TA100
TA1537
TA153B
TA98
Activation
System
PCB-lnduced
rat liver S9
Chemical
Information
Concentration bested:
36 umol/plate
10 mM (suspension assay)
2 mmol/kg l.p. injection
female NMfll mice
Results
Negative
Negative
Negative
Comments
1 . Standard plate
incorporation test
was conducted. No
precautions were
taken to prevent
excessive evapo-
ration of EDC.
Nestnan et al.,
1980
vO
i
Salmonella/ TA1535 PCB-lnduced
nicroaone assay TA100 rat liver S9
(plate test and TA1537 mix.
and desiccator TA1536
exposure). TA98
Source: Herck Co.
Darmstadt, FRG
Purity: Not given.
Stated that samples
had correct melting
point and elemental
analysis.
Concentration tested:
Up to 9 mg/plate (91
IIDOI) In desiccators.
10 mg/plate In plate tests.
Source: Chem Service
Purity: Not given
Solvent: DHSO
Negative in
standard test.
Positive in
desiccator testing
in strain TA1535.
1. Stated that
maximum yield with
TA100 is 20
revertants above
background (i.e.,
negative). For
TA1535 a doubling
of mutant colonies
observed. No
other data pre-
sented.
2. Cannot adequately
evaluate results.
-------
TABLE 9-27 (cont.)
Teat
Reference System
Stolzenberg and Salmonella/
Hlne, 1980 micro3onie
assay (plate test)
Activation
Strain System
TA100 PCS- induced
rat liver S9
nix (2 ng
protein/0.5 ml)
Chemical
Information
Concentration tested:
Up to 10 |imol/plate
Source: Aldrich
Chemical Co.
Purity: 99% pure
Results
Negative
Comments
1. All compounds
tested In trip-
licate with and
without S9 mix.
2. Experimental
values minus
background
revertants.
Revertants
Barber et al . , Salmonella/
1981 oicrosone assay
(vapor exposure)
TA1535 PCB-induced
TA100 rat liver S9
TA1538 nix
TA98
Concentration tested:
Up to 231.8 (imol/plate
as determined by GLC
analysis of distilled
water samples.
umol/plate -S9 +S9
10"1 0 0
1 0 0
10 15 No
Toxic
Negative in
standard plate.
test. Positive
in desiccator
testing in strains
TA1535 and TA100.
growth
1 . Bacteria exposed in
gas tight exposure
chambers .
2. Plastic plates
round to absorb
Source: Eastman Kodak Co.
Purity: 99.98*
dibromomethane
In parallel
experiment. Thus,
glass plates used
Tor all other*
testing.
3. Weak positive
result. 0.002
revertanta/nmol
in TA1535 with or
without activation.
0.001 revertants/
nmol in TA100 with or
without activation.
4. Revertants selected
from each experiment
and tested to ensure
that they were
actually his .
-------
plate incorporation tests could not be performed because of the volatility of the
test agent. Positive and negative control tests were conducted for these
experiments and indicated the systems were working properly. Principe et al.
(1981) conducted a spot test using strains TA1535, TA1537, TA1538, TA98, and
TA100. A positive response was observed for TA1535 when a triangular shaped
paper disc soaked with 100 u£ EDC was placed on the agar in the presence of S9
from Aroclor 1254-induced rat liver (i.e., 103 revertants on the treated plate
vs. 33 revertants for the negative control). Negative responses were obtained
with the other strains. A negative response was also obtained in plate
incorporation tests conducted with TA100 and TA1535 at doses up to 100 [ill/plate.
A standard assay was conducted; no precautions were taken to prevent excessive
evaporation of the EDC. Similarly in forward mutation tests conducted with
Streptomyces coelicolor and Aspergillus nidulans negative responses were
obtained at doses up to 100 and 500 uH/plate in plate incorporation and spot
tests. There was a 100$ survival in the test conducted with ^. coelicolor and a
60$ reduction in cell survival at the highest dose with A. nidulans. Thus, these
tests are judged to provide less than adequate conditions for assessing the
mutagenicity of EDC because insufficient exposure to the test organisms may have
occurred.
McCann et al. (1975) exposed Salmonella strains TA1525, TA100, and TA98 to
EDC (Aldrich, stated to be highest purity available) concentrations as high as 13
rag/plate (131 (imol/plate). An equivocal response was observed in TA100 (0.19
revertants/umol). However, reproducible dose-related response curves were not
obtained. The presence of an exogenous S9 mix metabolic activation system (from
rat livers induced with either phenobarbital or Aroclor 1254) did not increase
the response. Chloroacetic acid, which is a known mammalian metabolite of EDC,
and the putative intermediates (i.e., chloroethanol and chloroacetaldehyde; see
9-137
-------
section 9.1.3.3A), were also tested for their tnutagenic potential. Chloroacetic
acid was negative, but chloroethanol yielded a marginally positive and
chloroacetaldehyde a strong positive result (0.06 and 7^6 revertants/nraol,
respectively, in TA100). The authors speculated that the mutagenic response of
EDC may be due to relatively inefficient conversion of chloroethanol to
chloroacetaldehyde in the in vitro system.
Two reports (Rannug, 1976; Rannug and Ramel, 1977) compared the
mutagenicity of EDC with that of EDC tar, a complex mixture formed during the
manufacture of vinyl chloride from either acetylene, ethylene, or a mixture of
the two. The mixture is called CDC tar because EDC is a major component
comprising about 30% of the mass. The sample of EDC tested in these studies was
from British Drug House (BDH) Chemicals, Ltd. (purity not given). Salmonella
tester strain TA1535 was dosed up to 45 ^mol/plate in the presence or absence of
an exogenous metabolic activation system (from livers of male strain R Wistar
rats) with and without NADP. Positive (twofold) increases in revertant
frequencies over background (13*0 + 1.76) were observed in the plates receiving
the highest dose (21.8 + 3*06) in tests conducted without metabolic activation.
However, with activation, a stronger positive response was observed. The
revertant count was elevated tenfold over the spontaneous level (132.8 + 8.35 vs.
15.5 + 1.21). The response to EDC was independent of the presence of NADP; in
contrast, the mutagenicity of EDC tars was increased with metabolic activation
only in the presence of NADP. It was concluded that the mutagenicity of EDC tar
cannot be ascribed primarily to EDC, because EDC and EDC tar have different
requirements for metabolic activation. In addition, the concentration of EDC
present in EDC tar at the highest dose tested would yield only a marginal
positive response if tested alone, while in fact a strong response was observed
for the EDC tar.
9-138
-------
Following these observations, Rannug et al. (1978) tested Salmonella strain
TA1535 with EDC (BDH Chemicals Ltd.; although the purity was not reported, the
material was checked by glass capillary chromatography using a flame ionization
detector) at doses up to 60 iiraol/plate. Tests were conducted with and without an
exogenous rat liver metabolic activation system. The components of the
activation system were varied in order to study the mechanism of activation of
EDC. Phenobarbital-induced liver fractions from Sprague-Dawley or R strain
Wistar rats were prepared with and without an NADPH-generating system and with
and without glutathione transferases A, B, and C. It was observed that EDC is
activated by the cytosol fraction of liver homogenates; thus, mixed-function
oxygenases are not involved. The activation was NADPH-independent but required
glutathione (GSH) A or C S-transferase enzyme activity. The extent of activation
was found to be dependent on the rat strain used (liver fractions from R strain
rats were more effective than those from Sprague-Dawley rats) and the handling of
the extract (e.g., storage on ice or freezing reduced the effectiveness of the
activation system). The report by McCann et al. (1975) (described earlier in
this section) that EDC was not metabolized to a mutagenic intermediate may be due
to differences in the exogenous activation system used (S9 mix rather than
cytosol fraction) in the two studies. Rannug et al. (1978) hypothesized that the
mutagenicity of EDC after metabolic activation was caused by the formation of a
highly reactive half sulfur mustard, S-(2-chloroethyl)-L-cysteine (see section
9.1.3-3.B). This compound (>99? purity) was found to be more strongly mutagenic
in TA1535 than is EDC. At 0.2 umol/plate, 12.8 + 1.5 and 176.8 + 11.1 revertants
were observed per plate after treatment with EDC and S-(2-chloroethyl)-
L-cysteine, respectively. At 5.0 |imol/plate the observed numbers of revertants
per plate were 13.0 + 1.1 and 1951 (only one plate tested), respectively. Thus,
S-(2-chloroethyl)-L-cysteine gives a strong mutagenic effect at low doses where
no effect can be seen for EDC.
9-139
-------
In intact mammals most GSH conjugates are normally excreted in bile. Rannug
and Bieje (1979) reasoned that if EDC were raetabolically activated by conjugation
with glutathione to form a half sulfur mustard, rautagenic products in the bile
would be produced in EDC-treated mammals or perfused livers. To test this
hypothesis, an EDC concentration of 0.1 mi (1.3 mmol) were perfused through R
strain Wistar rat livers for up to 1 hours. Bile was collected right after
addition of EDC and at 15 and 30 minutes, as well as 1 „ 1.5, 1.75, 2, 3, and 4
hours later. The bile was then added directly to top agar or diluted about 5 to
10 times in sterile water and added to top agar containing Salmonella strain
TA1535 for plate incorporation tests. Positive responses were obtained. The
greatest response (15-60 revertants per plate, compared to 7-10 in negative
controls) was reached about 15 minutes to 1 hour after addition of EDC. In a
second experiment, 80 mg/kg EDC was given to male CBA mice intraperitoneally.
The animals were sacrificed, their livers removed, and bile collected 30 and 60
minutes later for mutagenicity testing with TA1535 in plate incorporation tests.
An increase in revertants (greater than twofold) was observed for bile collected
30 minutes after treatment compared to bile from negative control animals (28.8 +
27 revertants and 11.3 ± 1.1 revertants, respectively). The positive responses
obtained with bile from the perfused rat livers and intact mouse livers are
consistent with the hypothesis that EDC is activated by conjugation with gluta-
thione.
Four studies have been reported in which EDC was found to be negative in the
standard Salmonella/microsome assay plate incorporation tests (King et al.,
1979; Nestmann et al., 1980; Stolzenberg and Mine, 1980; Barber et al., 1981).
The maximum doses employed in the first three studies were 36 umol/plate, 91
umol/plate, and 10 ^mol/plate, respectively; the fourth study did not report the
doses used. The doses reported in these studies therefore encompass a range
-------
at which positive responses have been reported in other studies. Except for the
report by Stolzenberg and Hine (1980), the negative Salmonella plate
incorporation studies were conducted with appropriate positive controls. Each
test was conducted with and without PCB-induced rat liver S9 mix. It should be
pointed out, however, that the positive controls, which require activation, were
not structually similar to EDC. Therefore, these positive controls may not be
able to determine the effectiveness of the components in the S9 mix necessary for
EDC activation. King et al. (1979) also reported negative responses when E. coli
K12/3^3/113 was tested in either a liquid suspension test or an intrasanguineous
host-mediated assay.
Two of the negative studies (i.e., Nestmann et al., 1980; Barber et al.,
1981) reported that although EDC did not induce mutations in standard plate
incorporation tests, positive responses were obtained when the studies were
conducted in air tight exposure chambers. Nestmann et al. (1980) exposed
Salmonella strains TA1535 and TA100 to doses from 3 to 9 rag/plate (30 to 91
umol/plate) in desiccators. It was reported that this treatment yielded positive
results, at least for TA1535 in which there was a doubling in the number of
mutant colonies over the control. It was stated that concentrations were tested
up to levels where cell-killing was observed, but no data are given and insuffi-
cient detail is provided to allow the results to be adequately evaluated.
Barber et al. (1981) exposed Salmonella tester strains TA1535, TA98, and
TA100 to four levels of EDC vapors (Eastman Kodak Co., 99.98$ pure) in a 3.1-
liter airtight exposure chamber. These exposures resulted in estimated plate
concentrations ranging from 31.8 to 231.8 umol/plate as determined by gas liquid
chromatography analysis of distilled water samples placed in the exposure
chamber. Linear, dose-related increases in revertant counts were observed for
TA1535 and TA100. The mutagenicity of EDC in these two strains was 0.002 and
-------
0.001 revertants/nmol, respectively. No difference in revertant counts or
potency of EDC was noted in comparisons of tests done with and without metabolic
activation. The positive results were obtained by Nestmann et al. (1980) and
Barber et al. (1981) only when tests were conducted in airtight exposure
chambers. This suggests that standard mutagenicity testing of EDC (bp 83°-84°)
may not provide an adequate assessment of its mutagenic potential due to
excessive evaporation.
In summary, the positive responses obtained without metabolic activation
indicate that EDC is a mutagen in bacteria. The positive responses obtained with
metabolic activation indicate that one or more of its metabolites are more potent
mutagens. The negative findings reported in bacterial tests are not considered
to contradict the reported positive results, because the negative results may
have been due to excessive evaporation of EDC or to inadequacy of the S9
activation system.
9.1.1.2. EUCARYOTIC TEST SYSTEMS — In addition to causing gene mutations
in bacteria, EDC also has been shown to cause gene mutations in eukaryotes.
9.4.1.2.1. Higher Plants — Two reports were evaluated concerning the
ability of EDC to induce mutations in higher plants (Table 9-28). Ehrenberg et
al. (197*0 treated barley seeds (variety Bonus) with 30.3 mmol EDC (Merck, purity
not given) for 24 hours and scored for sterile spikelets at maturity or
chlorophyll mutations in about 600 spike progenies in the subsequent generation.
The dose level tested corresponds to the LDg_. EDC treated kernels had an
increased incidence of chlorophyll mutations (6.8$) compared to untreated
controls (0.06$).
Kirichek (1974) exposed eight varieties of pea seeds (100 each) to gaseous
EDC (purity and concentration not given) for 4 hours. The germination of treated
9-142
-------
TABLE 9-28
Summary of Hutagenielty Testing of EDC: Higher Plants
Reference
Ehrenberg et al . ,
1974
Teat
System
Segregating
chlorophyll
(gene) mutations
in barley
Chemical
Information
Concentration tested:
200 seeds treated
with 30.3 0001/21
hours (LD,.) in a
closed vessel.
Results Comments
Positive response. 1. About 600 spike progeny were tested.
(6-8J mutants from
treated progeny vs.
0.06) mutants from
control progeny).
Source: Nerck Co.
Darmstadt, PRG
Purity: Not given
Kiricheck, 1974
\o
i
Visible mutations
In peas, 8 varieties
Concentration tested:
EDC (concentration not
reported) or water
(negative controls)
vapors for 4 hours.
Source: Not reported
Purity: Not reported
Reported postive.
5.42-28.13* of
seeds reported
to be mutated.
1. English translation of Russian article.
2. Germination of the treated seed varied
with the variety tested from IS) to 58*
compared to 100J germination of the
control seeds.
3. Control mutation frequency not given.
1. Putative mutations not characterized.
5. Not possible to adequately evaluate
results.
-------
seeds differed with the variety tested but was reduced (15-50$ germination)
compared to negative control seeds exposed to water vapor for 4 hours (100$
germination). From 5.4$ to 28.13$ of the seeds were reported to be mutated. It
is reported that two times as many morphological mutants were induced as chloro-
phyll mutations. The author considered this a positive response, but the muta-
tion frequency for negative controls was not given. This limitation of the
report plus a lack of information concerning the characterization of the putative
mutations precludes an adequate evaluation of the results.
9.4.1.2.2. Insects — Four studies were evaluated concerning the ability of
EDC to cause mutations in Drosophila melanogaster (Table 9-29). These studies
demonstrated the ability of EDC to cause sex-linked recessive lethal mutations
(Shakarnis, 1969, 1970; King et al., 1979). The fourth study demonstrated the
induction of somatic cell mutations by EDC (Nylander et al., 1979).
Shakarnis (1969i 1970) performed two experiments to assess the ability of
EDC (purity not given) to cause sex-linked recessive lethal mutations in
Drosophila. In the first study (Shakarnis 1969), adult females from a radio-
sensitive strain (Canton S) were exposed to 0.07$ EDC gas for 4 or 8 hours at 24°-
25°C. Immediately after treatment the females were mated to Muller-5 or In wa B
males. Fertility of treated females was reduced 47$ by the 1-hour treatment and
91$ by the 8-hour treatment. The F_ progeny were scored for lethality (measured
as the absence of wild-type males). A statistically significant (p<0.05) time-
related increase in the incidence of sex-linked recessive lethals was observed.
The frequencies of lethals were 0.3$ (negative controls), 3*2$ (4-hour
treatment) and 5-9% (8-hour treatment). In his second study, Shakarnis (1970)
exposed females from a radiostable strain (D-32) of Drosophila melanogaster to
0.07$ EDC vapors (source and purity not reported) for 1 or 6 hours. Immediately
9-144
-------
TABLE 9-29
Summary of Mutagenlcity Testing of EDC: Gene Mutation Teats in Insects
Reference
Shakarnia, 1969
Test
System
Drosophlla
nelanogaster
Chemical
Information Results
Concentration tested: Postive response
virgin 3-day -old
Comments
1. Huller-5 or In wa B males.
rsex-linked
recessive lethal
test
Canton S females
exposed to 0.079
EDC gas for M or B
hours at 2U-25°C.
Source: Not given
Purity: Not given
2. Fertility of treated females
reduced significantly (V7) after
4 hour treatment and 91J after
6 hour treatment).
3. Canton S strain reported to be
sensitive.
\o
i
XT
-------
TABLE 9-29 (cont.)
VO
I
Test Chemical
Reference System Information Results
Shakarnls, 1970 Prosophliia Concentration tested: Positive response 1.
melanogaster virgin 3-day-old
sex-linked radios table D-32 2.
recessive lethal strain females
test exposed to 0.07* EDC
gas for 4 or 6 hours
at 24-25°C. 3.
Source: Not given
Purity: Not given
Duration of
Treatment
(h) with Number of
0.07* EDC Chromosomes Scored
Control 1904
4 2205
6 2362
Comments
Nuller-5 males.
Experiment conducted
twice for
4 hour exposure and three times
for 6 hour exposure.
4 hour treatment did
significantly reduce
but 6 hour treatment
by 50*.
Lethal Nutations
No. *
1 0.05
46 2.00
7B 3.30
(p<0.05)
not
fertility
reduced It
-------
TABLE 9-29 (cont.)
so
I
Test Chemical
Reference System Information Results
King et al., Drosophlla Concentration tested: Positive response 1.
1979 melanogaster 50 mM solution of EDC
sex-linked in 5% sucrose fed to 2.
recessive lethal 1- to 2-day-old Berlin K
test males for 3 days (near
Source: Merck Co.
Darmstadt, PRO
Purity: Not given but
stated to have
correct melting
point (SIC) and
elemental
analysis
Cone. Days after Number of
(mM) Brood Treatment Chromosomes Scored
0 I-III 0-9 22,048
50 I 0-3 1,185
II 4-6 1,179
III 7-9 156
I-III 0-9 2,520
Comments
Base females.
Lethal frequency Increased for all
broods. Greatest effect in brood
II, which corresponds to the
speraatid stage of spermatogenesis.
Lethal Mutations
No. \ p
47 0.21
6 0.51
41 3.48 <0.01
2 1.28
49 1.94 <0.01
-------
TABLE 9-29 (cont.)
Reference
Nylander et al.,
1976
Teat
System
Drosophlla
melanogaater
somatic cell
mutations so
- |j Soie 19
stocks.
Chemical
Information Results
Concentration tested: Positive response
0.1J and 0.5* EDC In
food at 25°C and 75*
humidity during larval
development.
Source: Fisher
Scientific Co.
Comments
1. Mutations at £ locus scored in
files of stable and unstable
genotype. Both have same phenotype
Instability thought to be caused
by transposable genetic element.
2. Survival of treated flies reduced
significantly In both genetically
Purity: Not given
stable (342 reduction for 0.If and
77S reduction for 0.5% dose) and
genetically unstable stocks (86J
reduction for 0.51 dose).
I
oo
Treatment
Control
Stable
Unstable
0.01}
Stable
Unstable
0.05$
Stable
Unstable
Number
Hales
Scored
M441
5363
6260
2889
610
201
Number
with
Sectors
2
I
263
271
44
50
J
0.045
0.075
4.20
9.2B
7.21
24. 8B
t Value Differences
Within Genotypes
—
—
18.748
24.31a
11.463
13. 12a
t Value Differences
Between Genotypes
0.60
9.l6a
5.66a
p<0.001
-------
after treatment they were mated to Muller 5 males and the F2 progeny scored for
sex-linked recessive lethals. The 4-hour treatment did not significantly reduce
fertility but the 6-hour treatment reduced it by 50%. As in the first study, a
statistically significant (p<0.05) time-related increase in sex-linked recessive
lethals was observed. A frequency of 0.05% lethals was observed in the negative
controls, and frequencies of 2.00% and 3-30? lethals were observed in offspring
from the 4-hour and 6-hour treatments, respectively. This corresponds to the
mutagenic effect induced by an acute dose of two to three thousand (2000 to 3000)
rad of x-rays.
King et al. (1979) also studied the ability of EDC to cause sex-linked
recessive lethals in Drosophila. One to two day old Berlin K males were fed a 50
mM solution of EDC (Merck and Co., purity not given but correct melting point and
elemental analysis reported) in 5% sucrose for 3 days. This dose approximated
the LD,-Q. The males were immediately mated to virgin Base females every 3 days
for a total of 9 days to determine if EDC preferentially damaged particular
stages in spermatogenesis. Progeny of the F_ generation were scored for
lethality. The lethal frequency was found to be elevated for all three broods
(0.51$, 3.48?, and 1.28$, respectively, for broods I, II, and III) compared to
the negative controls (0.21$). The highest lethal frequency (3.48$) was found in
brood II (p<0.01), which corresponds primarily to the spermatid stage of
spermatogenesis.
Mylander et al. (1979) raised flies on food containing 0.1? and 0.5$ EDC
(Fisher Scientific Co., purity not given) during larval development and scored F
progeny for the induction of somatic cell sex-linked mutations in the eye. A
genetically unstable stock (so z w+) and a genetically stable stock
(^ Dp w+ e ) both having the same phenotype were used in these studies and
mated to attached X females. Mutations result in the expression of normal (dark
red) eye pigment in adult treated males. The genetic instability of 50 £ w+ is
9-149
-------
caused by the insertion of a transposable genetic element. The survival of flies
raised on the EDC-treated food was significantly reduced compared to the negative
control values (e.g., 77$ reduction for the stable stock and 86% reduction of the
unstable stock at 0.5% EDO). Statistically significant increases in somatic cell
mutations occurring in both stocks were observed at both the low and high doses
(p<0.001).
The positive responses in Drosophila indicate that EDO is capable of causing
both somatic cell and heritable germinal mutations in a raulticellular eukaryote.
9.1.1.2.3. Mammals
9.4.1.2.3.1. In Vitro. Tan and Hsie (1981) found that EDC (Matheson,
Coleman and Bell, purity not given) caused a dose-related increase in HGPRT
mutations in cultured Chinese hamster ovary (CHO) cells (Table 9-30). CHO-K.-BH^
cells were exposed in suspension culture for a period of 5 hours to EDC
concentrations ranging up to 3 mM (30$ survival) in tests with exogenous rat
liver S9 mix for metabolic activation and concentrations ranging up to 50 mM (50%
survival) in tests conducted without metabolic activation. Positive responses
were observed both in tests with (5 mutants/10 cells/mmol) and without
(1 mutant/10 cells/mmol) metabolic activation (p<0.01). EDC was detected as a
mutagen in studies not requiring metabolic activation only at high doses, but the
induced mutation frequency was increased about tenfold over control values. Only
about a fourfold additional increase in mutagenicity was noted with metabolic
activation but excessive toxicity precluded testing at concentrations greater
than 3mM (i.e., S9 mix increased mutagenic activity by approximately fourfold and
cytotoxic activity from 5 to 25-fold). The metabolic activation system was only
effective in the presence of NADP. This contrasts with Rannug (1976) who found
metabolic activation of EDC to be NADP-independent.
9-150
-------
TABLE 9-30
Summary of Hutagenlclty Testing of EDC: Mammalian Syaterns
Reference
Tan and Hsie,
1981
Test
System
Chinese hamster
ovary cell HGPRT
gene mutation
assay
Chemical
Information
Concentration tested:
Up to 3 mM in tests
with rat liver S9 mix
and up to 50 mH in
Results
Positive response ,
60 vs. 3 mutants/10
clonable cells for
50 and 0 mM EDC
Comments
1. Mutagenic activity of EDC without and
with metabolic activation calculated
to be 1 and 5 mutants/ 10 cells/
mmol, respectively.
Gocke et al., 1983
House Spot Test
C57Bl/6JHan (F)
X
T-stock (M)
tests without
metabolic activation.
LD5Q is 1 mH with
activation and 6 mH
without activation.
One l.p. injection 10
days after conception
of 300 mg/kg EDC in
olive oil.
Source:
Merck Co.
Darmstadt, FRG
without activation.>
28 vs. 3 mutants/ID
clonable cells for
1.5 and 0 nM EDC
with activation.
7/110H offspring
had spots compared
to 2/812 In the oil
controls and 3/2161
in the cumulated
controls.
2. S9 nix increases mutagenlcity by
about fourfold and cytotoxiclty
5- to 25-fold.
3. NADP required in S9 mix for
metabolic activation.
1. Data suggestive of a positive response.
ui
Purity: Not given
-------
9.4.1.2.3.2. In Vivo. Gocke et al. (1983) conducted a mouse spot test to
evaluate the ability of EDC to induce somatic cell mutations in developing mice.
One 300 mg/kg intraperitoneal injection of EDC (Merck, purity not given) was
administered to C57 Bl/6JHan females 10 days after mating with T-stock males.
Seven out of the resulting 1104 offspring had mutant coat color spots compared to
2/812 in the controls and 3/2161 in the combined untreated controls. The data
show a significant (p=0.03) effect against the cumulated controls, but no
significance (p=0.l8) against the oil control. These data are suggestive of a
positive response.
The consistency of positive results obtained in higher plants, Drosophila,
and cultured mammalian cells demonstrates that EDC causes gene mutations in
higher eukaryotes.
9.4.2. Chromosome Aberration Studies. Five studies were evaluated regarding
the ability of EDC to cause chromosome aberrations (Table 9-31). One was an
abstract of testing EDC for its ability to cause chromosome breakage in Allium
root tips and cultured human lymphocytes (Kristoffersson, 1974). Two were
Drosophila melanogaster X chromosome nondisjunction tests (Shakarnis, 1969,
1970), and two were raicronucleus tests (King et al., 1979; Jenssen and Ramel,
1980).
In the paper by Kristoffersson (1974) EDC (source, purity, and concentra-
tion not given) was reported to cause C-raitoses in Allium root tip cells but not
to cause chromosome breaks in Allium root tip cells or human lymphocytes.
Because the report was an abstract, insufficient information was available to
substantiate the reported results. However, the induction of C-mitoses in Allium
root tip cells suggests that EDC can interfere with the mitotic spindle
apparatus.
9-152
-------
TABLE 9-31
Summary of Mutagenicity Testing of EDO: Chromosomal Aberrations Tests
Reference
Test
System
Chemical
Information Results
Comments
Shakarnis, 1969
Drosophila
melanogaster
X chromosome
nondisjunction.
Canton S (F)
X
Muller-5 (M)
Concentration tested:
virgin Canton S females
exposed to 0.07$ EDC
in 1.5 & desiccator
for 1 and 8 hours at
24-25°C.
Source: Not given
Purity: Not given
vo
.^
ui
Statistically
significantly
(p<0.05) increases
in exceptional
female progeny reported
for 4-hour exposure
and male and female
progeny for 8-hour
exposure. However,
test Judged to be
invalid.
The study was poorly
designed in that the
Canton S stock did not
possess appropriate
genetic markers to
rule out non-virginity
as the cause for the
"exceptional" progeny.
The control rate was
much lower than the
historical background
rate.
0.07$ EDC
Duration of
Treatment
(h)
Control
4
8
Normal
No. Females
5,848
24,125
8,437
Progeny
No. Males
5,727
21,297
7,773
Exceptional
Females
No. $
0 0
8 0.033
15 O.l8a
Progeny
Males
No. $
0 0
3 0.01
7 0.093
p<0.05
-------
TABLE 9-31 (cont.)
Reference
Test
System
Chemical
Information Results
Comments
Shakarnis, 1970
vO
i
VJl
Drosophila
melanogaster
X chromosome
nondisjunction.
Canton S (F)
X
Muller -5 (M)
Concentration tested:
3-day-old females
from radiostable D-32
strain exposed to 0.0751
EDC in a 1.5 £
desiccator for 4 or 6
hours at 24-25°C.
Source: Not given
Purity: Not given
Incidence of nondis-
junction greater in
treated group than
in control. Increase
not statistically
significant. Test
judged to be invalid.
See comments for
previous study.
0.07} EDC
Duration of
Treatment
(h)
Control
4
6
Exceptional Progeny
Normal Progeny
No. Females
2172
3584
4034
No. Males
2205
3401
4090
Females
No. %
0
1
4
0
0.03
0.10
Males
No. %
1
1
3
0.03
0.03
0.07
-------
TABLE 9-31 (cont.)
Reference
King et al. ,
1979
Test
System
Micronucleus test:
NMRI mice
Chemical
Information Results
Concentration tested: Negative
2 i.p. injections of
4 mmol/kg (400 mg/kg)
Comments
1 . 1 , 000 polychromatic
erythrocytes
analyzed/animal .
VJ1
given 2<4 hours apart.
4 animals/dose
sacrificed after
second injection.
Source:
Merck Co.
Darmstadt, FRG
Purity: Not specified,
but melting
point and ele-
mental analysis
were correct
2. Frequency of micro-
nuclei not given.
Jenssen and Ramel,
1980
Micronucleus test:
CBA mice
Concentration tested:
single i.p. injection
100 mg/kg.
Source:
BDH Chemicals,
Ltd.
Purity: Not given
Negative.
(0.15 + 0.11) poly-
cromatic erythrocytes
with micronuclei in
controls vs. 0.17 ±
0.10 in treated
animals).
-------
Data perhaps consistent with this possibility, but not conclusively showing
non-disjunction, are found in studies by Shakarnis (1969, 1970) using Drosophila
melanogaster. Females were exposed to 0.07$ EDC vapors (source and purity not
given). In the first study (1969) a statistically significant (p<0.05) increase
in exceptional F.. progeny, indicative of meiotic nondisjunction, was reported
after a 1-hour treatment (0.03$ exceptional females) and 8-hour treatment (0.18$
exceptional females and 0.09$ exceptional males) compared to the negative
control value (0$; 0/11575 progeny). In the second study, the incidence of
exceptional progeny was elevated in the treated groups but not significantly so.
However, both studies were flawed by an experimental design which did not allow
discrimination between progeny resulting from nondisjunction compared to those
from non-virgin females.
Micronucleus tests were performed by King et al. (1979) and Jenssen and
Ramel (1980). Both studies reported negative results. King et al. (1979)
injected NMRI mice with two intraperitoneal injections of 4 mmol/kg (100 mg/kg)
EDC (Merck Co., purity not specified but melting point and chemical analysis
reported to be correct). The authors state this corresponds to an "approximate
lethal dose" (the LD, for intraperitoneal injections of EDC in mice is 250
lo
mg/kg). The injections were given 24-hours apart; the animals were killed 6-
hours after the second injection, and bone marrow smears were made. One thousand
polychromatic erythrocytes (PCEs) were analyzed per animal. Frequencies of
micronuclei were not given, but the results were evaluated to be negative by the
authors.
Jenssen and Ramel (1980) also reported negative results in a micronucleus
test. CBA mice were given a single intraperitoneal injection of EDC (BDH
Chemicals Ltds., England, purity not given) at a dosage of 100 mg/kg. The
animals were sacrificed the next day and PCEs (number not given) were scored for
9-156
-------
micronuclei. The frequencies of PCEs with micronuclei were 0.15 +. 0.14 in the
controls and 0.17 +_ 0.10 in treated animals.
The equivocal X-chromosome loss test in Drosophila (Shakarnis, 1969) may
suggest that EDC can cause meiotic nondisjunction. A properly designed
experiment should be conducted to determine this. The negative responses
obtained in the raicronucleus tests may indicate that EDC does not cause
chromosomal damage in mice. However, because EDC has not been adequately tested
for its ability to cause structural chomosomal aberrations, it would be
appropriate to perform mammalian in vitro and iji vivo cytogenetic tests. Such
testing is required before a judgment can be made on the ability of EDC to cause
chromosomal aberrations.
9.^.3* Other Evidence of DNA Damage. Three other tests have been conducted on
the genotoxicity of EDC. These tests do not measure mutagenic events per se in
that they do not demonstrate the induction of heritable (i.e., somatic or
germinal) genetic alterations, but positive results in these test systems
suggest that DNA has been damaged. Such test systems provide supporting evidence
useful for assessing genetic risk.
9.4.3.1. BACTERIAL TEST SYSTEMS
9.4.3.1.1. PolA Assay — Ethylene dichloride has been reported positive in
the polA assay which measures toxicity associated with unrepaired damage in DNA
(Table 9-32). Brem et al. (1974) soaked sterile filter disks with 10 \ii (80
jijnol) EDC. These were centered on the agar surface of petri dishes covered with
bacteria (one set with polA* strain, the other with polA~ strain). After
incubation the plates were scored for differential inhibition of growth. An 8-mm
zone of growth inhibition was observed in the polA* strain compared to a 9-mm
zone of inhibition for the polA~ strain. These responses were said to be
9-157
-------
TABLE 9-32
Summary of Mutagenicity Testing of EDC: PolA Assay
Reference
Bren et al . ,
1974
Test
System
polA differential
cell killing
assay
Strain
E. coll
polA
polA*
Activation
System
None
Chemical
Information
Concentration tested:
10 \it on filter disk
Results
Questionable
positive
Comments
1. All assays carried out In
duplicate on at least
three different occasions.
Only a small difference
in diameter (i.e., 1 mm)
was noted between the
zones of killing in the
two strains. Thus, the
A*/A~ ratio may be
misleading.
Pol A Zone of Inhibition
vO
CD
EDC
HHS
Chloramphenicol
A*
no
8
"45
28
A"
nun
9
51
28
A*/A~
1.26
1.44
1.00
-------
reproducible. The ratio between the zones of inhibition (polA XpolA") was 1.26
which is interpreted by the authors to be a positive response. However, it
should be noted that 1 mm is not a big difference, and the ratio may be
misleading.
9.U.3.2. EUKARYOTIC TEST SYSTEMS
9.1.3.2.1. Unscheduled DNA Synthesis — One test has been performed to
assess the ability of EDC to cause unscheduled DNA synthesis (Perocco and Prodi
1981). Although demonstration that a chemical causes unscheduled DNA synthesis
does not provide a measurement of its mutagenicity. per se, it does indicate that
the material damages DNA. Perocco and Prodi (1981) collected blood samples from
healthy humans, separated the lymphocytes, and cultured 5 x 10 of them in 0.2 m£
medium for 4 hours at 37°C in the presence or absence of EDC (Carlo Erban, Milan,
Italy or Merck-Schuchardt, Darmstadt, FRG, 97%-99% pure). The tests were
conducted both in the presence and in the absence of PCB-induced rat liver S9
mix. A comparison was made between treated and untreated cells for scheduled DNA
synthesis (i.e., DNA replication) and unscheduled DNA synthesis. No difference
was noted between the groups with respect to scheduled DNA synthesis measured as
dpm of [3H] deoxythymidylic acid (TdR) after U hours of culture (2661 + 57 dpm in
untreated cells compared to 2287 + 60 dpm in cells treated with 5 [ii/mSL [0.06 mM]
EDC). Subsequently 2.5, 5, and 10 \ii/mi (0.03, 0.06 and 0.1 mM) EDC was added to
cells which were cultured in 10 mM hydroxyurea to suppress scheduled DNA
synthesis. The amount of unscheduled DNA synthesis was estimated by measuring
dpm from incorporated [3H]TdR U hours later. At 10 pi/ml EDC 483 + 37 and 532 +
21 dpm were counted without and with exogenous metabolic activation,
respectively. Both values were lower than corresponding negative controls of 715
i 24 and 612 +_ 26 dpm, respectively. No positive controls were run to ensure that
the system was working properly although testing of chloromethyl methyl ether
9-159
-------
(CMME) with activation resulted in a doubling of dpras over the corresponding
negative control values (1320 + 57 at 5 ul/ml CMME versus 612 + 26 untreated).
The authors calculated an effective DMA repair value (r) for each chemical based
on the control and experimental values with and without metabolic activation.
Ethylene dichloride was evaluated by the authors as positive in the test, but
they did not state their criteria for classifying a chemical as positive. It is
important to note that none of the experimental values from cells treated with
EDC without metabolic activation had higher dpm values than the controls.
Furthermore, although two out of three experimental values were greater than the
controls with metabolic activation (673 ± 45 at 5 uS,/m£ and 630 +3*4 at 2.5 ufc/mJl
compared to control value of 612 + 26) the increases were not statistically
significant. The positive finding reported in this work is therefore judged to
be inconclusive.
9.4.3.2.2. Detection of DNA Adducts — Reitz et al. (1982) compared the
pharmacokinetics of EDC administered to Osborne-Mendel rats after inhalation and
after exposure by gavage. (See section 9.1.3, especially 9.1.3.5.) As part of
their study, DNA alkylation was measured in bacteria at EDC cytosol
concentrations corresponding to those used in the DNA binding study (Table 9-33).
14
Two gram aliquots of TA1535 were incubated with 7.06 umol [ C] EDC/mfc (sp. act.
= 3.2 mCi/mmol) and varying amounts of cytosol. DNA alkylation values at cytosol
concentrations of 2.2, 7.8, 27, and 71$ were 8.65, 27, 107, and 137 dpra/mg
purified DNA, respectively. This corresponds to 4, 12.5, 49, and 64 alkylations
x 10 DNA nucleotides. The corresponding reversion frequencies were 4.6 + 0.82,
23.5 + 3.0, 80.2 ± 9.6, and 111 + 2.6, respectively (n = 3 in each case). A
direct correlation between the degree of alkylation and an increase in mutation
frequency was indicated by linear regression analysis (r = 0.9976). In the DNA
alkylation studies with rats [ C] EDC (sp. act. = 0.32 mCi/mmol) was
administered to groups of three animals by gavage (150 mg/kg) or
9-160
-------
TABLE 9-33
Summary of Mutagenicity Testing of EDC: DMA Binding Studies
vo
i
Activation
Reference Test System Strains System
Reitzetal., DNA alkylation TA1535 Phenobarbital-
1982 and mutagenesis induced rat > Cytosol
in Salmonella liver cytoaol 2.2
7.8
27
71
DNA alkylation Osborne- NA
in rats Mendel rat Route of
Exposure
Gavage
150 mg/kg
Inhalation
150 ppm,
6 hours
Chemical Information
Revertants
4.6 + 0.82
23.5 + 3.0
80.2 7 9.6
111 * 2.6
Tissue
Liver
Spleen
Kidney
Stomach
Liver
Spleen
Kidney
Stomach
Alkylation x 10~6
Nucleotides
4
12.5
49
64
Alkylation x 10~6
Nucleotides (means
from 2 experiments
21.3
5.8
17.4
14.9
8.2
1.8
5.2
2.8
13.9
2.5
14.5
6.7
3.3
1.8
2.0
1.9
Comments
1. Bacteria incubated with
7.06 |iool EDC/md.
2. Significant correlation
between degree of alkylation
and increased reversion
frequency (r = 0.9976).
3. Rats sacrificed 4 hours post
gavage or immediately after
Inhalation.
4. Sp. act. 3.2 mCi/mmol for
bacteria; 0.32 mCi/mmol
for rats.
-------
inhalation (150 ppm, 6 hours). The animals were subsequently sacrificed and DMA
was extracted from the liver, spleen, kidney, and stomach for measurement of DNA
alkylation. Overall, three to five times more DNA alkylation was present after
gavage than after inhalation. The values ranged from 5.8 * 0.7 to 23.1 ± 7.1
alkylation/10 nucleotides for gavage versus 1.8 + 0.3 to 8.2 + 3-3
alkylation/106 nucleotides for inhalation. Under the conditions of test used in
the experiments by Reitz et al. (1982) EDC exposure resulted in a similar degree
of adduct formation in both rats and bacteria. Because DNA is the genetic
material in both bacteria and rats, these data predict that mutations were
induced in the rat at the exposures used. This is in keeping with the positive
responses in other eukaryotes including Drosophila and cultured CHO cells. DNA
alkylation was not measured in rat gonads so it is not possible to estimate what
the heritable genetic risk might be.
9.4.1. Summary and Conclusions. Ethylene dichloride (EDC) has been shown to
cause gene mutations in bacteria, plants, Drosophila, and cultured Chinese
hamster ovary (CHO) cells. Positive responses were observed in bacterial and CHO
cells in tests that did not require an exogenous metabolic activation system.
Stronger positive responses were found when hepatic metabolic activation systems
were incorporated. Based on these positive findings in different test systems
representing a wide range of organisms, EDC is judged to be capable of causing
gene mutations.
EDC has been reported to cause meiotic chromosomal nondisjunction in
Drosophila. The induction of meiotic nondisjunction is a significant genotoxic
effect; however, insufficient data are provided to be able to evaluate this test
adequately. A positive response in a properly conducted test is needed to permit
a judgment on the validity of this report. With respect to its ability to cause
structural chromosomal aberrations, sufficient testing has also not been
9-162
-------
performed. There is only one study on the ability of EDC to cause structural
chromosomal aberrations (i.e., in Allium root tip cells and human lymphocytes).
Because this study was reported in an abstract, the author's conclusions are
unsubstantiated. Even though negative results are reported from micronucleus
tests, additional information is needed to draw conclusions on the ability of EDC
to cause chromosomal aberrations. For example, it would be appropriate to test
EDC in ^n vitro and iri vivo mammalian cytogenetic assays. A sister chromatid
exchange assay would also be useful in assessing the ability of EDC to cause
chromosome damage.
There are no available data on the ability of EDC to damage DNA in mammalian
germ cells. Thus, studies on the ability of EDC to reach germinal tissue would
be appropriate to determine whether EDC has the potential to cause heritable
mutations which may contribute to the genetic disease burden. The finding that
EDC causes heritable mutations in Drosophila and alkylates DNA in several somatic
tissues in the rat reinforces the need for further germ cell studies in mammals.
Based on the weight-of-evidence, EDC is judged to be a mutagen that may have
the potential to cause adverse effects in humans.
9-163
-------
9.5. CARCINOGENICITY
The purpose of this section is to provide an evaluation of the likelihood
that ethylene dichloride (EDC) (1,2-dichloroethane) is a human carcinogen and,
on the assumption that it is a human carcinogen, to provide a basis for estima-
ting its public health impact, including a potency evaluation in relation to
other carcinogens. The evaluation of carcinogenicity depends heavily on animal
bloassays and epidemiologic evidence. However, other factors, including muta-
genicity, metabolism (particularly in relation to interaction with DNA), and
pharmacoki.netic behavior, have an important bearing on both the qualitative and
quantitative assessment of carcinogenicity. The available information on these
subjects is reviewed in other sections of this document and used in this evalu-
ation as appropriate. This section presents an evaluation of the animal bio-
assays, the human epidemiologic evidence, the quantitative aspects of assessment,
and finally, a summary and conclusions dealing with all of the relevant aspects
of the carcinogenicity of EDC.
9.5.1. Animal Studies. The carcinogenic potential of EDC has been investigat-
ed in a number of studies in which EDC was administered to rats and mice via
various routes of administration. The following studies will be discussed:
one gavage study performed by the National Cancer Institute (1978) in which
EDC was administered to rats and mice; two inhalation studies, including one
by Spencer et al. (1951) using rats, and one bioassay by Maltoni et al. (1980)
in which EDC was administered to rats and mice; one intraperitoneal study by
Theiss et al. (1977) in which EDC was administered to mice; and one skin-
painting study by Van Duuren et al. ( 1979) in which EDC was administered to
mice.
9.5.1.1. NATIONAL CANCER INSTITUTE (1978) RAT STUDY ~ Hazleton Labora-
tories America Inc., Vienna, Virginia, under the sponsorship of the NCI, con-
9-164
-------
ducted a bioassay of 1,2-dichloroethane (EDC) using Osborne-Mendel rats. The
NCI published a final report of the bioassay in 1978. Their results were also
reported by Weisburger (1977), the International Agency for Research on Cancer
(IARC, 1979), and Ward (1980).
Technical-grade EDC, obtained for this study from the Dow Chemical Company,
Midland, Michigan, was tested for its purity. Gas-liquid chromatography re-
vealed a purity of 98% to 99% EDC with 4 to 10 minor peaks (Ward, 1980). How-
ever, in the NCI broassay a purity of greater than 90% was reported, with only
two unidentified minor contaminants. Recently this discrepancy was resolved
when the original sample of EDC was reanalyzed, at which time the EDC was
found to be greater than 99% pure, with several unidentified contaminants.
This analysis was also performed by the National Institute for Occupational
Safety and Health (NIOSH) after completion of the bioassay; the sample contained
about 99% EDC, with chloroform as the major contaminant (Hooper et al., 1980).
Solutions of EDC were prepared in corn oil and administered by oral
intubation to 200 Osborne-Mendel rats starting at 8 weeks of age. The design
summary of this experiment is given in Table 9-34. On the basis of a sub-
chronic EDC study, 50 rats of each sex were used for each of two dose levels
in the chronic study. The maximum tolerated dose (MTD) was determined to be 95
mg/kg/day for both males and females; the second dose was one-half the MTD
(47 mg/kg/day).
Twenty animals of each sex served as untreated controls; an equal number
were given the vehicle (corn oil) by gavage. Because of the inadequacies of the
subchronic studies, the initial doses administered to the test animals were
found to be inappropriate. Early signs of toxicity necessitated several changes
in the dosages (Table 9-34). Anitnal weight and food consumption per cage were
obtained weekly for the first LO weeks and monthly thereafter. Animals were
9-165
-------
TABLE 9-34.
DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC) GAVAGE
EXPERIMENT IN OSBORNE-MENDEL RATS
Group
Males
Untreated control
Vehicle-control
Lou-dose
High-dosea
Females
Untreated control
Vehicle-control
Low-dose
High-dosea
Initial
number of
animals
20
20
50
50
20
20
50
50
1,2-dichloro-
e thane
dosage3
0
50
75
50
5QC
0
100
150
100
10QC
0
0
50
75
50
50=
0
100
150
100
100C
0
Observation period
Treated
(weeks)
78
7
10
18
34
_•_
7
10
18
34
—
78
7
10
18
34
—
7
10
18
34
— f *
Untreated
(weeks)
106
32
—
9
32
—
9
23
106
32
9
32
—
9
15
Time-weighted
average dosage
over a 78-week
periodb
0
47
™"™
95
— —
_•«
0
47
~ ~~
95
— — —
^^^
aDosage, given in mg/kg body weight, was administered by gavage five consecutive
days per week.
^Time-weighted average dosage = (dosage x weeks received)/78 weeks.
cThese dosages were cyclically administered with a pattern of one dosage-free
week followed by 4 weeks (5 days per week) of dosage at the level indicated.
"All animals in this group died before the bioassay was terminated.
SOURCE: NCI, 1978.
9-166
-------
checked daily for mortality. Weight depression was observed in both groups
exposed to EDC. By SO weeks, the weight depression averaged 12% in high-dose
rats (Figure 9-3). Mortality was early and severe in dosed animals, especial-
ly those given the highest doses. The mean survival was approximately 55 weeks
on test for high-dose males and females (Figure 9-9 and Table 9-35). The
early deaths were usually not due to cancer; rather, the toxic effects of EDC
appeared to be responsible for these deaths. Rats dying early had a variety of
lesions, including bronchopneumonia and endocardial thrombosis, which may have
contributed to early death. The pneumonia may have been the result of a viral,
bacterial, or mycoplasmal infection, and the exposure to the chemical may have
increased the tendency to develop severe pulmonary lesions, which would lead to
death. For the high-dose male rats, 50% (25/50) were alive at week 55 and 16%
(8/50) were alive at week 75. Survival was higher in the other groups; 52%
(26/50) of the rats in the low-dose group lived at least 82 weeks, and 50%
(10/20) in the vehicle-control group lived at least 72 weeks. In the high-dose
female rats, 50% (25/50) were alive at week 57; 20% (10/50) of the rats in the
low-dose group were alive at week 85. Despite the sacrifice of five females at
week 57, 65% (13/22) of the untreated control group survived until the end of
the study.
A gross necropsy was performed on each animal that died during the experi-
ment or was killed at the end. A total of 28 organs, plus any tissues contain-
ing visible lesions, were fixed in 10% buffered formalin, embedded in paraplast,
and sectioned at 5 y for slides. Hematoxylin and eosin stain were used rou-
tinely, and other stains were employed when necessary. Diagnoses of tumors
and other lesions were coded according to the Systematized Nomenclature of
Pathology (SNOP) of the College of American Pathologists (1965).
9-167
-------
750
600-
2
I 450-
g
uu
> 300-
o
CD
z
150-
MALE RATS
Untreated Control
Vehicle-Control
Low-Dose
High-Dose
•750
-600
-450
-300
-150
I
15 30 45 60 75
TIMEONTEST(Weeks)
90
105
120
750
| 600-
03
w
(S
I 450-1
O
2
300-
uj 150-
2
750
FEMALE RATS
Untreated Control
Vehicle-Control
Low-Dose
High-Dose
- 600
-450
-300
-150
\
\
15
30
45 60 75
TIME ON TEST (Weeks)
90
I
105
120
Figure 9-S. Growch curves for male and female Osborne-Mendel rats
administered EDC by gavage.
SOURCE: MCI, 1973.
9-168
-------
1 n
-J 0.8-
C
^<
>
QC
—5
w 0.6-
u.
0
>
j 0.4-
CQ
CQ
0
£ 0.2-
-
0.0 -
MALE RATS
\_ \ ''--,_, L|
'~"L. '--l 1
1 1 1^1
i '• — . _ V
1 , • j
• * • "" "L S
U^L ! '-
<-, i
\ i '--,
\ L. ';
— • — « n '
L— '"'
Untreated Control V "'-•._ i-._.
Vehicle-Control i "'-,
Low-Dose | "l;
High-Dose "L--L_ '--,_
' l ' l ' i ' i ' i ' i ' '
- 1.0
-0.8
-
-0.6
- 0.4
-0.2
-
0.0
0 15 30 45 60 75 90 105 120
TIME ON TEST (Weeks)
_J
<
>
>
cc
Z)
to
u.
0
>
^
_i
CO
<
0
cc
Q.
1 n
0.8-
•
0.6 — '
-
0.4-
0.2-
rrM AI i n ATr>
--J=o_ _ ~ '~I I 1
"Li~, \. h
~I ^ ' ! ' .
— ^ __ ~ ~ ~"»_ '
L_^___ -, !
i_
*^» ' — i
i V
T. [^ |
i ~ "i ! __
"'•i ~!
T
-I
I -I
Untreated Control "^
Vehicle-Control 1,
•-i -i_
Low-Dose "*•— •
L, ^
High-Dose ~" ^
' '< ' : ' ! ' 1 ' I '
-0.8
-
-0.6
-
-0.4
-0.2
~
15 30 45 60 75
TIMEONTEST(Weeks)
90
105 120
Figure 9-9. Survival comparisons for
racs administered EDC by -savage.
30URCE: :;CI, 19 73.
T.aie and female Osborne-Mendel
-------
TABLE 9-35. TERMINAL SURVIVAL OF OSBORNE-MENDEL RATS
TREATED WITH 1,2-DICHLOROETHANE (EDC)
Males Females
Weeks in Animals alive Weeks in Animals alive
Group study at end of study study at end of study
Untreated control 106 4/20a (20%) 106 13/20a (65%)
Vehicle-control 110 4/20 (20%) 110 8/20 (40%)
Low-dose 110 1/50 (2%) 101 1/50 (2%)
High-dose 101 0/50b (0%) 93 0/50*> (0%)
aFive rats were sacrificed at 75 weeks.
^All animals in this group died before the bioassay was terminated.
SOURCE: Adapted from NCI, 1978.
Squamous cell carcinomas of the forestomach occurred in 3/50 (6%) low-dose
males, 9/50 (18%) high-dose males, 0/50 (0%) high-dose females, and 1/49 (2%)
low-dose females (Table 9-36). None of these tumors occurred in the controls.
For male rats, the Cochran-Armitage Trend Test indicated a significant (p =
0.01) positive association between dosage and the incidence of squamous cell
carcinoma of the stomach. The Fisher Exact Test confirmed the significance
of these results with p values of 0.039 and 0.001 when comparisons were made
with the matched vehicle-control group and the pooled vehicle-control* group,
respectively.
Squamous cell carcinomas of the forestomach were first observed in high-
dose male rats at 51 weeks after oral intubation of EDC. These lesions were
*The pooled vehicle-control group combined the vehicle-controls from the
studies of 1,2-dichloroethane, 1,1,2-trichloroethane, and trichloroethane.
9-170
-------
characterized microscopically by acanthosis and hyperkeratosis in the super-
ficial area. The basal epithelial layer contained papillary cords and nests of
anaplastic squaraous epithelium supported by a dense band of fibrous connective
tissue. These carcinomas extended through the muscularis mucosa, submucosa,
muscular layers, and serosa, and in one high-dose male, raetastasized to adja-
cent tissues.
TABLE 9-36. SQUAMOUS CELL CARCINOMAS OF THE FORESTOMACH IN OSBORNE-MENDEL
RATS TREATED WITH 1,2-DlCHLOROETHANE (EDC)
Group
Untreated control
Matched vehicle-
control
Pooled vehicle-
control
Low-dose
High-dose
Males p value3
0/20 (0%)
0/20 (0%)
0/60 (0%)
3/50 (6%) NS
9/50 (18%)b 0.001
Females p value3
0/20 (0%)
0/20 (0%)
0/59 (0%)
1/49 (2%) NS
0/50 (0%) NS
ap values calculated using the Fisher Exact Test. Treated versus pooled
vehicle-control.
t"A squaraous cell carcinoma of the forestoraach metastasized in one male in this
group.
NS = not significant when p values are greater than 0.05.
SOURCE: Adapted from NCI, 1978.
Hemangiosarcomas were noted in some treated rats, as described in Table
9-37, but not in any of the controls. Low-dose males and females showed higher
incidences of hemangiosarcoma than high-dose animals. Tumors were observed in
several sites, including spleen, liver, adrenal glands, pancreas, large intes-
tine, subcutaneous tissue, and abdominal cavity. The Cochran-Armitage Trend
9-171
-------
Test indicated a significant (p = 0.021) positive association between dosage
and the incidence of hemangiosarcomas in male rats as compared to the pooled
vehicle-control rats; the trend was also positive in female rats (p = 0.042).
The Fisher Exact Test confirmed these findings, with statistically significant
p values for high-dose males versus pooled vehicle-controls (p = 0.016), for
low-dose males versus pooled vehicle-controls (p = 0.003), and for both low-
and high-dose females versus pooled vehicle-controls (p = 0.041).
TABLE 9-37. HEMANGIOSARCOMAS IN OSBORNE-MENDEL RATS TREATED WITH
1,2-DICHLOROETHANE (EDC)
Group
Untreated control
Matched vehicle-
control
Pooled vehicle-
control
Low-dose**
High-dosec
Males p value3
0/20 (0%)
0/20 (0%)
1/60 (2%)
9/50 (18%) 0.003
7/50 (14%)b 0.016
Females
0/20 (0%)
0/20 (0%)
0/59 (0%)
4/50 (8%)
4/50 (8%)
p value3
0.041
0.041
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
bonly 48 animals were examined for hemangiosarcomas of the large intestine.
cOnly 49 animals were examined for hemangiosarcomas of the spleen and adrenals
and 48 for hemangiosarcomas of the pancreas.
SOURCE: Adapted from NCI, 1978.
In addition to stomach carcinomas and hemangiosarcomas, EDC-treated female
rats showed significant increases in the incidence of mammary adenocarcinomas
(Table 9-38). Tumors were observed in the high-dose group as early as 20 weeks
after treatment. The Cochran-Armitage Trend Test detected a significant
9-172
-------
(p < 0.001) positive association betwen the dosage and the incidence of mammary
adenocarcinomas when compared with either control group. This tumor incidence
was significant when the high-dose group was compared with either the matched
vehicle-control group (p < 0.001) or the pooled vehicle-control group (p =
0.002) using the Fisher Exact Test. Historically, this tumor was observed in
4/200 (2%) of the vehicle-control females.
TABLE 9-38. ADENOCARCINOMAS OF THE MAMMARY GLAND IN FEMALE OSBORNE-MENDEL
RATS TREATED WITH 1,2-DICHLOROETHANE (EDC)
Adenocarcinoma of
Group the mammary gland p value3
Untreated control 2/20 (10%).
Matched vehicle-control 0/20 (0%)
Pooled vehicle-control 1/59 (2%)
Low-dose 1/50 (2%) NS
High-dose 18/50 (36%) 0.0008
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
NS = not significant.
SOURCE: Adapted from NCI, 1978.
An increased incidence of fibromas of the subcutaneous tissue was reported
in both high-dose (p = 0.007) and low-dose (p = 0.017) male rats when compared
to the pooled vehicle-control group.
In summary, a statistically significant increase in the incidence of squa-
mous cell carcinomas of the forestomach, hemangiosarcomas of the circulatory
system, and fibromas of the subcutaneous tissue occurred in male rats. There
9-173
-------
was also a statistically significant increase in the incidence of adenocarci-
nomas of the mammary gland and hemangiosarcomas of the circulatory system in
female rats.
9.5.1.2. NATIONAL CANCER INSTITUTE (1978) MOUSE STUDY — Hazleton Labora-
tories America Inc., under the sponsorship of the NCI, conducted a bioassay of
1,2-dichloroethane (EDC) using B6C3F1 mice. The results of this study were
published by the NCI (1978) and were also reported by Weisburger (1977), the
International Agency for Research em Cancer (IARC, 1979), and Ward (1980).
Technical-grade EDC (with the same purity as that described in the NCI
rat study) was administered to 200 B6C3F1 mice starting at 5 weeks of age. The
design summary for this experiment is given in Table 9-39. On the basis of
results of the subchronic studies, 50 mice of each sex were used at each of the
two dose levels for the chronic study. The MTD was determined to be 195 mg/kg/
day for male mice and 299 mg/kg/day for female mice. The second dose, which
was one-half the MTD, was determined to be 97 mg/kg/day for male mice and 149
mg/kg/day for female mice.
Twenty mice of each sex served as untreated controls, and an equal number
were given the vehicle (corn oil) by gavage. Because of inadequacies in the
subchronic studies, the initial doses administered to the test animals were
found to be inappropriate. Signs of toxicity in these animals early in the
study led to changes of the dosages several times (Table 9-40). Animal weights
and food consumption were recorded for the first 10 weeks and monthly thereaf-
ter. Animals were checked daily for mortality and signs of toxic effects. No
dose-related mean body weigtit depression was observed in male mice or low-dose
female mice (Figure 9-10). A depression in the mean body weight of the high-
dose female mice was apparent as early as week 15. The estimated probabilities
of survival for male and female mice in the control and EDC-dosed groups are
9-174
-------
Group
TABLE 9-39. DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC) GAVAGE
EXPERIMENT IN B6C3F1 MICE
Initial 1,2-dichloro- Observation period
number of ethane Treated Untreated Time-weighted
animals dosage3 • (weeks) (weeks) average dosage'1
Males
Untreated control
Vehicle-control
Low-dose
High-dose
Females
Untreated control
Vehicle-control
Low-dose
High-dose
a Dosage, given in
20
20 0
50 75
100
0
50 150
200
0
20
20 0
50 125
200
150
0
50 250
400
300
0
rag/kg body weight, was
days per week.
^T iine-weigh ted average dosage = (dosage
78
8
70
_«_
8
70
™»™
78
8
3
67
-»*—
8
3
67
administered by
x weeks received)
90
12
12
__—
13
91
32
13
.^—
13
gavage
0
97
™-"—
195
*
0
149
— —
299
~ ~ -~
five consecutive
weeks receiving chemical
SOURCE: Adapted from NCI, 1978.
9-175
-------
shown in Figure 9-11. Terminal survival of treated and control mice is shown
in Table 9-40. For male mice, no statistically significant association between
dosage and mortality was observed. In the high-dose group, 50% (25/50) of the
mice were alive at 84 weeks and 42% (21/50) survived until the end of the
study. In the low-dose group, however-, survival was low. By 24 weeks, 52%
(26/50) of the low-dose group and 55% (11/20) of the untreated control group
had died. In the high-dose group, 72% (36/50) of the animals died between
weeks 60 and 80. These deaths may have been tumor-related, since 69% (25/36)
had one or more tumors. Survival was high in the other groups; 68% (35/50) of
the low-dose and 80% (16/20) of the untreated control groups survived until the
end of the study.
TABLE 9-40. TERMINAL SURVIVAL OF B6C3F1 MICE TREATED WITH
1,2-DICHLOROETHANE (EDC)
Males
Group
Untreated control
Vehicle-control
Low— dose
High-dose
Weeks in
study
90
90
90
91
Animals alive
at end of study
7/20 (35%)
11/20 (55%)
11/50 (22%)
21/50 (42%)
Females
Weeks in
study
90
90
91
91
Animals alive
at end of study
16/20 (80%)
16/20 (80%)
34/50 (68%)
1/50 (2%)
SOURCE: Adapted from NCI, 1978.
A gross necropsy was performed on each animal that died during the experi-
ment or was killed at the end. Twenty-eight organs in all, plus any tissues
containing visible lesions, were fixed in 10% buffered formalin, embedded in
9-176
-------
DU —
1 40-
2
5
t-
0 30-
UJ
§
I 20-
00
z
^
^ 10^
(
-<:-^.
—*?'/ *^^ *^**^>^r-^
^^ ^^ -^X^X^ ^x * ^"^^r ** "^^"^
-^^" N * ^^
^/if
rT
f
1
Untreated Control
Vehicle-Control
P.
-— — — -— Low-uose
High-Dose MALE MICE
I I I I ' 1 i 1 i 1 ' 1 1
) 15 30 45 60 75 90 105 1:
-40
_
-30
~
-20
-10
- 0
10
TIME ON TEST (Weeks)
50
40-
30-
LU
Q 20-
O
GO
10-
Untreated Control
Vehicle-Control
Low-Dose
High-Dose
FEMALE MICE
-\ 1 1 1 1 1 1 r-
15 30 45 60 75
TIME ON TEST (Weeks)
90
T~
105
•50
-40
-30
-20
-10
0
120
Figure 9-10. Growth curves for male and female 86C3F1 mice
administered EDC by gavage.
SOURCE: Adapted from NCI, 1978.
9-177
-------
<
>
>
cr
t/5
LL.
0
H;
m
CD
O
1 n
0.8 -
_
0.6 -
0.4 -
-
0.2-
'- |~~!_ ~| _
L- -, i rt Is j
*~ *• L "*""' 1
1 ~~nVi L'
i '-^S I
1 K '., '.
*! • •— •- i
|__ ls T"L
iy-k V'
s t"
1
1
hi
— — Untreated Control
Vehicle-Control
Low-Dose
High-Dose MALE MICE
1 i 1 i 1 i 1 'I1!1!1
-0.8
__'
-0.6
-0.4
m
-0.2
_
0 0
0 15 30 45 60 75 90 105 120
TIME ON TEST (Weeks)
.0
^ 0.8-
^
>
tr
w 0.6-
ij
0
5 0.4-
co
CD
o
oc 0.2 J
a.
(
^^— 't
"" ^-^— i
1 '-,
1,
1
i
^
•"i
\
Untreated Control
i
Vehicle-Control 1
Low-Dose \
H,gh-Dose 'n^ FEMALE MICE
^™* ""
1 1 ' 1 ' 1 1 1 1
) 15 30 45 60 75 90 105 1
— 1 n
1 .w
-0.8
—
-06
-
-0.4
-0.2
20
TIME ON TEST (Weeks)
Figure 9-11. Survival comparisons for male and female 36C3F1 mice
administered EDC by gavage.
SOURCE: Adapted ;ron NCI, 1973.
9-178
-------
paraplast, and sectioned at 5 p for slides. Hematoxylin and eosin stain were
used routinely, with other stains employed as necessary. Diagnoses of tumors
and other lesions were coded according to the Systematized Nomenclature of
Pathology (SNOP) of the College of American Pathologists.
The histopathologic findings of the study concerning hepatocellular
carcinomas in mice are tabulated in Table 9-41. Hepatocellular carcinomas
occurred in 2/17 (12%) untreated control males, 1/19 (5%) low-dose males, and
12/48 (25%) high-dose males. A significant number of hepatocellular carcinomas
were observed in high-dose males. The Cochran-Armitage Trend Test indicated
a positive dose-response association when the high-dose group was compared with
either the matched vehicle-controls (p = 0.025) or the pooled vehicle-controls
(p = 0.006). The Fisher Exact Test supported this finding with a significant
(p = 0.009) comparison of the high-dose to the pooled control group.
TABLE 9-41. HEPATOCELLULAR CARCINOMAS IN B6C3F1 MICE TREATED
WITH 1,2-DICHLOROETHANE (EDC)
Group
Untreated controls
Matched vehicle-
controls
Pooled vehicle-
controls
Low-dose
High-dose
Males p value3
2/17 (12%)
1/19 (5%)
4/59 (7%)
6/47 (13%) NS
12/48 (25%) 0.009
Females
0/19 (0%)
1/20 (5%)
0/50 (0%)
1/47 (2%)
p value3
NS
NS
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
NS = not significant.
SOURCE: Adnpted from NCI, 1978.
c>-179
-------
A large number of alveolar/bronchiolar adenomas were found in mice
treated with EDC (Table 9-42); 31% in both male (15/48) and female (15/48)
high-dose mice. These adenomas were not observed in untreated or vehicle-
control males. For both sexes the Cochran-Armitage Trend Test showed a sig-
nificant (p = 0.005) positive dose-response association when the dosed groups
were compared to either control group. The Fisher Exact Test also indicated
that the high-dose group for both sexes had significantly higher (p = 0.016)
incidence rates than either of the two control groups. For female mice, the
Fisher Exact Test comparing the low-dose group to the pooled vehicle-control
group was also statistically significant (p = 0.046).
TABLE 9-42. ALVEOLAR/BRONCHIOLAR ADENOMAS IN B6C3F1 MICE TREATED
WITH 1,2-DICHLOROETHANE (EDC)
Group
Untreated controls
Matched vehicle-
controls
Pooled vehicle-
controls
Low-dose
High-doseb
Males p value3
0/20 (0%)
0/19 (0%)
0/59 (3%)
1/47 (2%) NS
15/48 (31%) 0.0025
Females
1/19 (5%)
1/20 (5%)
2/60 (3%)
7/50 (14%)
15/48 (31%)
p value3
0.046
0.0201
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
°In addition, one high-dose female mouse had an alveolar/bronchiolar carcinoma.
NS = not significant.
SOURCE: Adapted from NCI, 1978.
9-180
-------
Squamous cell carcinomas of the forestomach occurred in 1/19 (5%) vehicle-
control males, 1/46 (2%) low-dose females, and 5/48 (10%) high-dose females
(Table 9-43). For female mice, the Cochran-Armitage Trend Test indicated a
significant (p = 0.035) positive association between dosage and the incidence
of squamous cell carcinomas of the forestomach when comparing the dosed groups
to the pooled vehicle-controls. The Fisher Exact Test, however, was not
significant. The microscopic appearance of the stomach of mice was comparable
to that described for rats.
TABLE 9-43. SQUAMOUS CELL CARCINOMAS OF THE FORESTOMACH
IN B6C3F1 MICE TREATED WITH 1,2-DICHLOROETHANE (EDC)
Group
Untreated controls
Matched vehicle-
controls
Pooled vehicle-
controls
Low-dose
High-dose
Males p value3
0/20 (0%)
1/19 (5%)
1/59 (2%)
1/46 (2%) NS
2/50 (4%) NS
Females
0/20 (0%)
1/20 (5%)
1/60 (2%)
2/50 (4%)
5/48 (10%)
p value3
NS
NS
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
NS = not significant.
SOURCE: Adapted from NCI, 1978.
The incidence of adenocarcinomas of the mammary gland in female mice
treated with EDC is presented in Table 9-44. The Cochran-Armitage Trend Test
indicated a significant (p = 0.007) positive association between dosage and
9-181
-------
the Incidence of adonocarcinomas of the mammary gland when compared to the
pooled vehicle-controls. The Fisher Exact Test confirmed these results with a
significant (p £ 0.003) comparison of both high-dosu (7/48) and low-dose (9/50)
groups to the pooled vehicle-control groups (0/60).
TABLE 9-44. ADENOCARCINOMAS OF THE MAMMARY GLAND [N FEMALE B6C3F1
MICE TREATED WITH I,2-IHCHLOROETHANE (EDC)
Adenocarcinoma of
Group the mammary gland p value"1
Pooled vehicle-controls 0/60 (0%)
Matched vehicle-controls 0/20 (0%)
Low-dose 9/50 (18%) 0.0005
High-dose 7/48 (15%) 0.0026
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
SOURCE: Adapted from NCI, 1978.
Endometrial tumors observed in female mice are described in Table 9-45.
The Cochran-Armitage Trend Test indicated a significant (p = 0.017) positive
association between dosage iml combined incidence. The Fisher Exact Test
showed a statistically significant (p = 0.014) incidence In the high-dose
(5/47) group.
In summary, the NC [ study in B6C3F1 mice demonstrated a statistically
significant increase i.i incidences of hepatocellular carcinomas .ind alveolar/
bronchiolar adenomas in male mice and a statistically significant increase in
incidences of alveolar/bronchlolar adenomas, mammary carcinomas, and endometrial
tumors in female mii-f.
9-182
-------
TABLE 9-45. ENDOMETRIAL POLYP OR ENDOMETRIAL STROMAL SARCOMAS IN
FEMALE B6C3F1 MICE TREATED WITH 1,2-DICHLOROETHANE (EDC)
Group
Endometrial polyp or
endometrial stromal sarcomas
p valuea
Fooled vehicle-controls
Matched vehicle-controls
Low-dose
High-dose
0/60 (0.0%)
0/20 (0.0%)
5/49 (10.0%)
5/47 (11.0%)
0.016
0.014
ap values calculated using the Fisher Exact Test (one-tailed). Treated versus
pooled vehicle-control.
SOURCE: Adapted from NCI, 1978.
9.5.1.3. SPENCER ET AL. (1951) RAT STUDY — An early inhalation study
conducted by Spencer et al. (1951) found no evidence of carcinogenic activity
when 15 male and 15 female Wistar rats were exposed 151 times during a 212-day
period to EDC at 200 ppra for 7 hours per exposure.
9.5.1.4. MALTONI ET AL. (1980) RAT STUDY — A more recent inhalation study
conducted by Maltoni et al. (1980) in Sprague-Dawley rats provided no evidence
of carcinogenicity after lifetime exposure to EDC. The EDC used in this study
was supplied by Montedison, and had a purity of 99.82%, with five contaminants
(Table 9-46). The design of the experiment is given in Table 9-47.
Maltoni et al. exposed four groups of 12-week-old Sprague-Dawley rats
(each group consisting of 180 rats of both sexes) to EDC concentrations of 250-
150 ppm, 50 ppm, 10 ppm, and 5 ppm, respectively, 7 hours per day, 5 days per
week, for 78 weeks. After several days of 250 ppm exposure, the rats began to
exhibit severe toxic effects, and the concentration was reduced to 150 ppm. Two
9-183
-------
TABLE 9-46. CHARACTERIZATION OF 1,2-DICHLOROETHANE (EDC) INHALATION
EXPERIMENT IN SPRAGUE-DAWLEY RATS
Component Purity
1,2-Dichloroethane 99.82%
1,1-Dichloroethane 0.02%
Carbon tetrachloride 0.02%
Trichloroethylene 0.02%
Perchloroethylene 0.03%
Benzene 0.09%
SOURCE: Maltoni et al. , 1980.
groups, composed of 180 rats per group, served as controls. One of the two
control groups was kept in an exposure chamber under the same conditions and
for the same length of time as the exposed rats. At the end of the treatment
period, the animals were allowed to live until spontaneous death. Animals
were weighed every 2 weeks during the treatment period and every 8 weeks there-
after. All detectable gross pathologic changes were recorded. A complete
autopsy was performed on each animal, with histopathologic examinations conduc-
ted on the following: brain, Zymbal glands, retrobulbar glands, tntersr.apular
brown fat, salivary gland, tongue, lungs, thymus, diaphragm, liver, pancreas,
kidneys, spleen, stomach, various segments of the intestine, bladder, gonads,
lymph nodes (axillary, inguinal, and mesenteric), and any other organ with
pathologic lesions.
The extent of mortality varied with the different groups, but there appears
to be no direct relationship between mortality and exposure to EDC (Table 9-A8).
9-184
-------
TABLE 9-47. DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC)
EXPERIMENT IN SPRAGUE-DAWLEY RATS3
Group
I
II
III
IV
V
VI
Concentration
250-150 pprac
50 ppra
10 ppm
5 ppm
Controls in chambers
Controls
Sex
M
F
M
F
M
F
M
F
M
F
M
F
Animals'3
Number
90
90
90
90
90
90
90
90
90
90
90
90
aExposed 7 hours/day, 5 days/week, for 78 weeks.
bSprague-Dawley rats, 12 weeks old at start.
cAfter a few weeks the dose was reduced to 150 ppm, because of high toxicity
at the 250 ppm level.
SOURCE: Maltoni et al., 1980.
9-185
-------
TABLE 9-48. SURVIVAL OF SPRAGUE-DAWLEY RATS EXPOSED TO EDC AT 52 AND 104 WEEKS3
oo
Dose
Group (ppm)
I 250-15Qb
II 50
III 10
IV 5
V Controls
in chambers
VI Controls
Sex
M
F
M
F
M
F
M
F
M
F
M
F
Initial
numbers
90
90
90
90
90
90
90
90
90
90
90
90
Survivors at
52 weeks of age
Number
79
84
87
87
81
87
89
90
80
79
83
88
Percent
87.8
93.3
96.7
96.7
90.0
96.7
98.9
100.0
88.9
87.8
92.2
97.8
Survivors at
104 weeks of age
Number
10
21
17
29
13
26
45
48
12
22
16
36
Percent
11.1
23.3
18.9
32.2
14.4
28.9
50.0
53.3
13.3
24.4
17.8
40.0
Survivors
52 weeks
start of
Number
67
79
70
84
70
81
75
85
64
73
72
84
after
from
study
Percent
74.4
87.8
77.8
93.3
77.8
90.0
83.3
94.4
71.1
81.1
80.0
93.3
aRats were 12 weeks old at the start of the experiment.
bAfter a few weeks the dose was reduced to 150 ppm because of high toxicity at the 250 ppm level.
SOURCE: Maltoni et al., 1980.
-------
The highest survival rate was observed in both males and females of the group
exposed to EDC at 5 ppm. In females the highest mortality rate was observed in
the control group in the chamber and in the group exposed to EDC at 250-150
ppm. The survival rates at 52 and 104 weeks of age are reported in Table 9-48.
At these ages the overall survival rates were 93.9% and 27.3%, respectively.
The survival rates after 52 weeks from the start of the experiment are also
given in Table 9-48. The overall survival rate was 83.7%.
The results of histopathologir analysis are shown in Table 9-49. No
statistically significant increase in the incidence of any specific type of
tumor was found in the treated rats when compared with controls. There was,
however, an increased incidence of mammary tumors in some rats in Group IV,
particularly when compared with the control group in the chamber (Table 9-50).
It appears that the increase in mammary tumors in some of the treated
groups, particularly when compared to controls in the chamber, was not due to
malignant tumors, but to fibromas and fibroadenomas. The increased incidence
of mammary fibromas and fibroadenomas is statistically significant in groups
exposed to EDC at 250-150 ppm, 50 ppm, and 5 ppm, when compared to controls
in the chamber, but not significant when compared to controls outside of the
chamber. Although the author stated that the onset of fibromas and fibroade-
nomas was age-correlated, the incidences of these tumors observed in the vari-
ous groups were probably due instead to the differences in survival rates
within the groups. The highest difference in incidence was found between the
control groups in the chamber, which had low survival rates, and the groups
treated with EDC at 5 ppm, which had high survival rates. The difference in
survival rates is thus seen to be related to the differences in incidence in
the two control groups.
9-1S7
-------
IABI> 9-49. rilMDR INCIUhNO IN SPKAGMt-DAULKY KATS fcXPIISHI TO 1,2-DICHLOROF.THANK (KIIC)
Animals with
Mammary tin
Anlnvilh"
(•r.inp (rfnii i nt t ill. in s.'\
1 'SO-ISH p ,.,„<• M
).
M iii.l 1
1 1 Ml [ipm "I
h
M .in.) 1
VO
,L III Ml ppra M
00 H
OO M .iinl Y
IV S |.pm H
}
N and !•
V (.mil nil* In H
i li iinhi'r* !•
H .mil !•
VI (nnt ml* M
t
M anil t
1.H II
Niimhi-l
11 I ill
'III
'lit
IH.I
'in
'lit
IHll
til
111
IHll
411
911
IHll
9(1
9l>
IH'l
90
911
180
10811
( nt ri'i toil Tol il
84
40
174
40
Oil
IHll
X9
40
1/4
411
41)
|8ll
90
90
IHll
40
90
180
107)1
immhrr
II
'ii
hi
III
58
hH
S
ll
iK
II
h5
76
8
18
!th
S
52
">'
Percent™
12.1
57.7
JS.2
II. 1
64.4
17.8
5.h
'•7.8
2h.H
12.2
72.2
42.2
8.9
42.2
25.5
5.5
57.8
31.7
nors
Average
latency
time
92.9
78.6
81.1
H8.8
78.5
80.1)
60.8
79.4
77.5
110.2
81.2
87.1
85.5
81.1
81.6
42.1)
85.5
86.1
/ymhal gland carcinomas
Total
number
II
2
2
II
1
1
0
II
(I
0
2
2
,
1)
1
1
1)
1
Percent*1
2.2
I.I
1 . 1
0.6
—
2.2
I.I
I.I
0.6
I.I
0.6
Avrrngp
lal'-nry
time
78.0
78.0
86.0
86.1)
47.1)
47.0
78.0
78.0
74.0
74.0
Total
numhe r
0
0
0
1
2
1
4
0
4
2
A
8
1
3
6
0
3
1
tumorn
Leukemlas
Percent"
_.__
—
I.I
2.2
1.7
4.6
2.2
2.2
6.7
4.4
I.I
3.1
2.2
3.3
1.7
Avernge
latency
time
..__
fld. (1
74.5
76.3
74.0
74.0
111. 5
81.5
96.11
17.0
M.I
59.5
79.3
79.3
Nephroblastnmas
Total
numbe r
0
0
»
0
0
0
0
0
0
1
(I
1
0
0
0
(1
0
0
Average
latency
tine
Percent"
-------
TABIF 4-49. (rnnllnnnH)
Animals with tumr
Anglnsarcomas
Group Cnn« 4*«ll rat l»n SPK
I JSO-IVI ffaf H
F
•< ,v»d F
1 1 Sn rpm M
K
^l" H and F
00 III in ppn M
*£> t
M and 1
IV S ppn H
F
N and f
V GiiUrnll (n M
chamber*. F
M and F
VI Cnnlrnls 1
r
H anri F
l.lver
Aver-ir.-
Intent i
Tut il I INK*
nnmher Pen em n (wk)
-------
TABLE 1-49. (continued)
Animals with tumors
l.nmp Cum iMit r.il Inn Sc<
1 2SII-IMI |ipnp M
F
H .ind >•
I 1 Sll ppm H
F
M and F
VO
1 III Id ppm H
rx *
vD
O H .ind F
IV •) ppm M
|.
H and V
V GmirnK In H
iliamhrrs F
H and t
VI Cnntrols H
F
M and F
Hepatomaa
Average
latency
Tiu.il lime
number IVrc«nth (wk)"1
II —
II
II ... —
o — —
II
II — —
0
I)
II
0
o
0
0
0
o
0
0
Foreatomach epithelial
Total
number
0
0
0
1
0
1
0
1
1
1
0
1
2
1
1
2
1
3
Percent b
._ .
—
—
I.I
—
O.n
—
I.I
O.b
I.I
0.6
2.2
1.1
1.7
2.2
I.I
1.7
Average
latencv
time
...
...
60. 1)
60. 0
78.0
78.0
101. 1)
...
101.0
78.)
61.0
71.1
110.0
102.0
107.3
Skin carcinomas
Average
latency
Tut a 1 tine
number Percent11 (wk)d
0
1)
0
1 I.I th.O
0
1 0.6 36.0
1 I.I 94.0
1)
1 0.6 94.0
,1
o
„
0
0
0
1,
0
0
Subcutaneous aarcoaan
Total
number Percent1"
0
0
0
0
0
0
0
0
0
1 I.I
0
1 0.6
0
1 I.I
1 0.6
0
0
0
Average
latency
time
(„.)<
...
.__
—
_-.
—
...
30.0
10.0
—
7H.O
78.0
-------
TABLE 9-49. (continued)
Anlnu
ils with tumors
Kncephallc tunorB
(•rmip f'onrenlrHl Ion Sex
1 2VI-I5II ppme M
F
H and f
II 'ill i ppm H
F
H and f
V Controls In N
ih.imhi'rs F
M and >
V\ l.nntrul* N
t
H and F
Neuruhl iqtom.is
Avur.iRe
Inleiif y
Toi.il time
numhvr Percent11 (wk)d
1)
0
o
o
0
0
o
O
o
II — —
I)
0,
o
o — —
n
o — —
o
o
Other sites
Total
number
0
0
0
1
1
2
0
2
2
0
0
0
1
|
2
0
3
3
Percent"
_—_
1.1
I.I
I.I
1.1
2.2
I.I
I.I
I.I
1.3
1.7
Total
number
9
II
20
16
6
20
4
4
8
Ih
9
25
8
4
12
7
8
IS
Others
Benign
numbe r
7
5
12
14
}
17
2
3
5
15
5
20
7
2
9
7
5
12
Malignant
numbe r
2
6
8
0
3
3
2
1
3
1
4
5
1
2
3
0
3
3
Number
15
54
69
20
56
76
13
43
56
30
65
95
17
38
55
14
56
70
Perr«ntn
16.9
60.1)
38.5
22.2
62.2
42.2
14.6
47.8
31.3
33.3
72.2
52.8
18.9
42.2
30.6
15.6
62.2
38.9
Totals'
Number ol
HI ffprrnt
tumiirs/tufliiir-
beiirliiK nnfmals
1.2
2.0
1.9
1.6
1.7
1.7
I.I
l.n
1.5
1.4
1.8
1.7
1.3
1.9
1.7
1.1
1.7
1.6
Fxpnsiin by Inh.iI it Ion to Flic In air nt 250-151) ppm. 5U ppm, 10 ppm, and 5 ppm, 7 hours/day, 5 days/week,
fur 78 upi-ks. K.-sults after 148 week's (end of experiment).
"Spr.iK'ie-ltawluy r.its, 12 weeks old at stnrt.
blhe pere i'iil.ices rufer to the corrected numbers.
cAv<*r.i>>e •tf.f - onset of the first mammary tumor per .mlmal detected at the periodic control or at autopsy.
dAver.iKi' time from the itnrt nf the experiment to detection at the periodic control oc at autopsy.
eAfter A frw week's I he dr>
-------
TABLE 9-50. MAMMARY TUMORS IN SPRAGUB-DAWLEY RATS
EXPOSED TO 1,2-DICHLOROETHANE (EDC)
o
r-
Mammary tumors
Croup Concentration
I 250-150 ppod
II 50 ppm
III 10 ppm
IV 5 ppm
V Controls In
chambers
VI Controls
Total
Sex
M
F
M and F
M
F
M and F
M
F
M and F
M
F
M and F
M
f
M and F
M
F
M and F
Animals
Number
at start
90
90
180
90
90
180
90
90
180
90
90
180
90
90
180
90
90
180
1080
Corrected
number8
89
90
179
90
90
180
89
90
179
90
90
180
90
90
180
90
90
180
1078
Total
number
11
52
63
10
58
68
5
43
48
11
65
76
8
38
46
5
52
57
Percent b
12.3
57.7
35.2
11.1
64.4
37.8
5.6
47.8
26.8
12.2
72.2
42.2
8.9
42.2
25.5
5.5
57.8
31.7
Average
latency
time
("k>c
92.9
78.6
81.1
88.8
78.5
80.0
60.8
79.4
77.5
110.2
83.2
87.1
85.5
83.3
83.6
92.0
85.5
86.1
Number of
tumora/ tumor-
bearing animals
1.1
1.9
1.7
1.4
1.5
1.5
1.0
1.4
1.4
1.4
1.6
1.5
1.0
1.8
1.6
1.0
1.5
1.5
(continued on the following page)
-------
TAKI.F 9-SII. (conHini
I
vD
Mammary tnmorse
Hlstologlcal evaluation
Haimary tumors
C.ritiip Concent r.il Inn Sex
1 2 SO- ISO pin1' M
F
H .mil F
1 1 SO ppm M
r
M .111.1 F
III II) ppm M
1-
H .mil F
IV S ppm M
F
M riiiil F
V Com nils In M
chambers r
M *nd h
VI Controls M
r
H and r
Tnr.il
number
9
SO
S9
<,
SI
h2
S
IH
41
II
61)
/I
H
(S
4)
S
49
S4
Percent'
HI. 8
96. 1
91.6
til.H
Ol.'i
41.2
10(1. (1
MH.4
Hl.h
1llll.ll
92. 1
91.4
IIIU.O
9J.1
MI.S
Mill. II
94.2
9'.. 7
Nit m her
,
47
54
7
'.9
S6
1
11
)6
1 1
Sh
67
7
27
34
•j
',}
so
Per.entB
77.8
94.0
91. S
77. H
92.4
111. )
1,11.0
Hh.U
HI. 7
IIHI.II
91. 1
94.4
87. S
II. \
79.1
(,0.0
9S.9
92.6
Average
time
nsiiru hy Inlinl.illnn Hi I.IK In .ilr .it 2SO-ISIJ ppm, SO ppm. HI ppm, anil "> |>pm. 7 hours/d.iy, S tl.iys/uoi'
for /H ui'fks. Ki'sulls nfli-r I4H wcok« (enil ol .-tpHr Ini'nt).
s illvc afli-r 12 wucks, uhun the first tnnnr (a uiminnry crfrrlnnim) w.is observed.
''Tlii1 pt-ri unt.*Kt"« refer to C h<» currecLeil nunhfrs.
< Avur.u'e i^c >IL the onset of lliv flrht mammary tiimur per .inlnal iletc* ted .it the periodic (nntrul or nt
rfAflfr .1 few wi-vks the dose unadi>nnm.is, c.ir. Inoraas. sarcomas, cdrLlnoS'irconus) may
hi' pn-sunt In the same .inlm.il.
'The per i i-nin^i". refer to lulal number of animals hr.irlni; mammary tumors.
Erin- PITI i-nt.i|>,i's refer to total numher nt .inlm.iK ht-arlni; rn.imn.iry mmors, hi<:tulat;lcal ly examined.
SlUiKCr Haltonl et dl.. 1VHII.
-------
9.5.1.5. MALTONI ET AL. (1980) MOUSE STUDY — Four groups of Swiss mice
(180 mice of both sexes per group) were exposed to EDC, at the level of purity
previously described for the rat inhalation study, in concentrations of 250-150
ppm, 50 ppm, 10 ppra, and 5 ppra, respectively, 7 hours per day, 5 days per week,
for 78 weeks. After several days of 250 ppm exposure, the mire began to exhibit
severe toxic effects, and the concentration was reduced to 150 ppm. One group
of 249 mice served as controls. The design of the experiment is given in
Table 9-51.
At the end of the treatment period, the animals were allowed to live un-
til spontaneous death. The remainder of the procedure was the same as that
described previously for the Maltoni et al. (1980) rat study.
The survival rates for female mice were slightly lower in the group treated
with EDC at 250-150 ppm. The survival rates for mice at 52 and 78 weeks of age
are shown in Table 9-52. At 52 and 78 weeks, the overall survival rates were
82.4% and 45.9%, respectively. The survival rates after 52 weeks from the
start of the experiment are also given in Table 9-52. The overall survival
rate was 67.8%.
The results of histopathologic analysis of various tumors are shown in
Table 9-53. These results do not indicate a statistically significant increase
in the incidence of any specific type of tumor in the treated mice as compared
with controls.
In conclusion, although Maltoni et al. conducted extensive carcinogenicity
studies in rats and mice, no significant incidences of tumors were seen in any
of the target organs. Several factors may have contributed to these findings,
such as differences in the strains of rats used, and pharmacokinetic differences
in rates of formation and retention of reactive metabolites in the target organs
between gavage and inhalation routes of administration.
9-194
-------
TABLE 9-51. DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC)
EXPERIMENT IN SWISS MICE
Animals3
Group Concentration
I 250-150 ppmb
II 50 ppm
III 10 ppm
IV 5 ppm
V Control
Sex
M
F
M
F
M
F
M
F
M
F
Number
90
90
90
90
90
90
90
90
115
134
aSwiss mice, 11 weeks old at start. Exposed 7 hours/day, 5 days/week, for 78
weeks.
^After a few weeks the dose was reduced to 150 ppm, because of high toxicity
at the 250 ppm level.
SOURCE: Maltoni et al., 1980.
9-195
-------
TABLE 9-52. SURVIVAL OF SWISS MICE EXPOSED TO 1,2-DICHLOROETHANE (BOC) AT 52 and 78 WEEKS8
Survivors at
52 weeks
Croup
I
II
III
IV
V
Dose
(ppm) Sex
250-150 ppmb H
F
50 H
F
10 M
F
5 M
F
Control H
F
Initial
numbers
90
90
90
90
90
90
90
90
115
134
of
Number
56
75
68
83
74
86
54
84
91
127
age
Percent
62.2
83.3
75.6
92.2
82.2
95.6
60.0
93.3
79.1
94.8
Survivors at
78 weeks
of
Number
26
44
30
49
34
50
26
68
42
76
age
Percent
28.9
48.9
33.3
54.4
37.8
55.6
28.9
75.6
36.6
56.8
Survivors after
52 weeks from
start
Number
39
58
46
73
59
72
42
84
72
112
of study
Percent
43.3
64.4
51.1
81.1
65.6
80.0
46.7
93.3
62.6
83.6
"Mice were 11 weeks old at the start of the experiment.
bAfter a few weeks the dose was reduced to ISO ppm, because of high toxlclty at the 250 ppm level.
SOURCE: Maltont et al., 1980.
-------
TARI.F 9-%
TtfHIlK INCNIFNCF IN SWISS HICK
HI I ,2-niCIIUIROHHANF (KnC)
Mammary tumors
Anlnals"
Croup Cunrent mt 1 »n leu
I 2SH-\3« pprf M
F
H and ^
1 1 30 ppn H
F
H and F
III ID ppn H
F
H and t
IV 5 npm H
F
H and F
V Control;, M
F
M and F
Total
Number
at st.irt
90
911
180
911
9J
Ihl)
91)
90
18(1
9I>
9(1
ISO
115
134
249
969
Cnrrei tt
-------
TABLE 9-51. (continued)
I
I—1
00
Croup Concentration Sex
1 250-150 ppaf H
r
H and F
11 V) ppm H
F
N and F
III 10 ppm M
F
H and F
IV i ppm H
F
M and F
V Controls In H
chambera F
H and F
VI Controls N
F
H and F
Nephroblastomas
Average
latency
Total tine
number Percent (wk)'1
n ___ ___
0
I)
n __. __«
0
0
0
n — — — ___
o
n
0
o
o
o
0
0
0
U ™ ™
Animals with tumors
Kidney adenocarclnomas
Averse
latency
Total time
number Percent0 (wk)H
0 —
o
0
0
0
o
0
0 — -—
o
n _-. ___
1 I.I 62.0
1 0.6 62.0
n — — _—
o
Q __— ___
n ___ ___
o
n __._ «__
Liver
Average
latency
Total time
number Percent0 («k)d
O — — — —•
o — —
o
0 ___ __«
0
o
o
fl — — — — 1_
0 ™ —
o
f) .•__ ___
fl — — — — - -
o
n — — «»
o
0
0
o
Total
number
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
'
Other sit
Percent0
1.2
—
0.6
0.9
—
0.4
es
Average
latency
tine
40.0
40.0
121.0
131.0
1^8.0
69.U
69.11
(continued on the following page)
-------
TAK1F. 9-53. (continued)
vO
vO
AnRlomas
Animals
«lth tumors
and f IbroanKlomas
Liver
Crimp CnnciMiir.il Ion Sex
1 JSO-ISO ppm'' M
F
M and F
1 1 VI ppm N
F
H nnil K
III to ppm H
»
H and f
IV S pna H
K
H and F
V Cnntrnll In H
chambers F
H And F
Tut a I
number
(1
11
1)
II
1
1
0
O
0
0
0
II
n
0
0
Per<.entc
I.I
H.6
0.9
11.4
Average
latpnLy
time
—
R3.0
S3.0
__-
69. n
—
69.0
Total
number
0
0
0
|
1
i
0
1
1
0
0
n
,
0
1
Other sites
Percent0
I.I
I.I
1.1
1.1
0.6
...
0.9
0.4
Average
latency
time
76.0
83. 0
79. i
93.0
93.0
_..
76.0
76.0
Hepatomas
Average
latency
Total time
number Percent' (wk)''
0
o
o
0
o
0
0
o
o
o -— ---
o
4 3.5 82.0
A _ ___
4 1.6 82. 0
Forestnnurh vulthHIal
tunnrs
Total
number
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Average
latency
tine
Percent."1 (uk)d
...
(continued on the follm*Ing page)
-------
TABI-F. 9-51. (continued)
O
O
Croup ronrenlr.it Ion Sex
1 2511-150 ppra? M
F
H and t
1 1 5ll ppm H
F
H and F
III III ppm M
K
H and F
IV 5 ppm M
F
N and F
V rVintrnls In N
chambers F
M and F
Skin
Total
number
II
II
I)
I)
1
0
I)
0
0
1
"
1
1
2
epithelial tumors
Average
latency
time
l'ercentc (wk)d
___ _
—
___
I.I 101.0
ll.h 11)1. 0
___
—
I.I 79.0
ll.h 79.0
0.9 109.0
0.7 77.0
O.R 91. 1)
Animals
with tumors
Subcutaneous sarcomas
Total
number Percent0
0
0
11
0
0
0
1 I.I
1 I.I
2 I.I
0
0
(1
1 0.9
O
0 (1.4
Average
latency
lime
(wk)11
___
—
___.
—
77.0
25.0
51.0
lOh.O
Illh.O
Total
number
0
2
2
1
4
s
,
2
1
1
4
5
1
4
5
Others
Benign
number
0
2
2
0
1
1
,
1
2
0
3
1
1
2
1
Total'
Malignant
number
0
0
0
1
1
2
0
1
1
1
1
2
0
2
2
Number
2
II
11
9
15
24
12
17
29
4
19
23
14
27
41
Percent1"
2.4
12.9
7.7
III. A
17.2
14.0
11.5
19.1
Ih.l
5.7
21.1
14.5
12.4
20.1
In. ft
Number of
different
tumors/tumnr-
hearlng animals
1.0
1.0
1.0
1.2
I.I
1.2
I.I
1.0
I.I
1.0
1.0
1.0
1.4
1. 1
1.2
hxposun- hv lnli.Tl.it Ion to H)f In all Hi J 511- 150 ppm, 50 ppm, K) ppm, and 5 ppm, 7 houri/day, 5 ddys/wcrk,
fur ?H wei-k-. Krsuli- liter 119 weeks (cml ul exprr linrnt ).
(1swlsK rnlif, II wi>i*ks *ilil at start.
"AnlmiK illvo ilti-r l'> wi-i-ks, wliun tht* flr>.l I umor (n Ivnki-ml.i) w.i<. ohst-rvcd.
1 llu> pi'i 1 1 nl.ij't's refrr to llif t MTIIM tfil numlitTs.
time t rum tin- st.irt of the experiment to detection nt tin- periodic control or at autnpuy.
eAfti'r i I iiw wfeks tin1 dose was n ducpcl to 1 5
-------
9.5.1.6. THEISS ET AL. (1977) MOUSE STUDY — Theiss et al. (1977) con-
ducted a pulmonary tumor bloassay with EDC and other organic, contaminants of
drinking water in the United States. The compounds were injected intraperito-
neally into 6- to 8-week-old mice of the A/st strain. Each dose of reagent-
grade EDC, with tricaprylin as the vehicle, was injected into mice in groups
of 20, three times a week, for a total of 24 injections per mouse. Dose levels
of 20, 40, and 100 mg/kg (the maximum tolerated dose) were used.
The mice were sacrificed 24 weeks after the first injection, and their
lungs were placed in Tellyesniczky*s fluid. After 48 hours, the lungs were ex-
amined microscopically for surface adenomas, and the frequency of lung tumors
in each group was compared with that in a vehicle-treated control group by means
of the Student's t Test. The incidence of lung tumors increased with dose, but
but none of the groups had pulmonary adenoma responses that were significantly
greater (p > 0.05) than that of the vehicle-treated control mice (Table 9-54).
Because they found a nonsignificant elevation in pulmonary tumor response, the
authors suggested further investigation of the carcinogenic potential of EDC.
TABLE 9-54. PULMONARY TUMOR RESPONSE IN STRAIN A/st MICE INJECTED WITH
1,2-DICHLOROETHANE (EDC)
Dose/ Inject ion
(mg/kg)
Qa
20
40
100
Number of
injections
24
24
24
24
Fraction
surviving
46/50 (92%)
14/20 (70%)
16/20 (80%)
20/20 (100%)
Number of
tumors /mouse
0.39 + 0.06
0.21 + 0.06
0.44 + 0.11
0.75 + 0.17
aTricaprylin vehicle.
SOURCE: Theiss et al. , 1977.
9-201
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9.5.1.7. VAN DUUREN ET AL. (1979) MOUSE STUDY — Van Duuren et al. (1979)
conducted a bioassay of EDC and of a suspected metabolite, chloroacetatdehyde,
as initiators, promoters, and complete carcinogens using two-stage skin tests.
Female non-inbred ICR/Ha Swiss mice were used, with treatment beginning at 6 to
8 weeks of age. The experimental group consisted of 30 animals, exclusive of
the no-treatment groups or those receiving repeated application of phorbol
rayristate acetate (PMA). The results of this study are given in Table 9-55.
The compounds, in O.I raL and 0.2 mL acetone, were applied three times a week to
the dorsal skin. Skin lesions were diagnosed as papillomas when they reached
approximately 1 mm and persisted for 30 days or more.
All animals were examined daily and weighed monthly; findings were recor-
ded monthly. Animals in poor health or with large tumor masses were killed.
Animals were completely autopsied at the termination of the experiment or at
death. Tissue sections were fixed in 10% formalin, processed, blocked in par-
affin, and stained with heraatoxylin and eosin for pathologic diagnosis. The
results indicate that neither EDC nor chloroacetaldehyde induced a statistical-
ly significant increase in the incidence of carcinomas of the skin. This
result was probably due to the low dose levels used in the study. EDC was
found to induce a statistically significant increase in the incidence of benign
lung papillomas.
9.5.1.8. SUMMARY — The NCI study (1978), as well as the studies by
Theiss et al. (1977) and Van Duuren et al. (1979), show that, under appropriate
conditions, EDC can increase the incidence of tumors. The data obtained in
mutagenicity studies have also shown that, under appropriate experimental
conditions, EDC can cause DNA damage (Storer et al., 1982) and mutations
(Guengerich et al., 1980). In contrast, the lifetime inhalation study conducted
by Maltoni et al. (1980) showed no increased incidence of tumors.
9-202
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1A»I> 1-SS.
SKIN HIilASSAY lit I ,^-IMl HLIIKOI- CIIANI- (tl)C) AND (JILOROAO rALnKIIYIIK
N>
O
In 1 1 1 il Inn-pr-Jimtt Ion '
Dnsc Mivs tit Mice with
mj*/.ippl h it 1 mi/ 1 i rst papl 1 lom.is*'/
Compiii in*1 nmiKf* t nmir tnt.i I p»ipt 1 Inmds
MIC i "«.i> is; 1/1
Chloroiri-t ildphyeli' 1." IV 1/1(1)
HHA mm roll
(1211 mltH) II.IUI2S 141 I/ H> (1)
(/' (2)
ArPtniip (II. 1 mL) — —
No i rp.ilnpnt
(MID mli-e)
Kcpciti'd iippl i rat ton*1
Iliisv Days to Nice with Nil. ol mire
mi;/appllr.itlnn/ first fapl 1 lnnsc/ with distant
nniioi- tumil total pnplllnm.ii tnmnrs11
I2h.ll 0 2h IIIIIK
1 stnnvnh
•'iZ.lt II 17 InilK
1 s t otlil t h
l.ll (1 14 lung
II. 1 mL II II lung
i il iimn ill
0 III I" OR
*t itom.lrli
"Ml appllcHtlons wr« to tlw ilorial -skin hy mlrmplppl f i-. HIT. was .idmt nlntfivil onrc only In O. i nl. nrptune,
fnllnwrd 14 days later by *> UK of I'MA In <>..! ml. .icPt.ino three Hmei wci-kly. Chlnroan'trtlriohydp u.iv ndmlnls-
tervd unrc only In O.I ml niPtunr, fcillnuril 14 it.iy.*. liter hy 2.1) UK nf WA In O.I ml. ncetont- three times
weekly.
hKIT w,i- .id ml nlnli-rcd In II.j ml. Hrotnm*. (.Ill urn.K ctalripliydi* was .idrnl nl if ircrt three t Impo weekly
In IV. I nl. acetone.
rNnnihprs nf rolri" with •sqiMmiHif coll .-.ir.-lnniws .ire' Klven In pirenthcispn.
<*A1 I I"")! lnnnr Klvon only where siRnlflcant, I.e., p < O.!)1).
SOIIKCh- Adapted frnn V.in I n-n ct al., l*)7<>.
-------
The apparent discrepancies between inhalation and other routes of exposure
in the oncogenicity studies have led to considerable discussion of the conduct
and results of these studies (Maltoni et al., 1980; Hooper et al., 1980; Reitz
et al., 1982) (see discussion in section 9.5.3.). The NCI Clearing House
Committee review of the NCI bioassay report (1978) acknowledged several design
features which may have adversely affected the outcome of the experiment. For
example, the studies were conducted in rooms housing animals dosed with other
volatile carcinogens. It was concluded, however, that these shortcomings were
not significant enough to invalidate the findings. Other investigators (Hooper
et al., 1980) have concluded that unless unusual and unexpected synergisms
occurred, the presence of other animals in the test rooms would not have affected
the results.
Regardless of the explanations for the observed differences (Hooper et
al., 1980; Reitz et al., 1982), the data show both an ability of EDC to produce
neoplasms and an inhalation level at which no tumors are formed. This may be
due to the difference in rates of formation of reactive metabolites, rates of
formation of DNA-adduct, and rates of deactivation between the oral and inhala-
tion routes. Thus, the results indicate that EDC is carcinogenic in rats and
mice, and is also a mutagen.
9.5.2. Epidemiologic Studies. No epidemiologic studies have been published
regarding the carcinogenicity of EDC.
9.5.3. Risk Estimation from Animal Data. Evidence for the carcinogenicity
of EDC consists principally of positive long-term gavage studies in mice and
rats (NCI, 1978), a suggestive positive pulmonary tumor bioassay in mice (Theiss
et al. , 1977), and a suggestive positive two-stage mouse skin painting bioassay
(Van Duuren et al. , 1979). The NCI bioassay reported excess incidences of
forestoraach squamous cell carcinomas, circulatory system heraangiosarcomas, and
subcutaneous tissue fibroma^ in male rats, and in fi'nwlr> r^ts, mammary inland
9-204
-------
adenocarcinomas and hemangiosarcomas. In male mice, an excess Incidence of
hepatocellular carcinomas and bronchiolar adenomas were found, and in female
mice, bronchiolar adenomas, mammary carcinomas, and endometrial tumors.
Theiss et al. reported that their pulmonary tumor bioassay resulted in a dose-
related increased frequency of lung adenomas in mice, while Van Duuren et
al. found an increased incidence of benign lung papillomas in mice treated
with EDO by skin painting. In contrast to these positive reports, lifetime
inhalation studies in mice and rats by MaItonI et al. (1980) showed no excess
incidence of tumors. Part of the explanation for these differing results
may be the differences in the pharraacokinetics and metabolism of EDC for the
two routes of administration, as discussed below.
It is generally assumed that the carcinogenic potential of EDC is dependent
on the metabolism of the parent compound to nucleophilic metabolites (such as
chloroacetaldehyde, the half-sulfur mustard S-(2-chloroethyl)-GSH, and its
episulfonium ion) that extensively covalently bind to cellular lipids and
proteins with organ localization and binding intensity paralleling sites of
tumor formation in liver, lung, spleen, and forestomach (Reitz et al., 1982).
DNA adduct formation with EDC metabolites has also been demonstrated both in
vitro (Banerjee and Van Duuren, 1978, 1979; Banerjee et al. , 1980; Guengerich
et al., 1980) and in vivo (although at a low level) (Reitz et al., 1982; Storer
et al., 1982).
9.5.3.1. GENERAL OBSERVATIONS — For EDC, the positive results of the
NCI (1978) oral gavage lifetime studies in male Osborne-Mendel rats and male
B6C3F1 mice showing a statistically significant dose-related excess of tumors
(hemangiosarcomas in rats and hepatocellular carcinomas in mice) were select-
ed for risk assessment. Tumors were found at both "high" (maximum tolerated
dose) and "low" doses (one-half maximum tolerated dose), as summarized in
9-205
-------
Table 9-56.
The chronic oral dosage used in the NCI rat bioassay (95 and 47 mg/kg/
day) was well below (8- to 16-fold) the acute LD5Q for rats (680 to 770 mg/kg;
McCollister et al. , 1956; Smyth, 1956; Heppel et al., 1945). Similarly, the
chronic oral dosage in the NCI mouse bioassay (195 and 97 mg/kg/day) was well
below the acute LD5Q report for mice (489 and 413 mg/kg male and female,
respectively; Munson et al. , 1982). Nonetheless, signs of toxicity from
chronic administration of CDC (less weight gain, shortened life span) were
evident in the NCI bioassay for both rats and mice. Presumably EDC is sub-
jected to elimination processes during the 24-hour interval between doses (and
on weekends), and hence the chronic dosage of the NCI assays may not be
cumulative. EDC has a short half-life in rodents after acute oral administra-
tion (in corn oil) of 1 hour for a 150 mg/kg dose (Spreafico et al. , 1979,
1980); hence, the conditions of the NCI study could be represented kinetically
as repeated (multiple) acute exposures rather than chronic exposure in which
the administration is repeated at intervals (24 hours in this case) that are
shorter than the time required to clear all EDC from the body. Thus the
toxicity of EDC evident in the NCI gavage study of rats and mice may not be
due to the intact EDC molecule per se but to cumulative cellular damage from
covalent binding of reactive metabolites from EDC metabolism. It is of in-
terest that the Indices of toxicity (weight gain, depression, mortality) in
the NCI gavage study were not more severe for mice than for rats, although the
maximum and half-maximum "tolerated" time-weighted average daily dose was
twofold greater for mice than for rats on a mg/kg basis (Table 9-56). How-
ever, on a proportional surface area basis for the two species (rat, 500 g;
mouse, 33 g)''^ =6.1, the doses given to rats in terms of mg/antmal were
actually slightly greater than those given to mice (ratio, ratrmouse, 7.4;
9-206
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TABLE 9-56. INCIDENCE RATES OF HEMANGIOSARCOMAS IN THE CIRCULATORY SYSTEMS OF MALE
OSBORNE-MENDEL RATS AND OF HEPATOCELLULAR CARCINOMAS IN MALE B6C3F1 MICE
XO
O
Average
terminal
Species Sex weight (g)
Rats M 500
Mire M 33
Time-weighted average
gavage dosea
mg/kg/day
0
47
95b
0
97
195
mg/animal/day
0
23.5
47.5
0
3.2
6.4
Incidence
0/40
9/48
7/27
1/19
6/47
12/48
(0.0%)
(19%)
(26%)
(5%)
(13X)
(25%)
aGavage dose in corn oil administered 5 days/week with a time-weighted average dose of: dosage x weeks received
divided by all weeks receiving EDC. Mg/animal based on average terminal weights of rats (500 g) and mice (33 g).
bAll animals In this group died before the bioassay was terminated.
SOURCE: NCI, 1978.
-------
Table 9-56).
The fact that the NCI and Maltoni et al. carcinogenicity bioassays were
conducted using two different routes of EDC administration (oral and inhala-
tion, respectively), presents substantial interpretive difficulties, pharmaco-
kinetic and otherwise, for the evaluation of the positive results of the NCI
study and the negative findings of the Maltoni et al. study. A major reason
for these difficulties is evident from a consideration of the dosages used in
the two bioassays. The time-weighted average dose levels administered by
gavage per day to Osborne-Mendel rats in the NCI study were 47 and 95 mg/kg
(5 days/week for 78 weeks); the inhalation doses for rats (S-D) in the Maltoni
study were 150, 50, 10, and 5 ppm, 7 hours/day, 5 days/week, for 78 weeks. In
both assays higher doses (NCI, 150 mg/kg; Maltoni, 250 ppm, 6 hours) were
initiated but were reduced after a few weeks of treatment. The data of Reitz
et al. (1982) provide an approximate comparison of these doses. These inves-
tigators determined the assimilated dose of Osborne-Mendel rats given a single
oral administration of EDC (150 mg/kg) and a single inhalation exposure (150
ppm, 6 hours). These doses represent the highest levels of EDC administered
for any significant length of time in the two bioassays. The assimilated EDC
from the 150 rag/kg gavage dose was observed to be completely absorbed (150
mg/kg), while the inhalation exposure was estimated to represent an average
assimilated dose of 50.7 mg/kg, i.e., one-third of the oral dosage (Table
9-57). Furthermore, the assimilated dose from 150 ppm inhalation exposure to
rats (the highest dose level of the 150, 50, 10, and 5 ppm Maltoni et al.
bioassay), 50.7 mg/kg, is approximately equal to the lowest time-weighted dose
of the NCI bioassay of rats but is less than the lowest dose given to mice
(Table 9-56). Therefore, in respect to dosage levels of the NCI and Maltoni et
al. bioassays, it appears that the animals in the Maltoni study were subjected
9-208
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TABLE 9-57. DISPOSITION OF 14C-EDC IN RATS3
AFTER SINGLE ADMINISTRATIONS OF 150 MG/KG ORALLY
AND 150 PPM FOR 6 HOURS BY INHALATION
Oral Inhalation
mg/kg mg/kg
Assimilated doseb 152.3 ± 38.6 50.7 ± 13.4
Exhaled air 44.2 t 5.9 (29.0%) 0.9 ± 0.04 (1.8%)
(unchanged EDC)
Metabolized
C02
Urine
Feces
Carcass
Cage wash
8.2 ±
91.6 ±
2.6 t
4.6 ±
1.2 ±
108.2
1.2
34.4
1.5
1.5
0.6
(71.0%)
3.6 ±
42.8 ±
0.9 ±
2.2 ±
2.2 ±
49.8
0.7
11.9
0.3
0.3
0.3
(98.2%)
aOsborne-Mendel rats, 150 to 250 g.
^Radioactivity recovery in exhaled air, urine and feces, 48 hours after admin-
istration. The values given are means ± SD for 4 rats for each route of
exposure and are milligram equivalents of ll*C-EDC radioactivity.
SOURCE: Adapted from Reitz et al., 1982.
9-209
-------
to lower effective dose levels than those in the NCI mouse study and were, at
most, comparable to the low-dose group in the NCI rat study. Other evidence of
the effective dosage difference between the two bioassays is summarized in
Table 9-58 and discussed in section 9.5.3.2.5.
9.5.3.2. INTERSPECIES DOSE CONVERSION
9.5.3.2.1. General Considerations — This section discusses the metabolism,
pharmacokinetics, covalent binding, and toxicologic findings that are relevant
to the carcinogen risk assessment of EDC. Information on the metabolism
and kinetics is used to the extent possible for calculating the carcinogenic
risk for EDC.
Scaling of toxicologic effects, including carcinogenicity, among species
has been elaborated in several published papers (Freireich et al., 1966; Gillette,
1976; Krasovski, 1976; Dedrick, 1973; Dedrick and Bischoff, 1980;' and Gehring
et al., 1980). The major components requiring consideration in determining an
appropriate extrapolation base for scaling carcinogenicity data from laboratory
animals to man are: 1) toxicologic data, 2) metabolism and kinetics, and 3)
covalent binding. The published literature concerning the biological basis
for extrapolation of the dose-carcinogenic response relationship of laboratory
animals to man has been reviewed in a paper prepared by Davidson (1984) for
the Carcinogen Assessment Group and presented by Parker and Davidson (1984).
9.5.3.2.2. Toxicologic Data — Section 9.2 of this document outlines the
general features of the acute, subchronic, and chronic toxicities of EDC in
man and animals. However, comparative quantitative data across species on
specific indices of EDC toxicity are experimentally lacking. Gross measures
such as acute oral LD5Q are similar in rats and mice (680 to 770 mg/kg, rats;
413 to 489 mg/kg, mice) and reflect overwhelming CNS and cardiac depression
effects rather than specific organ toxicities (McCollister et al., 1956; Smyth
9-210
-------
TABLF. 9-58. COMPARISON OF THt: METABOLISM AND PMARMACOKINETICS
OK THE "HIGH" DOSKS IN RATS OF THE MALTONI ET AL. INHALATION
AND NCL (JAVAGK IUOASSAYS
Observation
Maltonl et al,
inhalation
150 ppm,
7 hours
5 days/week
NCI
gavage
150 mg/kga
5 days/week
Assimilated dose
ADC, pg/mL x min
Spreafico et al.
Re it 7. et al.
350b
2910
7297
4500
Balance study with
1"C-EUC, mg/kg
Keitz et al.
Fraction metabolized,
mg/kg
Reicz et al.
Peak blood levels, \tg/mL
50.7
49.8
150
108.2
Spreafico et al.
Reitz et al.
Time to peak blood level, hr
Spreafico et al.
Reitz et al.
Blood clearance time, hr
Spreafico et al.
Reitz et al.
Hepatic DNA covalent binding
gmole equiv E DC/mole DNA
Reitz et al.
lib
9.4
4 to 6
2.5 to 6
l.5b
1.5C
8.2d
3.3
66.8
30 to 40
0.6
0.25
5.6
5
21.3
L3.9
aTime-weinhted average for 78-weok treatment period was 95 mg/kg/day.
blnterpolation from the data of Spre-ifico et al. (1978, 1979, 1980), Table 9-56.
cRstimate from data of Keitz et al. (1982), Figure 9-16.
"Determined 4 hours after gavaye .me' immediately at termination of inhalation
9-2 1 I
-------
1956; Munson et al., 1982). Other toxicologic end points have been measured,
but not across species in comparative dose-related comprehensive studies.
Differences in toxicity according to species, strain, or sex have not been
reported for EDC.
9.5.3.2.3. Metabolism and Kinetics — Few data are available on the
metabolism and kinetics of CDC in man, but these kinetics have been studied in
mice and rats.
9.5.3.2.3.1. Metabolism. In mice and rats, EDC is extensively metabolized
after both oral and inhalation exposures. After oral and inhalation exposures
in rats, at 150 rag/kg and 150 ppra for 6 hours ("high" doses in the NCI and
Maltoni et al. carcinogenicity bioassays), 71% oE the oral dose and 98% of the
inhalation dose was metabolized (Table 9-57). However, as outlined below,
metabolism is dose-dependent, with proportionately greater percentages of the
dose metabolized at low doses and less at higher doses. Metabolism occurs by
multiple pathways in liver, microsomal, and cytosol functions. Microsomal P450
oxidation produces 2-chloroacetaldehyde, while cystosolic reactions of EDC with
glutathione are postulated to produce the half-sulfur mustard S-(2-chloro-
ethyl)-GSH and its episulfonium ion. Both chloroacetaldehyde and the half-
sulfur mustard are reactive metabolites that covalently bind to cellular
macroraolecules, a mechanism that is presumed to underlie the liver and other
organ tissue cytopathology observed from EDC exposure.
The cytosolic interaction of EDC with glutathione results in an activation
to a highly reactive half-mustard metabolite as well as to nonreactive gluta-
thione conjugates from "detoxification reactions." Johnson (1965) acutely
administered AO mg/kg EDC in corn oil to rats by gavage and observed an average
of 52% depletion of hepatic GSH within 2 hours (peak EDC blood concentration
after oral dosing of 150 mg/mL in corn oil, 40 minutes; Table 9-56). Reitz et
9-212
-------
al. (1982) assayed GSH levels in rats after a 150 mg/kg oral dose in corn oil.
After termination of a 150 ppra, 6-hour inhalation exposure, 4 hours after
exposure, hepatic GSH was depleted by 77% and 74%, respectively. These obser-
vations demonstrate the involvement and importance of cellular levels of GSH to
the cytotoxic potential of EDC, both for activation and for detoxification. It
would appear that the "high" doses in the carcinogenicity bioassays do not
completely overwhelm the availability of cellular GSH.
Species Comparison. The metabolism of EDC has been studied in only two
species, the rat and mouse. There is no evidence that the metabolic pathways
in these two species differ qualitatively, since urinary end-metabolites are
similar. In these two species, metabolism has been found to be capacity-
limited, with dose-dependent kinetics. There are, however, no direct compara-
tive balance studies (same laboratory, same time period) of the extent of
metabolism in these species, as are available, for example, for certain of the
halogenated ethylene compounds.
For the mouse, Yllner (1971b) provided data on the extent of metabolism
in this species (Figure 9-12); 10 years later, for the rat, Reitz et al. (1982)
found that for a gavage dose of 150 mg/kg (a dose approaching "saturation" for
rat and mouse), 108 rag/kg was metabolized (Table 9-57). Thus, for a 200 g rat,
21.6 rag of a 30-mg dose was metabolized. For a comparable dose (150 mg/kg),
Yllner found that 2.25 tng was metabolized by a 25 mg mouse. The ratio of
metabolism, rat:mouse, for this particular dose (150 mg/kg) is 9.6, while the
ratio of the surface areas is (200/25)^/3 = 4.0. Comparison of these data
suggests that metabolism of EDC in the rat and mouse is more associated with
the relative body weights of these species than with surface area. However,
for the reasons given, direct comparison of data from these two diverse studies
cannot be fully justified on experimental grounds.
9-213
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B
\0
to
25 50 75 100 125 150 175 200
DOSE
0 25 50 75 tOO 125 150 175 200
DOSE
Figure 9-12. Relationship in mice between dose of EDC (nig/kg) and the amount of EDC exhaled
unchanged (Panel A) and the amount of the dose metabolized to urinary metabolites plus CO2
(Panel B). The female NMRl mice weighed 20-30 g.
SOURCR: Adapted from Yllner, I97lb.
-------
9.5.3.2.3.2. Kinetics. Conclusive evidence exists that the pharmacoki-
netics of EDC in rats and mice are nonlinear. To detect and establish the
nonlinear nature of the pharmacokinetics of a compound, at least two, and
preferably more, dosage levels must be studied (Wagner, 1975). For EDC, the
studies of Yllner (1971b) in mice, and of Spreafico et al. (1978, 1979, 1980)
in rats meet this essential requirement, while the single-dose studies of
Reitz et al. (1982) do not.
EDC is completely and rapidly absorbed after oral administration (Yllner,
1971b; Spreafico et al., 1978, 1979, 1980; Reitz et al., 1982) with a first-
order constant, k , for an oral dose of 150 mg/kg of 0.11 minutes , indicating
95% absorption within 30 minutes. Yllner was the first to demonstrate both the
nonlinear nature of the pharmacokinetics of EDC after absorption and the sub-
stantial first-pass effect with EDC administration. Yllner administered ^C-
EDC intraperitoneally in olive oil to NMRI mice at four dose levels, 50, 100,
140, and 170 mg/kg. This dosage range approximated that of the NCI bioassay
in mice. Yllner quantitated ^C-radioactivity in urine and feces (as EDC
metabolites), as well as CO? (EDC metabolite) and unchanged C-EDC in ex-
haled air for 72 hours after administration. Total recovery of ^C-EDC
radioactivity ranged from 97% to 102% of the dose. The results are shown
plotted in Figure 9-12. With increased dose, the portion of the dose exhaled
as unchanged EDC (first-pass effect) increased in an exponential manner, so
that at a dose of 170 mg/kg, 45% of the dose was excreted as EDC. The remain-
ing portions of the doses were metabolized. Figure 9-12B shows the nonlinear
relationship between dose and metabolism, indicative of nonlinear Michaelis-
Menten kinetics for the metabolism of EDC, with "saturation" of metabolism
occurring in the mouse between 150 to 200 mg/kg administered dose. These
data can he used to estimate the fraction of the assimilated dose metabolized
-------
and contributing to tumorigenesis for the two dosage levels of the NCI mouse
bioassay.
Spreafico et al, (1978, 1979, 1980) investigated the pharraacokinetics
of CDC in rats after oral dosing, inhalation exposure, and intravenous bolus
administration at two (inhalation) or three dose levels (oral and intravenous
administration). These workers determined the blood EDC concentration-time
curves (C,t curve) after the various administrations and fitted the C,t data
to a linear two-compartment open kinetic model, thereby testing the linearity
of EDC pharraacokinetics. According to basic assumptions and definitions,
linear kinetics require that kinetic parameters (T 1/2, AUC, Vj, clearance,
slopes of semilog plots of blood concentration decay curves, etc.) be indepen-
dent of dose. Hence, these kinetic parameters, determined at two or more
dose levels in accordance to a linear kinetic model, provide a rigorous test
for nonlinearity (Wagner, 1975). If the magnitude of some or all of the
parameters changes with dose (and especially if there are dose-related trends),
then nonlinear kinetics are highly probable. If Michaelis-Menten kinetics
are operative, then one expects (I) that the percentage metabolism by the
Michaelis-Menten path will decrease with increase of dose (as in the data of
Yllner above), (2) that the area under the blood C,t curve will increase more
than proportionately with increase in dose, and (3) that semilog plots of
blood levels will curve inwardly with pseudolinear terminal portions, and
slopes of the pseudolinear terminal portion will decrease with increase in
dose.
Figure 9-13 presents the semilog plots of blood C,t curves obtained by
Spreafico et al. (1980) after oral administration of three dose levels of EDC
to rats. Also shown are adipose tissue, lung, and liver C,t curves. In each
case, the slope of the apparently linear decline curves decreases markedly
9-216
-------
vO
ro
o
7J
o
c
E
X
L
X
U
2.S-
1W
100
00
•0
00
E so
x
30
20
H>
25
56
1 I • ' • • I
75 tea
DOSE lag/kg)
125
ORAL
INHALAflON
•I
159
175
1 I
2B0
' I ' ' ' ' I
25 58
' I ' ' ' ' I '
75 188
125
i-r-r-r-
158
175
1 ' I ' ' ' ' I
2BB 225
258
i i i | i i
275
DOSE Ippn 6 hr. >
Figure 9-13. Relationship in Sprague-Dawley rats between the area under the blood EDC
concentration-time curve (AUC) and the gavage dose of EDC in corn oil (rag/kg) or Inhaled
doses of EDC (ppm for 6 hours).
SOURCE: Adapted from Spreafico et al., 1980.
-------
with increase in dose, in a manner inconsistent with first-order linear
kinetics but providing an excellent indication of the nonlinear dose-dependent
nature of the kinetics of EDC in rats. Immediately apparent also from Figure
9-13 is that, as a consequence of nonlinear kinetics, the higher the dose the
longer and more disproportionate is the time of clearance of EDC from the
blood and, therefore, from the body. Definitive evidence of the dose-depen-
dency of EDC kinetics is given by the linear kinetic parameters obtained by
Spreafico et al. by fitting their data to a linear two-compartment model, as
shown in Table 9-59. Both experimentally observed measures (peak blood
concentration, time to peak, areas under C,t curve) and calculated kinetic
parameters (half-life, volume distribution, clearance) are not independent of
dose, as required by linear kinetics, but rather increase with increase in
dose. Furthermore, the nonlinear dose-dependent nature of EDC pharmacoki-
netics is evident for all three routes of administration: oral, inhalation,
and intravenous. Spreafico et al. did not further analyze their data, al-
though it would have been appropriate for them to have fitted their data to a
first-order absorption model with parallel Michaelis-Menten kinetics, as well
as to a first-order elimination model.
The area under the blood C,t curve (AUC) is an experimental measure inde-
pendent of its assumption of any kinetic model. The AUC can be represented by
the definite integral
t
AUC = / C dt
o
The AUC is related to the amount of compound absorbed and, under certain
conditions, can be used to estimate the total internal dose. The AUCs deter-
mined from oral administration of EDC in corn oil vehicle and from inhalation
9-218
-------
I
CO
vO
TABLE 9-59. EVIDENCE OF NONLINEAR KINETICS FOR EDC IN RATS AFTEK GAVAGE, INTRAVENOUS,
AND INHALATION DOSAGE
Oral
Inhalation
Kinetic parameter
mg/kg dose3
25 50 150
ppra, 6-hr dose
50 250
Intravenous
mg/kg dose
25 50 150
Experimental determination'*
Peak blood cone. , pg/mL
Time to peak, hr
AUC, yg x mL~l x mln
Calculated from kinetic model**
Tl/2> mln
Vd, mL
Clearance, mL x min~l
13.2
-0.1
446
24.6
367
10.6
31.9
-0.2
1700
44.1
328
5.6
66.8
-0.6
7297
56.7
390
3.9
1.3 31.3 1.5 8.0
4 6 zero time
26 1023 9 54
12.7 22.1 7.3 9.5
231 239
22.0 17.5
38.1
59
14.1
162
8.0
aCavage dose administered In corn oil.
bThese parameters were experimental observations Independent of any kinetic model. The calculated parameters
were obtained by fitting the blood EDC concentration-time data to a linear two-compartment open model.
SOURCE: Adapted from Spreaflco et al., 1978, 1979, 1980.
-------
exposure at two or three dose Levels, respectively, to rats are shown plotted
against administered dose in Figure 9-L3. As expected from the dose-dependent
nature of EDC kinetics, the AUCs increase disproportionately with the dose
for both oral and inhalation exposures.
Consistent also with Michaelis-Menten dose-dependent kinetics for the
metabolism of EDC is the fact that when the logarithm of the AUCs is plotted
against the logarithm of the oral dose, a linear relationship is obtained as
shown In Figure 9-14. Since the AUCs represent the portion of the EDC dose
that reaches the systemic circulation, in the case of oral administration the
AUCs principally represent the absorbed dose (assumed to be complete) minus
the first-pass effect (primarily elimination of unchanged EDC via the lungs).
Comparison of AUCs for oral and intravenous doses (Table 9-59), a method used
to estimate first-pass effects, shows that for a 25 rag/kg oral dose of EDC,
the AUC (446 ug x mL~l x rain) is 25% less than for an identical intravenous
dose (595 ug x mL"1 x min). Hence, the AUCs after oral administration, like
those after intravenous administration, approximate the internal dose subject
to metabolism. Of interest, therefore, is a comparison of the AUCs (or inter-
nal dose) after oral and inhalation exposures to EDC, which were the condi-
tions of the NCI and Maltoni carcinogenicity bioassays. From Figure 9-13, it
is readily apparent that the AUCs for an oral dose of EDC of 100 mg/kg (the
"high" dose of the NCI study) and 150 ppm for 6 hours (the highest dose of the
Maltoni study) are approximately 10:1.
The disproportionate increase of AUCs, or internal dose subject to metabo-
lism, relative to linear increases in oral dosages of EDC, can be more clearly
seen If the ratio of the AUC to dose (AUC per unit dose) is plotted against
the administered dose, as shown in Figure 9-15. As the oral dose increases,
the internal dose approaches an asymptotic limit similar to the Michaelis-
9-220
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K>
8
x 6H
5-
CD
O
to
ts>
2-
1-
0
tf = 0.989
M=1.54
B=t25
1
3
In DOSE
Figure 9-14. Linear relationship for EDC between the logarithm of the area under the blood
C,t curve (AUC) and the logarithm of gavage dose in corn oil given to Sprague-Dawley rats.
The coefficient of regression, r2, is 0.989.
SOURCE: Adapted from Spreafico et al. , 1978, 1979, 1980.
-------
vO
M
NJ
M
20
40
60 80 100 120 140
DOSE (mg/Ug oral)
160
200
Figure 9-15. Relationship in Sprague-Dawley rats between gavage doses of EDC in corn oil
and the ratio from the area under the blood EDC concentration-time curve (ADC) divided by
the administered dose. The dashed line represents the limit approached in change of AUC
per unit dose as the administered dose is increased.
SOURCE: Adapted from Spreaflco et al., 1978, 1979, 1980.
-------
Menten expression. Assuming that the internal dose closely approximates or
is proportional to the portion of the administered dose metabolized, then
EUC metabolism in the rat approaches saturation at about 150 to 200 mg/kg,
as in the mouse. This relationship can be used to estimate the internal dose
and metabolism of EDC for the administered oral doses used in the NCI bioassay.
Reitz et al. (1980, 1982) also carried out an investigation of the phar-
macokinetics of EDC. As stated in Reitz et al. (1982), one objective was to
determine the absorbed dose of EDC in rats (Osborne-Mendel) and to characterize
its elimination after single exposures at a single dose level from inhalation
(150 ppm, 6 hours) and oral administration in corn oil (ISO mg/kg). These
doses were used because they represented the "highest levels of EDC adminis-
tered for any significant length of time in the two bioassays" (NCI and
Maltoni bioassays). Actually, the oral dose exceeds the time-weighted average
"high" dose for Osborne-Mendel rats of the NCI study (95 mg/kg, Table 9-56).
As shown by the data of Spreafico et al. (see above), the oral dose (150
mg/kg) is close to a saturating dose, and in comparison, the inhalation dose
(150 ppm, 6 hours) is considerably less (~L/10; Figure 9-13). Because
Reitz et al. did not examine the kinetics of multiple dose levels, they were
unable to determine the dose-dependent nonlinear nature of EDC kinetics.
However, these investigators fitted their blood C,t data to kinetic models
empirically. They found that their blood C,t curve, after inhalation expo-
sure, could be fitted to a linear two-compartment model with first-order elim-
ination, while the hlood C,t data after oral administration was "complex" and
was fitted to a two-compartment, first-order absorption model with Michaelis-
Menten elimination kinetics. Their blood C,t data are shown in Figure 9-16.
The Reitz et al. observations, because of their internal inconsistency, are
of limited interest and use; there is no reason to believe that the elimina-
9-221
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20 30 40 50 60 70 80
0.2
so
41-1
31
O
CD 11
B
O)
MINUTES
60 120 180 240 300 360
MINUTES
vO
I
ro
ro
Figure 9-16. Blood levels of EDC following exposure to F.DC by (A) Inhalation (150 ppm, 6
hours) or (B) gavage (150 mg/kg). The Inhalation C,t data were fitted to a linear two-com-
partment kinetic model, and the gavage C,t data were fitted to a nonlinear two-compartment
Nichaells-Nenten model. The solid line is the predicted blood concentration from the model
and the data point means of 4 rats ± SD. A goodness-of-fit was not reported for either
model. The calculated kinetic parameters are:
Inhalation
Linear model
C0 (peak blood level), 9.4 yg/mL
AUC, 2910 gg/mL x min
0.091 min
7.6 min
Vd, 425 mL/kg
T1/26
V 4
Gavage
Nonlinear model
Peak blood level, 30-40
AUC, 4500 pg/mL x min
Vm, 0.166 pg/mL x min
1C,,,, 1.96 ug/mL
Vd, 3400 mL/kg
ug/mL
SOURCE: Adapted from Reltz et al., 1982.
-------
tion kinetics of EDC differs with respect to the two routes of administration,
inhalation and oral. The data of Spreafico et al. show that the elimination
kinetics of EDC for all three routes of administration (inhalation, oral, and
intravenous), are dose-dependent, nonlinear kinetics.
Reitz et al. (1982), in balance studies in rats, compared the assimilated
or "absorbed" dose of EDC after oral (150 mg/kg in corn oil) or inhalation
(150 ppm, 6 hours) exposure to ^C-EDC. Radioactivity in exhaled air, urine,
feces, and carcass was determined for 48 hours after exposure. Total recovery
of EDC radioactivity averaged 101% for the oral dose; it was not possible to
determine recovery after inhalation exposure. The results are given in Table
9-57. The assimilated dose (152 mg/kg) found after oral administration repre-
sented complete absorption of EDC from the GI tract; the assimilated dose
(50.7 mg/kg) following inhalation exposure proved to be one-third of the oral
dose (Table 9-57). As found by Yllner (1981b) in mice and by Spreafico et al.
(1978, 1979, 1980) in rats, the data of Reitz et al. also indicate that the
percentage of the administered dose excreted by pulmonary excretion increased
with the dose and that the remaining percentage (metabolized) decreased,
suggesting dose-dependent Michaelis-Menten kinetics for metabolism. Reitz et
al. found that the major urinary metabolites of EDC in the rat appeared to be
identical in the same relative amounts, i.e., independent of the route of
exposure.
9.5.3.2.4. Covalent Binding — Covalent binding from both oxidative
microsomal and cytosolic metabolism of EDC has been demonstrated jln vitro
(Banerjee and Van Duuren, 1978, 1979; Banerjee et al., 1980; Sipes and Gan-
dolfi, 1980; Guengerich et al., 1980). Metabolites of EDC covalently bind
in vitro to added protein and DMA; binding from cytosolic metabolism depends
on the presence of GSH and requires GSH transferases.
9-225
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In vivo covalent binding from EDC metabolism has also been demonstrated
in Che rat. However, neither a dose-binding relationship nor turnover studies
are available for EDC; the studies conducted have only been at single, fixed-
dose levels. Theoretically, covalent binding might be expected to relate to
the dose-dependent metabolism of EDC.
Reitz et al. (1982) determined total tissue levels of covalent binding
and specific binding to DMA in rats exposed to 14C-EDC by the oral (150 mg/
kg) and Inhalation (150 ppra, 6 hours) routes, i.e., "high" doses of the
carcinogenicity bioassays. No striking differences between the two routes
were observed in the distribution of covalent binding and DNA binding among
the various organ tissues, although the tissue levels of covalent binding were
generally higher after inhalation exposure for total binding and after gavage
exposure for DNA binding. DNA isolated from the organ tissues was assessed
for covalent binding, and in general, higher levels of DNA alkylation (three-
to fivefold) were observed after gavage exposure, with highest values for
liver and kidney (Table 9-60). These DNA covalent binding studies are in good
accord with the differences in metabolism and pharmacokinetics (Table 9-58)
noted for the NCI gavage bioassay in rats (150 mg/kg "high" dose) and the
Maltoni et al. Inhalation bioassay (150 ppm, 7 hours "high" dose).
For mice, Storer et al. (1982) reported that DNA damage was produced in
vivo in the livers of male B6C3F1 mice following single oral doses of EDC at
200 mg/kg (the "high" dose of the NCI mouse bioassay). DNA was evaluated by
differental sedimentation of liver nuclei in high-density sucrose gradient.
Nuclei from EDC-treated mice isolated A hours after dosing sedimented more
slowly, and total DNA recovery from the nuclei was decreased slightly (16%)
as compared with untreated mice.
9.5.3.2.5. Comparison of Maltoni et al. and NCI Studies — The NCI (1978)
9-226
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TABLE 9-60. DNA COVALENT BINDING IN RATS EXPOSED TO 14C-EDC
Gavage Inhalation
150 ing/ kg 150 ppro, 6 hours
pmole equvalents bounds/mole DNA
Mean ± SD (n = 3)
Experiment 1
Liver
Spleen
Kidney
Stomach
21.
5.
17.
3
8
4
±
±
+
14.
7.
0.
2.
9
4
7
3
8.
1.
5.
2
8
2
+
±
±
2.
3.
0.
3.
8
3
7
7
Experiment 2
Liver 13.9 ± 2.1 3.3 ± 1.2
Spleen 2.5+0.3 1.8 ± 0.5
Kidney 14.5 ±6.2 2.0 ±0.3
Stomach 6.7 1.9
aAnimals were sacrificed 4 hours post gavage or immediately following inhalation
exposure.
SOURCE: Reitz et al. , 1982.
9-227
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lifetime gavage studies in rats and mice produced excess tumors, while the
Maltoni et al. (1980) lifetime inhalation study did not. The kinetic studies
of EDC disposition in rats after gavage and inhalation exposures, and the
metabolism studies of Reitz et al. (1982) provide partial explanations for the
contrasting results of the two carcinogenicity bioassays. The two EDC studies
differ in (1) assimilated or effective internal dose, (2) fraction of assimilated
dose metabolized to nucleophilic metabolites, and (3) peak blood levels and
duration of metabolism from a single daily dose.
Table 9-58 contrasts some metabolism and kinetic parameters of the "high"
doses of the Maltoni inhalation (150 ppm, 7 hours) and NCI gavage (150 rag/kg)
rat bioassays available from the studies of Spreafico et al. (1978, 1979, 1980)
and Reitz et al. (1982). While the data from these two Investigative groups
are from differing study designs and differ in some measures, they are never-
theless in surprisingly good agreement. Table 9-58 shows that the internal
dose assimilated by rats exposed to 150 ppm EDC is one-tenth to one-third less
than that from a 150 mg/kg gavage dose, and that it produces less than one-half
the metabolites of a 150 mg/kg gavage dose. Furthermore, peak blood levels
of EDC from a 150 mg/kg gavage dose are three- to fivefold greater, and are
achieved within one-half hour, as compared to 3 to 6 hours from inhalation
exposure. Since the rate of metabolism is proportional to blood level, these
observations suggest that the intensity of covalent binding may be expected
to be greater with the gavage dose, and this has been found to he the case;
covalent binding to DNA in vivo is three- to fivefold higher after gavage
exposure than after inhalation exposure.
Additionally, it can be noted that, contrary to what might be assumed, an
EDC inhalation exposure of 6 to 7 hours (150 ppm) does not produce a signifi-
cantly longer presence of EDC in the blood available for continuing liver and
9-228
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other organ metabolism than an oral gavage dose of 150 mg/kg. In both in-
stances, blood EDC is essentially eliminated (cleared) within 6 to 10 hours,
indicating that the "chronic" dosage in these bioassays is better described
as multiple single dose administration, since a given day's dose of CDC may
be expected to be eliminated from the body before the next day's dose.
However, the residual DNA covalent binding and general cellular covalent
binding, greater for the NCI gavage doses, may be cumulative and may exceed
the capacity of repair mechanisms with chronic EDC administration. In total,
these observations provide cogent reasons to conclude that the dose range of
the Maltoni inhalation study (5 to 150 ppm) may be insufficient to produce
measurable tumorigenesis. However, as will be discussed later in section
9.5.3.4.6, reasons other than those indicated here cannot be excluded.
9.5.3.2.6. Calculation of Human Equivalent Doses — The NCI carcinogen-
icity studies of EDC in mice and rats were performed by daily (5 days/week)
gavage dosing of EDC in corn oil. Experimental metabolism and pharmacokinetic
observations pertinent to the conditions of the NCI mouse bioassay are those
of Yllner (1971b), who administered acute doses of EDC in corn oil to mice by
gavage and determined at several dosage levels the fraction of the assimilated
dose metabolized. Pertinent studies in relation to the NCI rat bioassay are
the experimental kinetic observations of Spreafico et al. (1978, 1979, 1980)
who administered acute doses of EDC in corn oil to rats at several dosage
levels. The studies of both Yllner and Spreafico et al. included dose ranges
comparable to those of the NCI bioassays. However, the observations were not
made on "chronic" dosed animals, as in the NCI bioassays, although the experi-
mental evidence indicates that body clearance of EDC from rodents after a
single daily dose, while dose-dependent, is less than 12 hours for the highest
assay dose, i.e., less than the 24-hour dosing interval of the NCI bioassay.
9-229
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While there is little information on the metabolism or pharmacokinetics
of EDC in man, the pertinent experimental observations in animals may be
summarized as follows:
1. It is assumed that biochemical lesions precede the toxic effects of
EDC. Formation of reactive metabolites (P450 oxidation, chloroacetaldehyde;
GSH-mediated cytosolic reaction, S-(2-chloroethyl)-GSH) is followed by irre-
versible binding to proteins, lipids, and nucleic acids critical to cell func-
tion and resulting in toxic, rautagenic, or carcinogenic events.
2. After gavage administration in corn oil to mice or rats, EDC is
rapidly and completely absorbed.
3. After gavage administration, a portion of the administered dose
(increasing with the dose) is excreted via the lungs unchanged. Metabolism of
EDC is less than one-half saturation at the time-weighted average dose levels
•
of the NCI rat bioassay (47 and 95 mg/kg), but is at or near saturation for the
highest dose of the NCI mouse bioassay (195 mg/kg).
4. EDC is cleared from the body well within the bioassay dosing interval,
even at the highest bioassay doses. The clearance is by first-order pulmonary
excretion of unchanged EDC and by parallel dose-dependent Mlchaelis-Menten
metabolism kinetics. The end-metabolites (urinary metabolites) are more slowly
cleared than EDC itself. The rapid clearance of EDC, representing a fraction
of the 24-hour bioassay dosage interval, indicates that accumulation with
multiple doses would not occur; there is time between doses for repair of cellu-
lar damage from EDC metabolism, and also for repletion of cellular glutathione.
5. There is no evidence to suggest qualitative differences in the meta-
bolic pathways of EDC in the two species, mouse and rat, and no difference
resulting from the route of exposure (oral or inhalation) in the rat.
6. Covalent binding to cellular protein and lipids occurs in vivo after
9-230
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oral administration of EDC to the rat. DNA alkylation occurs not only at the
organ sites of tumors found in the NCI bioassay but also at other sites. There
is no information available on the turnover time or repair time of DNA adducts,
but DNA binding was evident 4 hours after oral dosing. DNA alkylation after
gavage is estimated at only 2 to 20 / 10& nucleotides.
Using this metabolic and pharmacokinetic information, the lifetime average
human equivalent doses for the NCI rat and mouse carcinogenicity bioassays of
EDC have been calculated as follows.
9.5.3.2.6.1. NCI Bioassay (Mouse): Liver Carcinomas. In this study, the
time-weighted average gavage dose levels of CDC were 195 and 97 mg/kg/day, or,
based on the average terminal weight of the male mice (33 g), 3.2 and 6.4
mg/male mouse/day (Table 9-56). Doses in this range given in corn oil have
been found to be completely absorbed by rodents, although a dose-related,
first-pass effect has been observed, leading to a portion of the dose being
excreted in exhaled air unchanged. From the data of Yllner (1971b), a dose-
metabolism curve for EDC administered to mice is available (Figure 9-12).
The metabolism of EUC in mice shows dose-dependent kinetics with the "high"
dose of the NCI bioassay (195 mg/kg) at saturation of metabolism (Figure
9-12); at this dose level, the amount of metabolites contributing to tumori-
genesis is 95 mg/kg. At the "low" dose level of the NCI bioassay (97 mg/kg),
the amount metabolized is 73 mg/kg. Hence, the fractions of the daily assim-
ilated dose metabolized by the mice (33 g) are:
Male mice
Assay dose level
time-weighted average Amount metabolized (AM)
mg/kg mg/kg mg/animal
195 95 3.14
97 73 2.41
9-23L
-------
The lifetime average daily exposure (LAE) of the mice is given by:
weeks/90 weeks) x (5 days/7 days) x AM, mg/animal/day
where AM is the amount of dose metabolized. For humans, the LAE is given
by:
LAEM x scaling factor
The interspecies scaling is assumed to be on a surface area basis (W?/3).
The scaling factor for mouse to humans is (70/0. 033)2/3 = 165. Table 9-61
presents the lifetime average human equivalent doses for the NCI mouse car-
cinogenicity bioassay of EDC, expressed in units of mg/day, mg/m^ surface
area/day, and mg/ kg/day.
TABLE 9-61. LIFETIME AVERAGE HUMAN EQUIVALENT DOSES
BASED ON THE NCI MOUSE BIOASSAY
Equivalent Lifetime average
Male mice human exposure dosage3
gavage dose
(time-weighted)
rag/kg mg/day (mg/m2)/day (mg/kg)/day
97 246.2 133.1 3.52
195 320.7 173.4 4.58
aWhere the terminal average weight of the male mouse is 0.033 kg; the average
weight for a human is assumed to be 70 kg with 1.85 m surface area.
9-232
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9.5.3.2.6.2. NCI ttioassay (Rat): Angiosarcomas. For this study, the
time-weighted average gavage doses ("high" and "low" doses) given to male
Osborne-Mendel rats in corn oil were as follows:
Gavage dose
male 0-M rat
mg/kg
(time-weighted) mg/animal/day
47 23.5
95 47.5
and the corresponding dose per single animal/day is given as calculated from
the average terminal weight of 500 g. Spreafico et al. (1978, 1979, 1980) and
Reitz et al. (1982) found that gavage doses in corn oil spanning this dosage
range were rapidly and completely absorbed, although a portion, depending on
dose, effects a first-pass of the liver and is excreted in exhaled air un-
changed. The area under the EDC blood C,t curve (AUC) represents EDC that
has reached the systemic circulation after oral absorption and first-pass pul-
monary excretion. The AUC represents the approximate internal dose available
for metabolism, and thus is equivalent (or nearly so) to the fraction of the
administered dose metabolized.
Data from Spreafico et al. (see Figure 9-15) and Reitz et al. (1982) can
be used to determine the fraction of the dose metabolized for the NCI bioassay
on rats. On the basis of Figure 9-15, the rate of change (i.e., the slope to
the curve) at dose levels 47, 95, and 150 mg/kg can be estimated to be 0.36,
0.17, and 0.094, respectively. Since the rate of change decreases as the dose
increases, it is reasonable to assume that the relative amount of EDC exhaled
unchanged is inversely proportional to these rates. Now, on the basis of
Reitz et al. (1982), 29% of EDC was exhaled unchanged after an administered
9-233
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dose of 150 mg/kg. Thus, the percentage unchanged at NCI doses, 47 and 95
rag/kg, are, respectively, 0.29 x 0.094/0.36 = 0.08, or 8%; and 0.029 x
0.094/0.17 = 0.16, or 16%. Therefore, the fractions of EUC metabolized
for the NCI study on rats are 92% at the dose 47 mg/kg and 84% at the dose
95 mg/kg. The amounts metabolized for the NCI EDO bioassay are as follows:
Male rats
Assay dose level
time-weighted average Amount metabolized (AM)
mg/kg mg/kg rag/animal
47 43.24 21.62
95 79.80 39.90
The lifetime average daily exposure (LAE) of the rats is given by:
LAER = (78 weeks/104 weeks) x (5 days/7 days) x AM, mg/animal/day
where 104 weeks is assumed to be the average life span for rats, and AM is the
amount of dose metabolized. For humans, the LAE is given by:
= LAEg x scaling factor
The interspecies scaling is assumed to be on a surface area basis
The scaling factor for rats to humans is (70/0. 5)2/3 = 26.96. Table 9-62
presents the lifetime average human equivalent doses for the NCI rat bioassay,
expressed in units of mg/day, mg/ia2 surface area/day, and rag/kg/day.
9-234
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TABLE 9-62. LIFETIME AVERAGE HUMAN EQUIVALENT IJOSCS
BASED OM THE NCI RAT BEOASSAY
Equivalent lifetime average
Gavage dose human exposure dosage3
(time-weighted)
mg/kg mg/day (mg/m2)/day (mg/kg)/day
47 312.25 168.78 4.46
95 576.27 311.50 8.23
aWhere the terminal average weight of the male rat is 0.5 kg; the average
weight for a human is assumed to be 70 kg with 1.85 m2 surface area.
9.5.3.3. CHOICE OF RISK MODEL
9.5.3.3.1. General Considerations — The unit risk estimate for EDC
represents an extrapolation below the dose range of experimental data. There
is currently no solid scientific basis for any mathematical extrapolation model
that relates exposure to cancer risk at the extremely low concentrations,
including the unit concentration given above, that must be dealt with in eval-
uating environmental hazards. For practical reasons the correspondingly low
levels of risk cannot be measured directly either by animal experiments or by
epidemiologic studies. Low-dose extrapolation must, therefore, be based on
current understanding of the mechanisms of carcinogenesis. At the present time
the dominant view of the carcinogenic process involves the concept that most
cancer-causing agents also cause irreversible damage to DMA. This position is
based in part on the fact that a very large proportion of agents that cause
cancer are also mutagenic. There is reason to expect that the quantal response
that is characteristic of mutagenesis is associated with a linear (at low
doses) nonthreshold dose-response relationship. Indeed, there is substantial
9-235
-------
evidence from mutagenicity studies with both ionizing radiation and a wide
variety of chemicals that this type oE dose-response model is the appropriate
one to use. This is particularly true at the lower end of the dose-response
curve; at high doses, there can be an upward curvature, probably reflecting the
effects of multistage processes on the mutagenic response. The low-dose linear
nonthreshold dose-response relationship is also consistent with the relatively
few epidemiologic studies of cancer responses to specific agents that contain
enough information to make the evaluation possible (e.g., radiation-induced
leukemia, breast and thyroid cancer, skin cancer induced by arsenic in drinking
water, liver cancer induced by aflatoxins in the diet). Some supporting evi-
dence also exists from animal experiments (e.g., the initiation stage of the
two-stage carcinogenesis model in rat liver and mouse skin).
Because its scientific basis, although limited, is the best of any of the
current mathematical extrapolation models, the nonthreshold model, which is
linear at low doses, has been adopted as the primary basis for risk extrapola-
tion to low levels of the dose-response relationship. The risk estimates made
with such a model should be regarded as conservative, representing a plausible
upper limit for the risk; i.e., the true risk is not likely to be higher than
the estimate, but it could be lower.
For several reasons, the risk estimate based on animal bioassays is only
an approximate indication of the absolute risk in populations exposed to known
carcinogen concentrations. First, there are important species differences in
uptake, metabolism, and organ distribution of carcinogens, as well as species
differences in target site susceptibility, immunological responses, hormone
function, dietary factors, and disease. Second, the concept of equivalent
doses for humans compared to animals on a mg/surface area basis is virtually
without experimental verification as regards carcinogenic response. Finally,
9-236
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human populations are variable with respect to genetic constitution and diet,
living environment, activity patterns, and other cultural factors.
The risk estimate can give a rough indication of the relative potency of
a given agent as compared with other carcinogens. Such estimates are, of
course, more reliable when the comparisons are based on studies in which the
test species, strain, sex, and routes of exposure are similar.
9.5.3.3.2. Mathematical Description of the Low-Dose Extrapolation Model -
The mathematical formulation- chosen to describe the linear (at low doses) non-
threshold dose-response relationship is the linearized multistage model. This
model employs enough arbitrary constants to be able to fit almost any mono-
tonically increasing dose-response data, and it incorporates a procedure for
estimating the largest possible linear slope (in the 95% confidence limit
sense) at low extrapolated doses that is consistent with the data at all dose
levels of the experiment.
Let P(d) represent the lifetime risk (probability) of cancer at dose d.
The multistage model has the form
P(d) = 1 - exp [-(q0
where
q^ > 0, i =
Equivalently,
Pt(d) = 1 - exp [-<
where
P (d) = P(d) - P(0)
c 1 - P(0)
9-237
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is Che extra risk over background rate at dose d or the effect of treatment.
The point estimate of the coefficients q^, 1 = 0, 1, 2, ..., k, and con-
sequently, the extra risk function, Pt(d), at any given dose d, is calculated
by maximizing the likelihood function of the data.
The point estimate and the 95% upper confidence limit of the extra risk,
Pt(d), are calculated by using the computer program, GLOBAL83, developed by
Howe (1983). At low doses, upper 95% confidence limits on the extra risk and
lower 95% confidence limits on the dose producing a given risk are determined
from a 95% upper confidence limit, q^, on parameter q^. Whenever q^ > 0, at
low doses the extra risk Pt(d) has approximately the form Pc(d) = qi x d.
Therefore, q^ x d is a 95% upper confidence limit on the extra risk and R/q?
Is a 95% lower confidence limit on the dose, producing an extra risk of R. Let
LQ be the maximum value of the log-likelihood function. The upper-limit, q^,
is calculated by increasing q^ to a value q^ such that when the log-likelihood
is remaximized subject to this fixed value, qj, for the linear coefficient,
the resulting maximum value of the log-likelihood LI satisfies the equation
2(L0 - LI) = 2.70554
where 2.70554 is the cumulative 90% point of the chi-square distribution with
one degree of freedom, which corresponds to a 95% upper-limit (one-sided).
This approach of computing the upper confidence limit for the extra risk Pt(d)
is an improvement on the Crump et al. (1977) model. The upper confidence limit
for the extra risk calculated at low doses is always linear. This is conceptu-
ally consistent with the linear nonthreshold concept discussed earlier. The
slope, q^, is taken as an upper bound of the potency of the chemical in
inducing cancer at low doses. (In the section calculating the risk estimates,
Pt(d) will be abbreviated as P.)
9-238
-------
In fitting the dose-response model, the number of terms in the polynomial
is chosen equal to (h-1), where h is the number of dose groups in the experi-
ment, including the control group.
Whenever the multistage model does not fit the data sufficiently well,
data at the highest dose is deleted, and the model is refit to the rest of the
data. This is continued until an acceptable fit to the data is obtained. To
determine whether or not a fit is acceptable, the chi-square statistic
2
X
is calculated where N^ is the number of animals in the i dose group, X( is
the number of animals in the i dose group with a tumor response, P^ is the
probability of a response in the ith dose group estimated by fitting the multi-
stage model to the data, and h is the number of remaining groups. The fit is
determined to be unacceptable whenever X^ is larger than the cumulative 99%
point of the chi-square distribution with f degrees of freedom, where f equals
the number of dose groups minus the number of non-zero multistage coefficients.
9.5.3.3.3. Adjustments for Less Than Lifespan Duration of Experiment —
If the duration of experiment Le is less than the natural life span of the test
animal L, the slope qi, or more generally the exponent g(d), is increased by
multiplying a factor (L/Lg) . We assume that if the average dose d is continued,
the age-specific rate of cancer will continue to increase as a constant func-
tion of the background rate. The age-specific rates for humans increase at
least by the third power of the age and often by a considerably higher power,
as demonstrated by Doll (1971). Thus, it is expected that the cumulative tumor
rate would increase by at least the third power of age. Using this fact, it is
assumed that the slope qp or more generally the exponent g(d), would also
9-239
-------
increase by at least the third power of age. As a result, if the slope qj
[or g(d)] is calculated at age Le, it is expected that if the experiment had
been continued for the full life span L at the given average exposure, the
* *)
slope qj [or g(d) ] would have been increased by at least (L/Lg) .
This adjustment is conceptually consistent with the proportional hazard
model proposed by Cox (1972) and the time-to-tumor model considered by Daffer
et al. (1980), where the probability of cancer by age t and at dose d is
given by
P(d,t) = 1 - exp [-f(t) x g(d)].
9.5.3.4. CALCULATION OF UNIT RISK
9.5.3.4.1. Definition of Unit Risk — This section deals with the unit
risk for EDC in air and water and the potency of EDC relative to other carcino-
gens that the CAG has evaluated. The unit risk estimate for an air or water
pollutant is defined as the incremental lifetime cancer risk occurring in a
hypothetical population in which all Individuals are exposed continuously from
birth throughout their lifetimes to a concentration of 1 yg/m-* of the
agent in the air they breathe, or to 1 yg/L i° tne water they drink. This
calculation is done to estimate in quantitative terms the impact of the agent
as a carcinogen. Unit risk estimates are used for two purposes: (1) to com-
pare the carcinogenic potency of several agents with each other, and (2) to
give a crude indication of the population risk that might be associated with
air or water exposure to these agents, if the actual exposures are known.
9.5.3.4.2. Unit Risk Estimates and Interpretation — The unit risk
estimate based on animal bioassays is only an approximate indication of the
absolute risk in populations exposed to known carcinogen concentrations.
There may be important differences in target site susceptibility, immunological
9-240
-------
responses, hormone function, dietary factors, and disease. In addition,
human populations are variable with respect to genetic constitution and diet,
living environment, activity patterns, and other cultural factors.
The unit risk estimate can give a rough indication of the relative potency
of a given agent compared with other carcinogens. The comparative potency of
different agents is more reliable when the comparison is based on studies in
the same test species, strain, and sex.
The quantitative aspect of carcinogen risk assessment is prepared because
of its possible value in estimating the potential magnitude of the public health
hazard. However, it should be recognized that the estimation of cancer risks to
humans at low levels of exposure is uncertain. At best, the linear extrapola-
tion model used here provides a rough but plausible estimate of the upper-limit
of risk; i.e., it is not likely that the true risk would be much more than the
estimated risk, but it could very well be considerably lower. The risk esti-
mates presented in subsequent sections should not be regarded as immutable
representations of the true cancer risks; however, the estimates presented may
be used to the extent that the concept of upper-risk limits is found to be useful.
9.5.3.A.3. Alternative Low-Dose Extrapolation Models — In addition to
the multistage model currently used by the CAG for low-dose extrapolation, two
more models, the probit and the Weibull, are also employed for purposes of
comparison. These models cover almost the entire spectrum of risk estimates
that could be generated from existing mathematical extrapolation models.
Generally statistical in character, these models are not derived from biolog-
ical arguments, except for Llie inul" ista^K model, which has been used to support
the somatic mutation hypothesis of carcinogenesis (Armitage and Doll, 1954;
Whittemore, 1978; Whittemore and Keller, 1978). The main difference among
these models is the rate ^t which the response function P(d) approaches ^e
9-241
-------
or P(0) as dose d decreases. For instance, the probit model would usually
predict a smaller risk at low doses than the multistage model because of the
difference of the decreasing rate in the low-dose region. However, it should
be noted that one could always artificially give the multistage model the same
(or even greater) rate of decrease as the probit model by making some dose
transformation and/or by assuming that some of the parameters in the multistage
model are zero. This, of course, is not reasonable without knowing, a priori,
what the carcinogenic process for the agent is.
9.5.3.4.4. Calculation of the EDC Carcinogenic Potency (Slope) — The
multistage model is used in connection with the most sensitive tumor sites,
and for purposes of comparison, other extrapolation models and data sets are
also used to estimate the carcinogenic potency of EDC.
Data from both rats and mice (NCI, 1978) are used to estimate the carcino-
genic potency of EDC. For rats, hemangiosarcomas in the circulatory system of
male rats were selected, because these tumors are the most sensitive and were
not located at the site of direct contact with the agent. Because of the high
mortality rate In the high-dose group, the cancer risk is calculated by using
two types of data:
1. Dichotomous data. The number of animals that survived at least 50
weeks was used as the denominator of the incidence rate (Table 9-63).
This data set serves as a basis for comparing multistage models with
the other two extrapolation models.
2. Time-to-death data (Table B-l, Appendix B). This data set is more
appropriate for use in the calculation of the carcinogenic potency
of EDC. In this calculation, it is assumed that animals with heraan-
giosarcomas were killed by the tumors. This assumption seems reason-
able since almost all of the animals with hemangiosarcomas died
before terminal sacrifice.
9-242
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For mice, hepatocellular carcinomas In male mice (Table 9-64) were used.
Again, this tumor site was selected because it is the most sensitive in mice.
Using the incidence data in Tables 9-63 and 9-64 and the corresponding human
equivalent doses, the maximum likelihood estimates of the parameters in each
of three extrapolation models are calculated and presented in Table A-l of
Appendix A. These models ca.n be used to calculate point estimates of risk at
given doses or point estimates of doses for given levels of risk. The 95%
upper-bound estimates of the risk at 1 mg/kg/day, calculated by various
models using different data sets, are presented in Table 9-65. From this
table, it is observed that the multistage model predicts a comparable risk on
the basis of either hemangiosarcomas in male rats or liver carcinomas in male
mice, while the probit and Wei bull models are extremely unstable and predict a
wide range of risk, depending on the data base used.
The slope, qp which is the 95% upper-bound estimate of the linear
coefficient in the multistage model, is also comparable when different data
sets are used. The values are presented in Table 9-65.
* 7
The CAG recommends that the slope estimate q^ = 9. L x 10 /mg/kg/day
be used to represent the carcinogenic potency of EDC by oral exposure. This
value is calculated on the basis of hemangiosarcomas in male rats using the
multistage model with the time-to-death factor. This value will be used to
estimate the unit risk of EDC by inhalation and drinking water.
9.5.3.4.5. Risk Associated with 1 ug/L of EDC in Drinking Water — It
is assumed that 100% of EDC in drinking water can be absorbed, and that the
water intake is 2 L/day. Under these assumptions, the daily dose from con-
sumption of water containing 1 ng/L (I ppb) of EUC is
d = I pg/L x 2 L/day x 10~3 mg/ug x 1/70 kg = 2.9 x 10-5 mg/kg/day
9-243
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TABLE 9-63. INCIDENCE RATES OF HEMANGIOSARCOMAS IN THE CIRCULATORY
SYSTEM OF MALE OSBORNE-MENDEL RATS
Experimental
dose
(mg/kg/day)
0
47
95
Human
equivalent
metabolized
dose
(mg/kg/day)3
0
4.46
8.23
Tumor
incidence
rates'3
0/40
9/48
7/27
aHuman equivalent doses are taken from Table 9-62.
''The number of animals that survived at least 50 weeks is used as the
denominator for the EDC-treated group.
SOURCE: NCI, 1978.
TABLE 9-64. INCIDENCE RATES OF HEPATOCELLULAR CARCINOMAS
IN MALE B6C3F1 MICE
Experimental
dose
(mg/kg/day)
0
97
195
Human
equivalent
metabolized
dose
(mg/kg/day)a
0
3.52
4.58
Tumor
incidence
rates
1/19
6/47
12/48
aHuraan equivalent doses are taken from Table 9-61,
SOURCE: NCI, 1978.
9-244
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TABLE 9-65. THE 95% UPPER-BOUND ESTIMATE OF RISK AT I MG/KG/DAY
USING THREE DIFFERENT MODELS3
Multistage
Data base (with time factor)*5 Probit Weibull
Hemangiosarcomas 6.0 x 10~2 2.81 x KT1 2.70 x 10~l
in male rats (8.7 x 10~2)
Hepatocellular carcinomas
in male mice 6.1 x 10~2 1.87 x 10~4 5.04 x 10~3
alt is assumed that, at low doses, a given dose is totally metabolized. There-
fore, the risk estimates presented here are corresponding to either adminis-
tered or metabolized dose.
''The corresponding 95% upper-bound estimates of the linear component in the
multistage model are:
q* = 6.1 x 10~2/mg/kg/day
on the basis of dichotomous data (hemangiosarcoma);
qj = 9.1 x 10~2/mg/kg/day
on the basis of time-to-death data (hemangiosarcoma);
q* = 6.2 x 10-2/mg/kg/day
on the basis of hepatocellular carcinomas.
9-245
-------
Therefore, the upper-bound estimate of the incremental risk associated with
1 ug/L of EDC in drinking water is
P = 9.1 x 10-2 x 2.9 x 10-5 = 2.6 x 1(T6
9.5.3.4.6. Risk Associated with 1 ng/m3 of EDC in Air — The only
available inhalation study (Maltoni et al., 1980) did not indicate any signif-
icant carcinogenic effects in either rats or mice. This seems contradictory
to the result of the NCI gavage study, in wh'ich EDC was shown to be
carcinogenic to both rats and mice. The seemingly discrepant results between
the two exposure routes might be ascribed to one or more of the following
causes:
1. The strains of test animals differ in responsiveness;
2. There is a difference in duration of exposure In the peak blood levels
of EDC, and therefore the target tissue levels of its intermediate
reactive metabolites are different for the doses in the two studies;
3. An artifact has been introduced by the intercurrent mortality.
One issue that is of interest to the risk assessment is to determine
whether or not the dose used in the inhalation study is too low to induce a
detectable tumor response in the number of animals tested, in view of the
fact that EDC is carcinogenic by oral route of exposure. Reitz et al. (1982)
indicates that rats exposed to EDC in air at 150 ppm for 6 hours had an assim-
ilated dose of 50.7 mg/kg and a metabolized dose of 49.8 ing/kg. On this basis,
the high-dose group (150 ppra to 250 ppm, 7 hours/day) in the Maltont et al.
study on rats should have assimilated and metabolized doses greater than 50.7
mg/kg/day and 49.8 mg/kg/day, respectively. Therefore, the Maltoni et al.
high-dose group should be at least as responsive as the NCI low-dose (47
mg/kg/day) group of rats in which 19% (9/48) of the male rats developed heman-
9-246
-------
giosarcomas. If the dose available to animals (assimilated or metabolized)
is the only factor that determines tumor response, one would expect to see at
least one tumor type in the Maltoni et al. study based on what was observed in
the NCI study. It is unlikely that 19% (9/48) of hemangiosarcomas would be
observed in one study and none in the other in which"more animals (79 male
rats survived at 52 weeks) were used in the experiment. The probability of
observing such an event is very small (p < 0.0001) if the total dose available
to animals is indeed the only factor that determines the tumor response.
Therefore, the discrepant results between the two exposure routes cannot be
totally explained by the difference in dose available to animals.
The observation of greater (three- to fivefold) peak blood concentrations
in the gavage study when compared to the inhalation study, together with dif-
ferences in the rates of formation, deactivation, and binding to macromolecules
between the two routes of exposure, may explain the differing response pattern.
The implication of the considerations just discussed is that, in addition
to the EDC kinetic information (oral vs. inhalation) on assimilated and metabo-
lized dose, it is important to determine whether strains of test animals,
duration of exposure, and/or route of exposure make significant differences in
the tumorigenic responses. If the strains of animals differ in responsiveness,
then the most sensitive animal strain (i.e., the one from the NCI gavage study)
should be used for potency estimation via inhalation. On the other hand, if
the route of exposure makes a difference in the tumorigenic response, it would
be more appropriate to use the inhalation study rather than the gavage study
to estimate the risk by inhalation. Since the reason for the discrepant results
between the two exposure routes is not known, both the NCI gavage study and
the negative inhalation study by Maltoni et al. (1980) are used to calculate
the risk for EDC by inhalation. The unit risk estimates calculated on the
9-247
-------
basis of these two data sets provide a range of risk estimates that reflect
several types of uncertainty, including strains and routes of exposure used in
the bioassays.
9.5.3.A.6.1. Unit Risk for Air Calculated on the Basis of Rat Gavage Data.
The daily dose due to 1 pg/m3 of EDC in air is
d = 1 ug/m3 x 20 m3/day x 10~3 mg/kg/70 kg
= 2.86 x 10~4 rag/kg/day
where 20 m3 is assumed to be the volume of daily air intake by a 70 kg person.
In this calculation, the EDC in air breathed is assumed to be 100% absorbed.
It is reasonable to assume 100% absorption and metabolism at low doses.
Thus, the upper-bound estimate of the incremental risk due to 1 yg/ra3 of EDC
in air is
P = 9.1 x ID'2 x 2.9 x 10"4 = 2.6 x lO'5
The information on EDC kinetics is not used in this derivation of unit
risk. Although Reitz et al. (1982) provides some information on the compara-
tive metabolism orally (at 150 mg/kg) and by inhalation (at 150 ppra, for 6
hours), this information cannot be appropriately used for route-to-route extrap-
olation because the relative amounts metabolized between the two routes of
exposure do not appear to be linearly related to the administered dose, as
evidenced by the data of Spreafico et al. on the blood concentration-time curve
(AUC). Using the Spreafico et al. data in Table 9-59 and the assumption that
the amount metabolized is linearly related to AUC, as the administered EDC dose
decreases, the amount metabolized, when administered by inhalation, decreases in
a greater proportion than the amount of EDC metabolized when administered by a
single bolus by the oral route. Therefore, if the comparative metabolism
9-248
-------
observed at high doses in Reitz et al. (1982) is used for route-to-route extrap-
olation, the inhalation risk at low doses (e.g., 1 yg/m^) would he over-
estimated.
9.5.3.4.6.2. Unit Risk for Air Calculated on the Basis of a Negative Rat
Inhalation Study. A negative inhalation study by Maltoni et al. (1980) can be
used to calculate an upper-bound estimate of risk. The data from male rats is
used to calculate an upper-bound estimate of risk because it was found to be
most sensitive in the NCI study. The smallest upper-bound estimate is calcu-
lated by using the information from the high-dose group in which 79 male rats
survived at 52 weeks. These animals were exposed to EDO in air at 150 ppm,
7 hours/day, 5 days/week, for 78 weeks. The lifetime average exposure is cal-
culated to be
d = (150 ppm x 7 hours/24 hours) x (5 days/7 days) x 78 weeks/104 weeks
= 23.44 ppm
or equivalently,
d = 23.44 ppra x 4,121 (pg/m3)/ppm
= 96,596.24 ug/m3
Assuming no "tumor" is observed among the 79 animals, the 95% upper limit
of the tumor probability is R = 0.037, calculated by solving the equation
(1 R)79 = 0.05. By using the one-hit model, R = 1 - exp(-b x d), the
slope, b, Is calculated as
b = [ - ln(l-R)]/d
= 3.9 x 10-7/(ug/ra3)
Thus, for male rats, the risk due to 1 pg/m3 of EDC in air is 3.9 x 10~7.
To convert this animal risk estimate into human risk, it is assumed that body
9-249
-------
burden per surface area is equivalent among species. For L yg/m3 of EDC in
air, Che total EDC uptake (body burden) of a rat weighing 0.35 kg is
1 ug/m3 x 0.224 m3/day
where 0.224 m3/day is the daily air intake for a 0.35 kg rat. The concentra-
tion for humans, C(ug/m3), which is equivalent to 1 jig/m3 for rats, is
calculated to be C = 0.38 ug/m3, by solving for C from the equation
20 m3/day x C(ug/m3) = 1 ug/m3 x 0.224 m3/day
(70 kg)2/3 (0.35 kg)2/3
where 20 m3/day is assumed to be the volume of air intake by a 70 kg person.
Therefore, the upper-bound estimate of incremental human cancer risk due to
1 ug/m3 of EDC in air is
P = 3.9 x 10-7/0.38
= 1.0 x 10~6
This risk estimate is 26 times smaller than that calculated on the basis of
the gavage study. As discussed previously, this upper-bound risk estimate is
appropriate only if the discrepant results in tumor response between the oral
and inhalation routes is due to the possible difference in the rates of forma-
tion or rates of deactivation or rates of covalent bond formation and if, in
combination, these results differ between oral versus inhalation routes of
exposure, but are not due to the strains of test animals.
9.5.3.5. COMPARISON OF POTENCY WITH OTHER COMPOUNDS — One of the uses
of quantitative potency estimates is to compare the potencies of various car-
cinogens. Figure 9-17 is a histogram representing the frequency distribution
of potency indices of 54 suspect carcinogens evaluated by the CAG. The data
9-250
-------
4th
QUARTILE
1 x
3rd
QUARTILE
10'1 4>
2nd
QUARTILE
I
MO*2 2 x
1s:
QUARTILE
1C'3
-1
1234 56
LOG OF POTENCY INDEX
Figure 9-17. Histogram representing the frequency distribution
of the potency indices of the 5A suspect carcinogens evaluated
by the Carcinogen Assessment Group.
9-251
-------
summarized by Che histogram are presented in Table 9-66. The potency index
is derived from q^, the 95% upper bound of the linear component in the
multistage model, and is expressed in terms of (mmol/kg/day)"1. Where human
data were available for an agent, they have been used to calculate the Index.
Where no human data were available, animal oral studies have been used in
preference to animal inhalation studies, since oral studies constitute the
majority of animal studies.
Based on available data concerning hemangiosarcomas in male rats (NCI,
1978), the potency index for EDC has been calculated as 9 x 10°. This figure
fc 9
is derived by multiplying the slope q^ = 9.1 x 10~^/mg/kg/day and the
molecular weight of EDC, 98.9. This places the potency index for EDC in the
fourth quartile of the 54 suspect carcinogens evaluated by the CAG.
The ranking of relative potency indices is subject to the uncertainties
involved in comparing a number of potency estimates for different chemicals
based on varying routes of exposure in different species, by means of data from
studies whose quality varies widely. All of the indices presented are based on
estimates of low-dose risk, using a low-dose linear extrapolation model. These
indices may not be appropriate for the comparison of potencies if linearity
does not exist at the low-dose range, or if comparison is to be made at the
high-dose range. If the latter is the case, then an index other than the one
calculated above may be more appropriate.
9.5.4. Summary
9.5.4.1. QUALITATIVE — Although seven cancer bioassay studies of EDC
have been reported, only the NCI bioassay in which EDC was administered to
rats and mice by gavage produced a clear positive tumorigenic response.
In the NCI (1978) rat study, EDC produced a statistically significant
increase in the incidence of squamous cell carcinomas of the forestomach,
9-252
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July 1985
TABLE 9-66. RELATIVE CARCINOGENIC POTENCIES AMONG 54 CHEMICALS EVALUATED BY THE CARCINOGEN ASSESSMENT GROUP
AS SUSPECT HUMAN CARCINOGENS
vO
I
Level
of evidence3
Compounds
Acrylonitrile
Aflatoxin B.
Aldrln
Allyl chloride
Arsenic
B[a]P
Benzene
Benzidene
Beryllium
1,3-Butadiene
Cadmium
Carbon tetrachloride
Chlordane
CAS Number
107-13-1
1162-65-8
309-00-2
107-05-1
7440-38-2
50-32-8
71-43-2
92-87-5
7440-41-7
106-99-0
7440-43-9
56-23-5
57-74-9
Humans
L
L
I
S
I
S
S
L
I
L
I
I
Animals
S
S
L
I
S
S
S
S
S
S
S
L
Grouping
based on
IARC
criteria
2A
2A
3
1
2B
1
1
2A
2B
2A
2B
3
Slopeb
(mg/kg/day)-l
0.24(W)
2900
11.4
1.19x10-2
15(H)
11.5
2.9xlO-2(W)
234(W)
2.6
l.OxHT'U)
6.1(U)
1. 30x10-!
1.61
Molecular
weight
53.1
312.3
369.4
76.5
149.8
252.3
78
184.2
9
54.1
112.4
153.8
409.8
Potency
lndexc
1x10+1
9xlO+5
4x10+3
9x10-'
2x10+3
3x10+3
2x10°
4xlO+4
2xlO+1
5x10°
7x10+2
2x10+'
7x10+2
Order of
magnitude
-------
TABLE 9-66. (continued)
I
NJ
Level
of evidence8
Compounds
Chlorinated ethanes
1 , 2-Dichloroethane
hexachloroethane
CAS Number Humans
107-06-2
67-72-1
1,1,2,2-Tetrachloroethane 79-34-5
1,1,2-Trichloroethane 79-00-5
Chloroform
Chromium VI
DDT
Dichlorobenzidine
1 , 1-Dichloroethylene
(Vinylidene chloride)
Dichloromethane
(Methylene chloride)
DieLdrln
2,4-Dinitrotoluene
Diphenylhydrazine
Epichlorohydrin
Bis(2-chloroethyl)ether
67-66-3
7440-47-3
50-29-3
91-94-1
75-35-4
75-09-2
60-57-1
121-14-2
122-66-7
106-89-8
111-44-4
I
I
I
I
I
S
I
I
I
I
I
I
I
I
I
Animals
S
L
L
L
S
S
S
S
L
L
S
S
S
S
S
Grouping
based on
1ARC
criteria
2B
3
3
3
2B
1
2B
2B
3
3
2B
2B
2B
2B
2B
Slopeb
(mg/kg/day)-1
9.1x10-2
1.42x10-2
0.20
5.73x10-2
7x10-2
41(W)
0.34
1.69
1.16(1)
6.3xlO-A(I)
30.4
0.31
0.77
9.9xlO-3
1.14
Molecular
weight
98.9
236.7
167.9
133.4
119.4
100
354.5
253.1
97
84.9
380.9
182
180
92.5
143
Potency
indexc
9x10°
3x10°
3xlO+1
8x10°
8x10°
4xlO+3
lxlO+2
4xlO+2
1x10+2
5x10-2
1x10**
6xlO+1
lxlO+2
9x10-!
2xlO+2
Order of
magnitude
(lOglQ
index)
•H
0
+1
+1
+ 1
+*
+2
+3
+2
-1
+4
+2
+2
0
+2
-------
TABLE 9-66. (continued)
I
tv.
Compounds
Bis(chloromethyl)ether
F.thylene dibromvde (EDB)
Elhylcne oxide
Keptrichlor
Hexachlorobenzene
HexachLorohutadlene
Hexachlorocyclohexane
technical grade
alpha isomer
beta tsomer
gamma isomur
Hexdchlorodlbenzodioxin
\ 1 l_k-' 1 ft! 1 1 ML' 1 " illlSt
(Nil 1 -.,ih-.'i i i ulii)
Li m ii. •
1)1 ,V 1 II ' 1 11111 Hi-
ll. !.l. • ,1110
1), < . \' • 1 • .1 IK-
N- 1. 1 r osii|", . !il I no
N-'i i : i>-..i-V i.ivlurc.i
CAS Number
542-88-1
106-93-4
75-21-8
76-44-8
118-74-1
87-68-3
319-84-6
319-85-7
58-89-9
34465-46-8
h2- *.-l(
.<>- ,- ,
4.''i •- >
'( - .
/ .'J- .- '
Level
of evidence3
Humans Animals
S S
I S
L S
I S
I S
1 L
I S
1 L
T L
1 S
S S
1 S
1 S
1 S
!
1 S
Grouping
based on
IARC
criteria
1
2B
2A
2B
2B
3
2B
3
3
2B
1
2H
2B
>B
in
in
Slopeb
(mg/kg/day)~l
9300(1)
41
3.5xlO-1U)
3.37
1.67
7.75xlO~2
4.75
11.12
1.84
1.33
6.2xlO+3
1.05(W)
2S.9(not b>
4 }.5(noL bv
ri.43
J.I3
i '.9
Molecular
weight
115
187.9
44.1
373.3
284.4
261
290.9
290.9
290.9
290.9
391
240.2
qf> 74.1
qf) 102.1
ISH.J
100.2
H7.I
Potency
index0
lxlO+6
8xlO+3
2xlO+1
lx!0+3
5xLO+-
2xlO+1
lxlO+3
3xlO+3
5xlO+2
4xlO+2
2xlO+6
2.5X1Q-1--2
2xlH+3
'ixl'if<
'i \ 1 1 1 ' -'
Mi 'J
txl"'(
Order of
magnitude
(logl()
Index)
+6
+4
+ 1
+3
+3
+ 1
+3
+ )
+ j
+ 3
+6
+2
+ 3
KI
r \
• :
' •
(rontinumt on t ho 11> I lowing
-------
TABLE 9-66. (continued)
vO
to
in
Level
of evidence3
Compounds CAS Number
N-ni t roso-N-me t hylu rea
N-nitroso-diphenylamine
PCBs
Phenols
2 , 4, 6-Trichlorophenol
Tetrachlorodibenzo-
p-dioxin (TCDD)
Tetrachloroethylene
Toxaphene
Trichloroethylene
Vinyl chloride
684-93-5
86-30-6
1336-36-3
88-06-2
1746-01-6
127-18-4
8001-35-2
79-01-6
75-01-4
Humans
I
I
I
I
I
I
1
I
S
Animals
S
S
S
S
S
L
S
L/S
S
Grouping
based on
IARC
criteria
2B
2B
2B
2B
2B
3
2B
3/2B
1
Slopeb
(mg/kg/day)-l
302.6
4.92xlO-3
4.34
1.99x10-2
1.56x10+5
5.1x10-2
1.13
l.lxlO-2
1.75x10-2(1)
Molecular
weight
103.1
198
324
197.4
322
165.8
414
131.4
62.5
Potency
indexc
3x1 0+*
1x10°
1X10+3
4x1 0°
5x1 0+7
8x10°
5xlO+2
1x10°
1x10°
Order of
magnitude
Uog1Q
index)
+4
0
+3
+1
+8
+ 1
+3
0
0
aS = Sufficient evidence; L ° Limited evidence; I = Inadequate evidence.
bAnimal slopes are 95% upper-bound slopes based on the linearized multistage model. They are calculated based on
animal oral studies, except for those Indicated by I (animal inhalation), U (human occupational exposure), and H
(human drinking water exposure). Human slopes are point estimates based on the linear nonthreshold model. Not all
of the carcinogenic potencies presented in this table represent the same degree of certainty. All are subject to
change as new evidence becomes available. The slope value is an upper bound in the sense that the true value (which
is unknown) is not likely to exceed the upper bound and may be much lower, with a lower bound approaching zero.
Thus, the use of the slope estimate in risk evaluations requires an appreciation for the implication of the upper
bound concept as well as the "weight of evidence" for the likelihood that the substance is a human carcinogen.
cThe potency index is a rounded-off slope in (mmol/kg/day)-1 and is calculated by multiplying the slopes in
(mg/kg/day)-l by the molecular weight of the compound.
-------
hemangiosarcomas of the circulatory system, and fibromas of subcutaneous tissue
in male rats. There was also a statistically significant increased incidence
of adenocarcinoraas of the mammary gland and hemangiosarcomas of the circulatory
system in female rats.
In the NCI (L978) mouse study, EDC produced a statistically significant
increased incidence of hepatocellular carcinomas and alveolar/bronchiolar
adenomas in male mice and a statistically significant increased incidence of
alveolar/bronchiolar adenomas, mammary carcinomas, and endometrial tumors in
female mice.
Two inhalation studies of EDC were conducted. The study by Spencer et
al. (1951) in Wistar rats showed no evidence of a positive response. However,
this study was inadequate to assess the carcinogenicity of EDC because the
experiment was conducted in a small number of animals for 212 days with L51
exposure days at 200 ppm. A second study by Maltoni et al. (1980), conducted
in both rats and mice, did not produce a statistically significant increase in
tumor incidences in any of the organ sites as compared to control animals.
The study by Theiss et al. (1977), a pulmonary tumor bioassay in which EDC
was administered intraperitoneally to strain A mice, produced an elevated, but
not a statistically significant, increase in the incidence of lung tumors in
treated animals.
In a study by Van Duuren et al. (1979), EDC was applied to the skin of
ICR/Ha mice to assess its carcinogenic potential as a skin turaorigen. This
study did not show a statistically significant increase in skin carcinomas;
however, an increased incidence of benign lung tumors in mice treated with the
high dose (126 mg/mouse) was reported.
No case reports or epidemiologic studies concerning EDC are available in
the published literature.
9-257
-------
9.5.4.2. QUANTITATIVE — Data from gavage studies using both rats and
mice have been used to estimate the carcinogenic potency of EDC. An evaluation
of the metabolism and kinetic data shows that there is no evidence to suggest
qualitative differences in the metabolic pathways of EOC in the mouse and
rat, and no difference resulting from the route of exposure (oral or inhalation)
in the rat.
Lifetime average human equivalent doses for the NCI rat and mouse carcino-
genicity bioassay can be calculated and are used in estimation of the cancer
potency. For rats, data on hemangiosarcoraas in the circulatory system are used.
For mice, data on hepatocellular carcinomas are used. The carcinogenic poten-
cies estimated on the basis of these two data sets are comparable when the
linearized multistage model is used. The upper-bound estimate of EDC potency
«t —O jt
(slope) is qi = 9.1 x 10 /mg/kg/day. This qi is calculated on the basis
of hetnangiosarcomas using the time-to-death data and an adjusted dose derived
from the metabolism/kinetic evaluation.
The upper-bound estimate of the Incremental cancer risk due to 1 yg/L
of EDC in drinking water is 2.6 x 10~6.
Because of the discrepant tumor responsiveness between oral and inhala-
tion assays, two upper-bound estimates of the incremental cancer risk due to
1 ug/m3 of EDC in air are calculated: 2.6 x 10~5 on the basis of the NCI
gavage study, and 1.0 x 10~6 on the basis of a negative inhalation study by
Maltoni et al. (1980). These two estimates reflect the various uncertainties
associated with the data used, including the uncertainties due to the strains
of test animals and routes of exposure used in the bioassays. If the strains
of test animals differ in responsiveness, the unit risk estimate, 2.6 x 10~5,
calculated from the positive response of the most sensitive strain of animals,
should be used. On the other hand, if the discrepant results between the
9-258
-------
gavage and inhalation studies are due solely to the difference in route of
exposure, than it would be more appropriate to use che unit risk estimate,
1.0 x 10~6, calculated on the basis of an inhalation study, even if it is a
negative study. Given the evidence currently available, there is no certain
scientific reason to explain why animals responded differently in the NCI
gavage study compared with the Maltoni et al. inhalation study.
9.5.5. Conclusions. There is evidence that EDC is a probable human carcino-
gen. This conclusion is based on: (1) multiple tumor types in both an oral
rat bioassay and an oral mouse bioassay; (2) suggestive evidence in two other
bioassays, one which showed an elevated increase in lung tumors with intraperi-
toneal injection and a second study which showed a significantly increased
Incidence of benign lung tumors with skin application of EDC; (3) demonstrated
evidence of reactive metabolites of EDC and formation of DNA adduct; and (4)
evidence that EDC is also a mutagen.
There is direct animal evidence for carcinogenicity via the oral (gavage)
route of exposure, but the only specific bioassay data for inhalation exposure
are a negative rat and mouse study. Because of the presence of distant site
tumors in the oral studies, it Is likely that EDC is also carcinogenic via
inhalation exposure. The negative findings in the rat and mouse inhalation
studies do not contradict the indirect, suggestive, and ancillary evidence for
the likelihood of EDC being carcinogenic via inhalation.
The U.S. Environmental Protection Agency is using, on an interim basis,
a proposed classification scheme for evaluating the weight-of-evidence for
carcinogenicity (U.S. EPA, 1984). Using this classification, the positive
findings in the oral rat and mouse studies would be considered as a sufficient
level of evidence in experimental animals. The sufficient level of animal
evidence, together with an absence of epidemiologic data, provides a basis for
9-259
-------
an overall welght-of-evidence ranking of Group B2, meaning that EDC is
"probably" carcinogenic in humans.
Applying the International Agency for Research on Cancer (IARC) classifi-
cation scheme, which EPA has used in the past, the positive findings in the
experimental animals and the absence of epideraiologic data provide an overall
weight-of-evidence ranking of Group 2TJ, meaning that EDC is "probably" carci-
nogenic in humans.
While the "probable" carclnogenicity conclusion obviously applies to
ingestion exposure since the positive evidence comes from gavage experiments,
it is likely that EDC is a probable carcinogen for humans via inhalation expo-
sure. In view of the evidence, it is prudent to consider EDC to be carcinogenic
via inhalation exposure, although the potency may vary from that estimated from
gavage. Research investigations to verify this assessment conclusion should be
implemented.
Assuming that EDC is carcinogenic for humans, upper-bound incremental unit
risks have been estimated for both ingestion and Inhalation exposure. The
development of these unit risk estimates is for the purpose of evaluating the
"what if" question: If EDC is carcinogenic for humans, what is the possible
magnitude of the public health impact? The upper-bound estimate of EDC's car-
JL —O
cinogenic potency is q, = 9.1 x 10 /rag/kg/day based on the occurrence of
heraangiosarcomas in the NCI gavage rat study. The upper-bound estimate of
the incremental cancer risk due to 1 pg/L of EDC in drinking water is 2.6 x
10~6, based on the previously mentioned potency value. Two upper-bound
estimates of the incremental cancer risk due to 1 [jg/ra-^ of EDC in air are
calculated: 2.6 x 10~5, on the basis of the gavage potency value and 1.0 x
10~6, on the basis of a negative inhalation study by Maltoni et al. (1980).
The inhalation unit risks are presented as two values in recognition of the
9-260
-------
uncertainties associated with the differing data that were used to estimate
the values. The upper-bound nature of these estimates is such that the true
risk is not likely to be exceeded and may be lower.
The potency index for EDC, defined as qj x molecular weight, lies in
the fourth quartile among 54 carcinogens evaluated by the Carcinogen Assessment
Group.
9-261
-------
APPENDIX A
COMPARISON AMONG DIFFERENT EXTRAPOLATION MODELS
Three models used for low-dose extrapolation, assuming the independent
background, are:
Multistage: P(d) - 1 - exp (-(q±d + . . . +
where q^ are non-negative parameters;
Probit: A + Bln(d)
P(d) = / f(x) dx
— OB
where f(.) is the standard normal probability density function;
Weibull: P(d) = 1 - exp [-bdk]
where b and k are non-negative parameters.
The maximum likelihood estimates (MLE) of the parameters in the multi-
stage model are calculated by means of the program GLOBAL83, which was developed
by Howe (1983). The MLE estimates of the parameters in the probit and Weibull
models are calculated by means of the program RISKS 1, which was developed by
Kovar and Krewski (1981).
Table A-l presents the MLE of parameters in each of the three models
that are applicable to a data set.
9-262
-------
TABLE A-l. MAXIMUM LIKELIHOOD ESTIMATES
OF THE PARAMETERS FOR EACH
OF THE THREE EXTRAPOLATION MODELS
BASED ON DIFFERENT DATA BASES
Data base
Multistage
ProbiC
Welbull
Hemanglosarcomas
in male rats
Hepatocellular
carcinomas
in male mice
q2
= 4.15 x 10
= 0
= 0.0
= 1.00 x 10
-2
-2
A = -1.48
B = 0.39
A = -4.27
B = 2.27
b = 8.46 x 10~2
k = 0.60
b = 5.70 x 10~4
k = 3.95
9-263
-------
APPENDIX B
RISK CALCULATION BASED ON TIME-TO-EVENT DATA
Because of the high mortality rate of rats in the high-dose group of
the NCI (1978) gavage study, it is more appropriate to use time-to-event
data (Table B-l) to calculate the potency of EDC. The probability of
cancer by time t at dose d is given by:
P(d,t) = 1 - exp [-f(t) x g(d)]
where g(d) is a polynomial in dose d, and f(t) is a function of time t. The
maximum likelihood estimate of the parameters and the asymptotic properties
of the incremental risk estimate were Investigated by Daffer et al. (1980).
Their approach to estimating the parameters resembles Cox's regression-life-
table approach (Cox 1972), in which the time function f(t) need not be
specified.
Using the data in Table B-l, the lifetime cancer risk is estimated to be
q* = 9.1 x lO'Vmg/kg/day
This value is the 95% upper bound of the risk calculated at t = 90 weeks.
Since the model fits the data very well up to 90 weeks but poorly beyond
90 weeks (overestimates), P(d, 90) is used to approximate the lifetime risk.
This seems reasonable because the median life span for control animals is also
less than 90 weeks (approximately 70% of the control animals died before 90
weeks).
9-264
-------
TABLE B-l. TIME-TO-DEATH IN WEEKS FROM HEMANGEOSARCOMAS
IN MALE OSBORNE-MENDEL RATS FED EDC BY GAVAGE
Control (vehicle and untreated): dose = 0
2B, 37, 50, 50, 53, 53, 53, 54, 57, 57, 57, 57, 57, 61, 61,
69, 71, 76, 78, 80, 80, 83, 85, 86, 87, 89, 90, 101, 101,
104, 106, 106, 106, 106, 106, 108, 110, 110, 110, 110.
Low-dose group: dose = 4.46 mg/kg/day
30, 34, 36, 51, 52, 52, 55, 55, 59, 61, 63, 65, 69, 73(H)a,
74(H), 75, 75, 76, 77, 77, 77(H), 77, 80, 81, 82, 82, 82, 84,
87(H), 89(H), 89, 89, 89, 90, 92, 92, 93(H), 95(H), 96, 97,
98, 99, 99, 102{H), 103, 103, 104, 104, 109(H), 110.
High-dose group: dose = 8.23 lag/kg/day
3, 8, 9, 9, 10, 10, 10, 10, 10, 11, 15, 15, 16, 20, 22, 33,
33, 33, 34, 37, 41, 48, 51, 52, 54, 57, 58, 59, 60, 61(H),
62, 63, 63, 68(H), 68(H), 71, 72, 73, 74, 74(H), 74, 74, 76(H),
76, 78(H), 83(H), 84, 89, 101.
aH indicates death from hemangiosarcoma.
9-265
-------
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