&EPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-600/8-82-003F
February 1984
Final Report
Research and Development
Health Assessment
Document for
1,1,1 -Trichloroethane
(Methyl Chloroform)
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EPA-600/8-82-003F
February 1984
Final Report
Health Assessment Document
for
1,1, 1 -Trichloroethane
(Methyl Chloroform)
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC 27711
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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 was originally developed for use by the Office of Air Quality
Planning and Standards to support decision-making regarding possible regula-
tion of methyl chloroform 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 critically 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 appropri-
ate, so that the nature of the adverse health responses is placed in per-
spective with observed environmental levels.
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TABLE OF CONTENTS
Page
LIST OF TABLES v
LIST OF FIGURES . vj
1. SUMMARY AND CONCLUSIONS 1-1
2. INTRODUCTION . 2-1
3. GENERAL BACKGROUND INFORMATION 3-1
3.1 CHEMICAL AND PHYSICAL PROPERTIES, ANALYTICAL METHODOLOGY 3-1
3.1.1 Chemical and Physical Properties 3-1
3.1.2 Analytical Methodology 3-2
3.1.2.1 Sampling and Sources of Error.. 3-5
3.1.2.2 Calibration 3-7
3.1.2.3 Standard Methods .. 3-9
3.2 PRODUCTION, USE, AND EMISSIONS 3-9
3.2.1 Production 3-9
3.2.2 Usage 3-11
3.2.3 Emissions .... 3-11
3.3 ATMOSPHERIC TRANSPORT, TRANSFORMATION AND FATE 3-13
3.3.1 Tropospheric Removal Mechanisms and Residence Time 3-13
3.3.2 Impact Upon the Ozone Layer 3-15
3.3.3 Laboratory Studies 3-17
3.4 MIXING RATIOS IN THE ATMOSPHERE .. 3-18
3.4.1 Global Atmospheric Distributions 3-18
3.5 LEVELS FOUND IN WATER 3-27
3. 6 REFERENCES. 3-28
4. METABOLIC FATE AND DISPOSITION 4-1
4.1 ABSORPTION, DISTRIBUTION AND ELIMINATION 4-1
4.1.1 Oral and Dermal Absorption 4-2
4.1.2 Pulmonary Uptake and Body Burden... 4-3
4.1.3 Tissue Distribution 4-11
4.1.4 Pulmonary Elimination 4-11
4.1.5 Elimination by Other Routes 4-13
4.2 BIOTRANSFORMATION 4-13
4.2.1 Magnitude of Methyl Chloroform Metabolism 4-13
4.2.2 Kinetics of Blood and Urine Metabolites. 4-20
4.2.3 Enzyme Pathways of Methyl Chloroform
Metabolism.... 4-22
4.3 SUMMARY AND CONCLUSIONS 4-27
4.4 REFERENCES 4-29
5. TOXIC EFFECTS 5-1
5.1 HEALTH EFFECTS IN HUMANS , 5-1
5.1.1 Experimental Studies 5-1
5.1.2 Occupational Studies 5-6
5.1.3 Accidental Exposure 5-8
5. 2 EFFECTS ON ANIMALS .- 5-11
i i i
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CONTENTS (cont.)
5.2.1 Acute and Subacute Effects 5-11
5.2.2 Nervous System and Behavior 5-16
5.2.3 Cardiovascular Effects 5-19
5.2.4 Dermal Effects 5-25
5.3 TERATOGENICITY, MUTAGENICITY, AND CARCINOGENICITY 5-25
5.3.1 Teratogenicity, Embryotoxicity, and Reproductive
Effects 5-26
5.3.1.1 Overview 5-26
5.3.1.2 Human Studies 5-28
5.3.1.3 Animal Studies 5-28
5.3.1.3.1 Rats 5-28
5.3.1.3.2 Mice 5-30
5.3.2 Mutagenicity 5-31
5.3.2.1 Gene Mutations in Bacteria... 5-32
5.3.2.2 Gene Mutations in Eukaryotes 5-42
5.3.2.3 Chromosomal Aberrations 5-44
5.3.2.4 Other Indicators of DNA Damage 5-46
5.3.2.5 Gonadal Effects 5-50
5.3.2.6 Summary and Conclusions 5-51
5.3.3 Evaluation of the Carcinogenicity of MC 5-53
5.3.3.1 Epidemiologic Studies 5-53
5.3.3.2 Animal Bioassays - Rats 5-53
5.3.3.2.1 National Cancer Institute
(1977) Rat Study 5-53
5.3.3.2.2 The Dow Chemical Company
(1978) Rat Study 5-57
5.3.3.3 Animal Bioassays - Mice 5-57
5.3.3.3.1 NCI (1977) Mouse Study 5-59
5.3.3.4 Cell Transformation Studies 5-60
5.3.3.5 Carcinogenicity of 1,4-Dioxane. 5-62
5.3.3.6 Summary and Conclusions 5-63
5.4 SUMMARY OF ADVERSE HEALTH EFFECTS AND LOWEST OBSERVED
EFFECTS LEVELS 5-65
5.4.1 Inhalation Exposure 5-65
5.4.1.1 Effects of Single Exposures 5-65
5.4.1.2 Effects of Intermittent or Prolonged
Exposures 5-70
5.4.2 Oral Exposure 5-72
5.4.3 Dermal Exposure 5-73
5.5 REFERENCES 5-75
IV
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LIST OF TABLES
3-1 Physical properties of 1,1,1-trichloroethane 3-1
3-2 Major producers of methyl chloroform 3-11
3-3 1978 emission losses to air 3-12
3-4 Relative efficiency of halocarbons in reducing
stratospheric ozone. 3_-,6
3-5 Ambient air mixing ratios of CH3CC13"measured" at'sites
around the worl d -
«j—J.y
4-1 Partition coefficients of methyl chloroform and other
solvents at 37°C
4-2 Estimated uptake of MC during a'single'i-hr'exposure
(average body weight, 77 kg; 67 kg lean body mass)
estimated uptake of MC during a single 8-hr exposure
(average body weight, 74 kg) 4 6
4-3 Mean values and SEM for 12 male subjects"at'rest"and"
exercise for 30-minute periods 4 7
4-4 Tissue content (rat) of methyl chloroform"(MC)'after
chronic inhalation exposure at 500 ppm... 4_10
4-5 Recovery Experiment with rats (3) intraperitonealIv
injected with 14C-MC(700 mg/kg) 4_15
4-6 Relation between inhalation exposure and urinary"metabolites'
of MC /, -,„
4-18
5-1 Subjective and physiological responses to a constantly
increasing methyl chloroform vapor concentration over
a period of 15 minutes 52
5-2 Subjective and physiological reponses"to"methyl'chloroform' "
vapor concentrations of 900 to 1000 ppm 5-3
5-3 Signs and symptoms of patients surviving intoxication'with" '
methyl chloroform
5-4 Acute toxicity of methyl chloroform! c"1?
5-5 Comparison of lethal and behavioral effects'of"MC'in
combination with ethanol 5 14
5-6 The relative hepatotoxic efficacy of "chlorinated" solvents!."" 5-15
5-7 Nervous system and behavioral effects of MC in laboratory
animals ; y
5-8 Left ventricular and hemodynamic effects of
methyl chloroform 5
5T9 Concentration of chemicals causing'cardiac"sensitization
and their physical properties 5_22
5-10 Oncogenic and teratogenic testing of methyl'chloroform 5-27
5-11 Mutagenicity testing of MC: Gene mutations in bacteria""" 5-33
c"J? Mutagenicity testing of MC: Gene mutations in eukaryotes!! " 5-43
5-13 Mutagenicity testing of MC: Mammalian in vivo cytogenetics
tests
5-14 Other tests of the genotoxic potential"of"methyl"chloroform" 5-47
b-15 Comparison of survival of control groups, methyl
chloroform-treated, and carbon tetrachloride-treated
(positive control) rats 5_55
5-16 Statistical analyses of the incidence'of"tumors"at'specific"
sites in matched controls and methyl chloroform-treated
rats _ ar.
5-46
v
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LIST OF TABLES (cont.)
5-17 Summary of NCI/NTP chloroethane bioassay results as of 1982.
5-18 Composition of the formulation of methyl chloroform
used in chronic inhalation studies in rats
5-19 Average frequency of tumor occurrences in rats treated with
methyl chloroform
5-20 Comparison of survival of control groups, methyl
chloroform-treated, and carbon tetrachloride-treated
(positive control) mice
5-21 Statistical analyses of the incidence of tumors at specific
sites in matched controls and methyl chloroform-treated
mice.
5-22 Summary of animal carcinogenicity studies for 1,4-dioxane..
5-23 Human fatalities associated with methyl chloroform
5-24 Non-lethal effects of methyl chloroform on humans
5-60
5-61
5-64
5-66
5-68
LIST OF FIGURES
3-1 Global distribution of methyl chloroform.
4-1 Absorption and pulmonary elimination of MC, and blood
concentration
4-2 Relationship between methyl chloroform concentration in
alveolar air and arterial blood
4-3 Postulated pathways of hepatic biotransformation of MC..
Page
3-24
4-4
4-8
4-23
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The EPA Office of Health and Environment Assessment (OHEA) is responsible
for the preparation of this health assessment document. The OHEA Environ-
mental Criteria and Assessment Office (ECAO/RTP) had overall responsibility
for coordination and direction of the document preparation and production
effort (Mark M. Greenberg, Project Manager).
The principal authors of Chapters 2, 3, 4, and 5 (exclusive of the
sections on mutagenicity and carcinogenicity) are:
Richard Carchman, Department of Pharmacology, The Medical College of
Virginia, Health Sciences Division, Virginia Commonwealth University,
Virginia
I. W. F. Davidson, Department of Physiology/Pharmacology, The Bowman Gray
School of Medicine, Wake Forest University, Winston Salem, North Carolina
Mark M. Greenberg, Environmental Criteria and Assessment Office, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina
Jean C. Parker, Environmental Criteria and Assessment Office, U.S. Environ-
mental Protection Agency, Research Triangle Park, North Carolina
Vernon Benignus, Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina
The OHEA Carcinogen Assessment Group (CAG) was responsible for preparation
of the section on carcinogenicity. Participating members of the CAG are listed
below (the principal author of the carcinogenicity materials is designated by *):
Roy E. Albert, M.D. (Chairman)
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.
David L. Bayliss, M.S.
Chao W. Chen, Ph.D.
Herman J. Gibb, B.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.
Robert E. McGaughy, Ph.D.
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 the section on mutagenicity. Participating members
of the REAG are listed below (the principal author of the mutagenicity section
is indicated by *):
Vicki Vaughan-Dellarco, Ph.D.
John R. Fowle III, Ph.D.*
Ernest R. Jackson, M.S.
K. S.- Lavappa, Ph.D.
Sheila L. Rosenthal, Ph.D.
Carol N. Sakai, Ph.D.
Peter E. Voytek, Ph.D.
vn
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Early drafts of this document were reviewed by EPA members of the Agency
Work Group on Solvents, as listed below:
Elizabeth L. Anderson
Charles H. Ris
Jean Parker
Hark Greenberg
Cynthia Sonich
Steve Lutkenhoff
James A. Stewart
Paul Price
William Lappenbush
Hugh Spitzer
David R. Patrick
Lois Jacob
Arnold Edelman
Josephine Brecher
Mike Ruggiero
Jan Jablonski
Charles Delos
Richard Johnson
Priscilla Holtzclaw
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Toxic Substances
Office of Toxic Substances
Office of Drinking Water
Consumer Product Safety Commission
Office of Air Quality Planning and Standards
Office of General Enforcement
Office of Toxic Integration
Office of Water Regulations and Standards
Office of Water Regulations and Standards
Office of Solid Waste
Office of Water Regulations and Standards
Office of Pesticide Programs
Office of Emergency and Remedial Response
ACKNOWLEDGEMENTS
We are grateful for the technical assistance received from Drs. Stephen
Nesnow, Kristien Mortelmans, and Ronald Spanggord concerning the mutagenicity
testing of methyl chloroform. Dr. Nesnow, Chief, Carcinogenesis and Metabolism
Branch, Genetic Toxicology Division, U.S. E.P.A. Environmental Research Center
contracted for and provided us with mass spectrometric analyses of methyl
chloroform samples tested by Snow et al. (1979).
Drs. Mortelmans and Spanggord provided us with unpublished data and
chemical analyses of MC samples tested at SRI International. The mutagenicity
section was reviewed by Drs. George Hoffmann and Daniel Straus.
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The following individuals were also asked to review an early draft of this
document and submit comments:
Dr. Donald Barnes
Office of Toxic Substances
U.S. Environmental Protection Agency
Dr. Joseph Borzelleca
Dept. of Pharmacology
The Medical College of Virginia
Virginia Commonwealth University
Richmond, VA 23298
All Members of the Interagency
Regulatory Liaison Group
Subcommittee on Organic Solvents
Dr. Herbert Cornish
Dept. of Environmental
University of Michigan
Ypsilanti, MI 48197
and Industrial Health
Dr. I. W. F. Davidson
Dept. of Physiology/Pharmacology
The Bowman Gray School of Medicine
Winston-Sal em, NC 27103
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center
New York, NY 10016
Dr. Lawrence Fishbein
National Center for Toxicological Research
Jefferson, AR 72079
Dr. Richard N. Hill
Office of Toxic Substances
U.S. Environmental Protection Agency
Dr. John G. Keller
P. 0. Box 10763
Research Triangle Park, NC 27709
Dr. John L. Laseter
Director, Environmental Affairs, Inc.
New Orleans, LA 70122
Dr. John Todhunter, Assistant Administrator
Office of Pesticides and Toxic Substances
U.S. Environmental Protection Agency
Dr. Herbert Wiser
Deputy Assistant Administrator
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
IX
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SCIENCE ADVISORY BOARD ENVIRONMENTAL HEALTH COMMITTEE
This document was independently peer-reviewed by the Environmental Health
Committee, U.S. Environmental Protection Agency Science Advisory Board.
Chairman, Environmental Health Committee
Dr. Roger 0. McClellan, Director of Inhalation Toxicity Research Institute,
Lovelace Biomedical and Environmental Research Institute, Albuquerque, New
Mexico 87115
Acting Director, Science Advisory Board
Dr. Terry F. Yosie, Science Advisory Board, U.S. EPA, Washington, DC 20460
Members
Dr. Herman E. Collier, Jr., President, Moravian College, Bethlehem,
Pennsylvania 18018
Dr. Morton Corn, Professor and Director, Division of Environmental Health
Engineering School of Hygiene and Public Health, The Johns Hopkins Univer-
sity, 615 N. Wolfe Street, Baltimore, Maryland 21205
Dr. John Doull, Professor of Pharmacology and Toxicology, University of
Kansas Medical Center, Kansas City, Kansas 66207
Dr. Edward F. Ferrand, Assistant Commissioner for Science and Technology,
New York City Department of Environmental Protection, 51 Astor Place, New
York, New York 10003
Dr. Herschel E. Griffin, Associate Director and Professor of Epidemiology,
Graduate School of Public Health, San Diego State University, San Diego,
California 92182
Dr. Jack, D. Hackney, Chief, Environmental Health Laboratories, Professor of
Medicine, Rancho Los Amigos Hospital Campus of the University of Southern
California, 7601 Imperial Highway, Downey, California 90242
Dr. D. Warner North, Principal, Decision Focus, Inc., 5 Palo Alto Square,
Palo Alto, California 94304
Dr. William J. Schull, Director and Professor of Population Genetics,
Center for Demographic and Population Genetics, School of Public Health,
University of Texas Health Science Center at Houston, Houston, Texas 77030
Dr. Michael J. Symons, Professor, Department of Biostatisties, School of
Public Health, University of North Carolina, Chapel Hill, North Carolina
27711
Dr. Sidney Weinhouse, Professor of Biochemistry, Senior Member, Fels
Research Institute, Temple University School of Medicine, Philadelphia,
Pennsylvania 19140
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Consultant
Dr. Bernard Weiss, Division of Toxicology, University of Rochester School
of Medicine, Rochester, New York 14642
XI
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ABSTRACT
Methyl chloroform (MC) is a volatile chlorinated hydrocarbon used exten-
sively as an industrial solvent and in consumer products. It has been detec-
ted in the ambient air of a variety of urban and non-urban areas of the United
States. Normally, background levels are in the range of 0.1 to 0.2 ppb (0.54 x
-3 -3 3
10 to 1.1 x 10 mg/m ). Levels in some urban areas have ranged up to
20 ppb (0.11 mg/m ). It has been less frequently detected in water, generally
at levels of 1 ppb or less. In certain instances involving contamination of
groundwater, much higher levels have been reported.
The weight of available evidence obtained from both human and animal data
suggest that long-term exposure to environmental levels of MC poses no serious
health concern to the general population. No teratogenic potential has been
demonstrated for MC in studies conducted to date in rodent species. Available
data are inadequate for reaching firm conclusions about its mutagenic poten-
tial in humans. Because of the limited usefulness of the animal bioassays
conducted to date, it is not possible to classify MC in regard to its carcin-
ogenic potential in humans.
The no-observed-effect level for short-term exposure of humans is in the
3
range of 350 to 500 ppm (1,890 to 2,700 mg/m ).
xii
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1. EXECUTIVE SUMMARY
1,1,1-Trichloroethane (methyl chloroform, MC) is a volatile chlorinated
hydrocarbon. Since its commercial introduction, it has been used increasingly
as an industrial solvent and in consumer products, such as spot removers.
Production of MC in the United States is estimated to have increased from
121,000 metric tons in 1970 to 315,000 in 1980. It is estimated that approx-
imately 88 percent of annual production in the United States is released
largely to the atmosphere through dispersive use. There are no known natural
sources of emissions of MC.
Methyl chloroform has been detected in the ambient (natural environment)
air of a variety of urban and non-urban areas of the United States and other
regions of the world. Levels range from trace amounts in rural areas to about
20 parts per billion (ppb) or 0.108 mg/m in some large urban centers. Nor-
mally background levels of MC are in the range of 0.1 to 0.2 ppb (0.54 x 10~3
-3 3
to 1.08 x 10 mg/m ). It has less frequently been detected in water. It is
not soluble to any appreciable extent but has been monitored in some surface
and drinking waters, generally at levels of 1 ppb or less. In certain in-
stances involving contamination of groundwater, much higher levels have been
reported.
In the troposphere, a region of the atmosphere extending from ground
level to as high as 15 kilometers, MC is removed to a substantial extent
through reaction with hydroxyl radicals. Based on current knowledge of its
reaction kinetics, the lifetime of MC in the troposphere is in the range of 5
to 10 years. This time period allows a portion of the MC to be conveyed to
the stratosphere where it, along with other compounds, may participate in
ozone (03) destruction pathways. It has been hypothesized that MC and other
compounds that add to the chlorine burden in the stratosphere may contribute
to the effects of global 03 depletion. If depletion occurs, it may result in
a possible increased incidence of non-malignant forms of skin cancer due to
increases in the amount of biologically-damaging radiation reaching the earth's
surface. The extent to which past, current, and future emissions of MC contrib-
ute to 03 depletion can only be realistically estimated by assessing the
interrelationships between all the principal reactions involved in both the
formation and destruction of atmospheric 0_. The extent and direction to
•j
which actual global 0^ levels have changed over the years is not estimable
with available measurement methods.
1-1
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Because MC is primarily an air contaminant, inhalation is the principal
and most rapid route of exposure. It is estimated that an 8-hour exposure to
the TWA* of 350 parts per million (ppm) or 1,890 mg/m will result in about 2
grams of MC absorbed into the body of an average-si zed 70 kg man. The total
amount absorbed increased in direct proportion to inspired air concentrations
and to the length of exposure and physical activity. Once body equilibrium or
steady-state has been attained, no further uptake is possible. There is strong
evidence that MC will partition selectively into lipid-tissues upon chronic or
long-term exposure to even low ambient air concentrations, until steady-state
is attained. Because of its lipophilic nature, MC is expected to cross mem-
brane barriers in the body and diffuse into the brain and the colostrum of
nursing mothers, as well as into the fetus during pregnancy. Unlike other
chlorinated solvents such as trichloro- and tetrachloroethylene, MC is only
metabolized in humans to a limited extent, about 6 percent or less of the total
retained dose. Although metabolism of MC is affected by other chemicals and
drugs, there is no evidence that it enhances its own metabolism. The primary
route of elimination from the body is via the lungs, through which MC is exhaled
in unchanged form along with a metabolite, carbon dioxide. The only identified
urinary metabolites are trichloroethanol and trichloroacetic acid.
The likelihood of adverse health effects resulting from chronic exposure
to the ambient air levels commonly encountered appears to be extremely low
based on presently available data. It is estimated that a no-observed-effect-
**
level (NOEL) for short-term exposure of humans to MC is in the range of approx-
imately 350 to 500 ppm (1,890 to 2,700 mg/m ). This NOEL is many orders of
magnitude higher than the highest levels of MC (20 ppb; 0.108 mg/m ) measured in
the ambient air of urban areas. Based upon available human data, the estimated
TWA (Time Weighted Average): is defined as the time-weighted average
concentration for a normal 8-hour workday and a 40-hour workweek to which
nearly all workers may be exposed repeatedly, day after day, without adverse
effect.
**
NOEL: is defined as the lowest exposure level at which there are no statis-
tically significant increases in frequency or severity of effects between
the exposed population and its appropriate control.
1-2
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relationship between acute effects and single short-term exposures
is as follows:
100 ppm, ,,
(540 mg/m )
350-500 ppm „
(1,890 - 2,700 mg/m )
1,000 ppm ~
(5,400 mg/nO
1,900 - 2,650 _
(10,260 -14,310 mg/m )
> 5,000 ppm-
(27,000 mg/m )
Apparent odor threshold
Obvious odor, slight changes
in perception
Disturbances of equilibrium
Lightheadedness, irritation
of the throat
Onset of narcosis
In the range of the NOEL no significant abnormal blood chemistry or organ
function decrements have been noted. The main health effects are symptoms of
neurological dysfunction observed at higher exposure levels. These symptoms
were qualitatively diagnosed by subjects' impaired performance of clinical-level
cognitive and manual tasks. More extensive human and laboratory animal data
would be needed before firm conclusions about adverse health responses to
low-level exposures to PC could be drawn.
Similarly, 1C has not demonstrated any teratogenic potential in the...
studies conducted to date in rodent species. Commercially available samples
of MC are genotoxic to mouse hepatocytes and are weakly mutagem'c in Salmonel la
under treatment conditions where sufficient exposure is ensured. The available
data are inadequate, however, for reaching firm conclusions regarding the ability
of MC to cause gene mutations in other organisms; however, the possibility that
this substance, its associated stabilizing materials, or its metabolites may
have mutagem'c effects in humans has not been eliminated.
On the basis of animal bioassays performed to date and in the absence of
epidemiological information, it is not possible to classify MC as to its car-
cinogenic potential in humans.
The weight of available evidence obtained from both human and animal data
suggest that long-term exposure to environmental levels of MC poses no serious
health concern to the general population. One must recognize, however, that
as new information becomes available, further re-evaluation of the health
consequences of exposure may become necessary.
1-3
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directed toward elucidation of subjective responses, CNS effects, and the
pharfnacokinetic parameters of MC exposure. These studies have established
that vapor inhalation is the principal route by which MC enters into the body.
It is widely distributed into all organ systems and is metabolized to a limited
extent. MC is eliminated from the body primarily as the parent compound via
the lungs, but metabolites are excreted mostly in the urine. Epidemiological
studies provide some information about the impact of MC on human health, but
it is necessary'to rely on animal studies to assess any indications of poten-
tially harmful effects for chronic low-level exposures. These studies are
reviewed in chapters 4 and 5.
Of chief concern regarding the potential impact of MC on human health
are: (1) narcosis effects associated with acute high-level exposures and (2)
any mutagenic or carcinogenic effects potentially associated with chronic
low-level exposures. This document reviews evidence regarding acute narcosis
effects and, also, summarizes available mutagenicity evidence and peer-reviewed
results of animal bioassay studies that relate to the carcinogenic potential
of MC. A definitive evaluation of the carcinogenicity of MC is being deferred
until the results of a recently-completed National Toxicology Program bioassay
in rats and mice undergo complete peer review.
Permissible levels of MC in the working environment have been established
in various countries. The U.S. Occupational Safety and Health Administration
(OSHA) health standard requires that a worker's exposure to MC at no time
exceed a TWA of 350 ppm (1,890 mg/m3) in the workplace air in any work shift
of a 40-hr week.
2-2
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3. GENERAL BACKGROUND INFORMATION
3.1 CHEMICAL AND PHYSICAL PROPERTIES, ANALYTICAL METHODOLOGY
3.1.1 Chemical and Physical Properties
1,1,1-Trichloroethane (CH-CC1 ), also called methyl chloroform (MC), is a
•J O
colorless nonflammable liquid which has a characteristic odor. Its line
formula is:
H Cl
H-C—C-C1
H Cl
Chemical Formula C H Cl
Chemical Abstracts Service Registry Number 71-55-6
Synonyms include:
Ethane, 1,1,1-Trichloro
Methyl Chloroform
Methyltrichloromethane
Trichloroethane
1,1,1-Trichloroethane
a-Trichloroethane
Table 3-1 shows some of its important chemical and physical properties.
TABLE 3-1. PHYSICAL PROPERTIES OF 1,1,1-TRICHLOROETHANE
Solubility in water @ 25°C 0.44 gm/lOOgm
Boi 1 i ng poi nt @ 760 torr 74°C
Vapor pressure @ 20°C 100 torr
Vapor density (air = 1) . 4. 6
Molecular weight . 133.41 ^
1 ppm 5.4 mg/m
In the atmosphere, MC is subject to free radical attack. Reaction with
hydroxyl radicals is the principal way in which MC is scavenged from the
atmosphere. Photooxidation products of MC identified via laboratory experi-
mentation include hydrogen chloride, carbon oxides, phosgene, and acetyl
chloride (Spence and Hanst, 1978; Christiansen et al., 1972). The tropospheric
photooxidation products are numerous and rapidly undergo further reactions
3-1
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(U.S. Environmental Protection Agency, 1975). Data discussed in Section 3.3
indicate that MC is relatively stable in the troposphere and that significant
amounts are conveyed to the stratosphere.
The volatilization of MC from water in laboratory studies was investi-
gated by Dilling and coworkers (Dilling et al. , 1975; Dilling, 1977). A
half-life of from 17 to 30 minutes was observed for MC dissolved in water in a
laboratory beaker. These investigators determined that degradation proceeded
with a 6-month half-life and that the rate of degradation was not increased
significantly upon exposure to sunlight. In the preliminary studies of Wood
et al. (1981), degradation of MC was observed under anaerobic conditions in
the presence of natural water samples. Sediment water samples obtained from
the Florida everglades were placed in sealed serum bottles and spiked with 200
ug of MC. Degradation was observed to proceed via 1,1-dichloroethane; this
intermediate suggests the mechanism involves reductive dehalogenation of MC.
Under these conditions, the observed half-life of MC was about 16 days.
Degradation of MC was not observed in autoclaved controls.
Anhydrous MC is generally noncorrosive, but in the presence of water it
can react to form hydrochloric acid, which is corrosive to metals (Keil,
1979). The addition of epoxides can neutralize the generated acid (Keil,
1979). Anhydrous MC, when heated to 360° to 440°C, decomposes to 1,1-dichloro-
ethylene and hydrqgen chloride. When MC is heated in the presence of water at
temperatures between 75° and 160°C, it decomposes to acetyl chloride, acetic
acid, and acetic anhydride upon contact with metallic chlorides or sulfuric
acid. Noweir et al. (1972) have observed that when MC comes in contact with
iron, copper, zinc, or aluminum at elevated temperatures, a small amount of
phosgene is produced. During welding operations, the amount of hydrogen
chloride produced, in contrast to phosgene, may be sufficient to provide
adequate warning to minimize exposure (Rinzema and Silverstein, 1973).
Bonse and Henschler (1976) have considered the general chemical reactivity
of MC. By virtue of the electron-inductive effects of the chlorine substitu-
ents, destabilization occurs at the C-C bond. Radical reaction mechanisms
would be favored as a result of -the delocalization of the unpaired electron
drawn from the C-C bond.
3.1.2 Analytical Methodology
To detect very low levels of methyl chloroform in ambient air, sophisti-
cated analytical techniques have been employed. The two most generally useful
3-2
-------
methods for detection and analysis of MC have been gas chromatography with
electron capture detection (GC-ECD) and gas chromatography-mass spectrometry
(GC-MS). Both systems have a lower limit of detection on the order of a few
parts per trillion (ppt). The utility of GC-ECD over GC-MS is that it can be
used in the field to provide nearly continuous measurements through inter-
mittent sampling (every 15 to 20 minutes).
In a comparison between GC-ECD and GC-MS, Cronn et al. (1976) judged
GC-ECD to be superior in precision. Standards for four halocarbon compounds,
measured by GC-ECD, had coefficients of variation ranging from 0.5 to 3.5
percent. Of 11 halocarbon compounds measured by GC-MS, coefficients of vari-
ation ranged from 4 to 19 percent. Temperature programming or isothermal
operation of the gas chromatograph yielded comparable results (Cronn et al.,
1977a). The precision with temperature-programming was 4.3 percent versus 3.5
percent with isothermal operation for MC.
A close agreement between the levels of MC and other halocarbons detected
by both GC-ECD and GC-MS (on the same ambient air samples) was obtained by
Russell and Shadoff (1977). A difference of 10 ppt was reported between the
systems. Air samples were sorbed onto Porapak N porous polymer and desorbed
onto the column by heating the collection tube rapidly to 200°C. Ambient air
mixing ratios measured by this method were in the range of 90 to 110 ppt.
The electron capture detector, as well as the mass spectrometer in the
selective ion monitoring mode (Cronn and Harsch, 1979a; Pellizzari, 1974), is
specific in the detection and quantisation of many halogenated hydrocarbons;
nonhalogenated hydrocarbons cannot be detected. Thus, a high background level
of hydrocarbons in ambient air samples does not preclude analysis of trace
quantities of MC. In the electron capture detector, MC is ionized by primary
electrons released from an internal radioactive source. The net result is
removal of electrons from the gaseous mixture with substitution by negative
ions of greater mass. The measured effect is a net decrease in ion current
because the mobility of the ions is less than that of the electrons.
Samples of tropospheric air were analyzed by Cronn and coworkers (1977b)
both by preconcentrating the samples according to the method of Rasmussen et
al. (1977) and by direct injection. The detection limit was 3 ppt (16.2 x
10 mg/m ). The precision of the method using preconcentration was ±4 percent.
The internal consistency, between direct GC-ECD analysis of MC and using the
preconcentration method was reported to be good (Cronn et al., 1977b).
3-3
-------
A detection limit of 2 ppt (10.8 x 10~6mg/m ) for MC was achieved by
Harsch and Cronn (1978) with a low pressure sample-transfer technique. Pre-
cision was ±7 percent with the Rasmussen et al. (1977) preconcentration
technique. Calibration was accomplished by use of secondary standards that
were compared to static dilutions of pure and commercially prepared mixtures
of MC (10 ppm; 54 mg/m3) in helium. Robinson (1978) reported a detection
limit of 6 ppt with a precision of ±4.2 percent with secondary standards in a
system using dual GC-ECD.
Pellizzari and Bunch (1979) reported an estimated detection limit of
12.45 ppt (66 x 10~6 mg/m3) using high resolution gas chromatography/mass
spectrometry. The detection limit was calculated on the basis of the break-
through volume for 2.2 grams of Tenax GC, at 21°C. The accuracy of analysis
was reported at ± 30 percent. Similar results were also reported by Krost et
al.(1982). Sources of error are discussed in section 3.1.2.1.
The relatively high volatility of MC was used to advantage by Piet et al.
(1978) in measuring the content of MC in water samples. A direct headspace
method was used with MC volatilizing into the air above the surface of the
water sample in a sealed container. A GC-ECD system was used for separation
and quantisation. The systematic error of the method was reported to be less
than 5 percent. The detection limit for MC was 0.05 ug per liter. The 0V 225
capillary GC column provided good separation of MC from CCl^.
Otson and Williams (1982) have described a modified purge and trap tech-
nique for evaluation of volatile organic pollutants in water. The detection
limits reported for MC was 0.5 pg/1, using either flame ionization detection
or electron capture. Tenax GC was used as packing for the combined trap/
chromatographic column. Purging efficiency was close to 100 percent.
Lionel et al. (1981) provided additional evidence that aeration can be
used successfully to purge compounds such as MC from water. Experimental
techniques suggest that at appropriate air flow, over 90 percent of MC can be
purged from water.
The purging efficiency from samples of sediment and fish tissue spiked
with MC was investigated by Hiatt (1981). Samples were sonicated, frozen, and
then subjected to three analytical methods: vacuum extraction, direct purge
and trap of diluted sample, and modified purge and trap with thermal desorp-
tion. For MC, vacuum extraction appeared to result in the highest recovery.
These preliminary data do suggest that vacuum extraction has application to
environmental analysis of similar samples.
3-4
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Development of analytical procedures for quantisation of MC and other
compounds from biological media such as mother's milk has been evaluated by
Pellizzari et al. (1982). All volatile compounds were recovered by warming
the milk and purging with helium. Vapors were then trapped on a Tenax cart-
ridge, followed by thermal desorption and analysis by GC-MS.
3.1.2.1 Sampling and Sources of Error—The National Academy of Sciences
(1978) has reviewed common approaches used to sample ambient air for trace gas
analysis. These approaches include:
1.
2.
3.
4.
Ambient pressure samples. An evacuated chamber is opened an.d
allowed to fill until it has reached ambient pressure at the
sampling location. If filling is conducted at high altitude,
contamination of the low pressure sample is likely when samples
are returned to ground level.
Pump pressure samples. A mechanical pump is used to fill a
stainless steel or glass container to a positive pressure
relative to the surrounding atmosphere.
Adsorption on molecular sieves, activated charcoal, or other
materials. Sorbents that have been used to collect MC include
Porapak Q, Tenax GC, and Chromosorbs 101, 102, and 103.
Cryogenic sampling. Air samples are transferred to a loop held
at the temperature of liquid oxygen. Components, including MC,
are concentrated by freezeout while other gases (oxygen, nitrogen)
pass through.
In the analysis of whole air samples of compounds such as MC, which are
present in ambient air in the ppt range, small uncontrolled errors in the
system could result in inaccurate results. Errors in the analysis can occur
during sampling, in the gas chromatograph, in the EC detector, and in cali-
bration of the instruments.
Rasmussen and Khali! (1981a) reported that, in an inter!aboratory com-
parison of two samples of MC in ambient air, a majority of the 19 partici-
pating laboratories reported values lower than those determined by the authors.
It was concluded that common standards are needed if atmospheric measurements
from different laboratories are to be pooled.
Singh and coworkers (1978a) used electrochemically-polished stainless
steel sampling vessels. The sampling vessels were flushed with ultra-pure
helium until background contamination was reduced to less than 2 to 3 percent
of the expected background concentration of a given trace constituent. Cronn
3-5
-------
and coworkers similarly employed electropolished stainless steel containers
whose interior surfaces had been passivated by electropolishing (Cronn and
Robinson, 1979; Cronn et al. , 1976; 1977).
Harsch and Cronn (1978) have evaluated sample transfer techniques. Most
analytical procedures involve the collection of pressurized air samples.
Sample transfer under positive pressure minimizes serious contamination possi-
bilities posed by laboratory air containing many times the ambient mixing
ratios of most halocarbons. However, collection of low pressure samples, such
as stratospheric air samples, is a specialized situation in which careful
precautions against sample contamination are necessary. A technique appli-
cable to collection of whole air samples at altitudes up to 21 kilometers
showed a 7 percent standard deviation when the MC mixing ratio was 105 ppt
(Harsch and Cronn, 1978). Electropolished sampling bottles were evacuated to
30 microns prior to sample collection. Transfer of contents to a freezeout
loop (Rasmussen et al., 1977) immersed in liquid 02 was made by vacuum
assist.1 Preconcentration was followed by injection onto a temperature-pro-
grammed column. The detection limit for MC with the GC-ECD procedure was,2
ppt (10.8 x 10-6mg/m3). To minimize the number of potential sources of leaks,
system components were silver soldered. The technique was applicable when
gross halocarbon levels were not present in laboratory air. However, it was
noted that if laboratory air contained ppb levels of halocarbons, high-boiling
halocarbons were adsorbed onto the surfaces of the fittings of the sampling
containers and associated plumbing during times when the system was exposed to
laboratory air.
The EC detector can be a source of error because water vapor and oxygen
may cause reduced sensitivity for certain halocarbons. Lillian and Singh
(1974) used an ascarite trap between the GC column and the detector to absorb
sample moisture. Carrier gas for the GC is purified by passage through traps
containing activated charcoal, anhydrous calcium sulfate, and an ascarite or
molecular sieve (Cronn, 1980a). Oxygen contamination in the detector can be
minimized by preconcentration of air samples on porous polymers (Russell and
Shadoff, 1977). Significant oxygen contamination is often caused by fitting
leaks in the GC rather than by carrier gas contamination.
A freezeout concentration method was employed by Rasmussen et al. (1977)
to determine atmospheric levels of MC in the presence of other trace vapors.
The detection limit of MC was reported to be 0.8 ppt (4.3 x 10- mg/m ) for
3-6
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500 ml aliquots of ambient air when measured by GC-ECD. Precision was 4.3
percent. Standards were prepared by static dilutions in helium. During the
procedure, the oven of the GC was cooled to -10°C. When freezeout was complete,
the loop containing the concentrated air sample was immersed in heated water
and the carrier gas swept the contents of the sample loop onto the column.
Pellizzari and Bunch (1979) reported the use of Tenax GC, a porous polymer
based on 2,6-diphenyl-p-phenylene oxide, to absorb MC from ambient air.
Recovery was made by thermal desorption and helium purging into a freezeout
trap. Included among the inherent analytical errors were (1) the ability to
accurately determine the breakthrough volume, (2) the percent recovery froiji
the sampling cartridge after a period of storage, and (3) the reproducibility
of thermal desorption from the cartridge and its introduction into the analy-
tical system. To minimize loss of sample, cartridge samplers should be en-
closed in cartridge holders and placed in a second container that can be
sealed, protected from light, and stored at 0°C. In an analytical system
using Tenax GC as the absorbent, Pellizzari and coworkers (Krost et al., 1982)
_ 5 3
reported a detection limit for MC of 12.4 ppt (6.7 x 10 mg/m ). Analysis of
the breakthrough volumes at various temperatures suggests that Tenax GC is not
a particularly effective absorbent for MC. Singh et al. (1982) have cautioned
that absorption of MC on Tenax may not reliably reflect ambient air levels
since measurements by some investigators have been reported as less than
background (100 to 200 ppt; 0.54 x 10"3 to 10.8 x lo"3 mg/m3).
3.1.2.2 Calibration—In general, calibration of the instrumentation for
GC-ECD analysis of methyl chloroform has involved static, multiple dilutions
of pure material in the ppm range to ppt levels (Cronn et al., 1976; 1977a,b;
Harsch and Cronn, 1978; Lillian and Singh, 1974; Singh et al., 1977a). Singh
et al. (1977b) have cautioned that, in some cases, multiple dilutions of
materials to ppt levels are tedious and prone to inaccuracies. Calibration
problems were cited by The World Meteorological Organization (1982) as a
factor responsible for the discrepancies in tropospheric measurements by
different investigators. In order to overcome the difficulties in generating
low-ppb primary standards of MC, Singh et al., (1981) has reported that perme-
ation tubes offer the most accurate means. Permeation tubes were conditioned
for at least two weeks and, during standard generation, were kept in holders
maintained at 70 ± 0.1°C. Error in the permeation rate (980 mg/min) was ± 15
percent. A reproducible means of generating low-ppb primary standards was
3-7
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judged essential since there are considerable difficulties in storing long-
term secondary standards. Various investigators have used GC-ECD systems to
quantitate MC in ambient air samples (Cronn et al., 1976; 1977; Lillian and
Singh, 1974; Rasmussen et al., 1977; Singh et al., 1977c). An interlaboratory
calibration (Cronn et al., 1976) of six real ambient air samples gave a preci-
sion (percent standard deviation) of 11.4 ppt. A similar comparison calibra-
tion was reported by Singh et al., (1981). An overall accuracy of ± 15 percent
was reported for the preparation of secondary standards (Singh et al, 1982).
Rasmussen and Khali! (1981a) reported that primary standards of MC (~ 139
and 52 ppt) remained stable for up to about one year, even though the pressure
in the master tanks had decreased significantly. More recently, Rasmussen and
Khali! (1982) have found MC to be stable up to 2 years in internally passi-
vated (SUMMA®) stainless steel flasks.
Because the ionization efficiency of MC in the EC detector is only 20
percent, use of dual EC detectors in series have been used to achieve higher
accuracy (Lillian and Singh, 1974). The dual detection system, as employed by
Singh et al. (1977c), enables MC to be quantitated coulometrically according
to the following equation:
Coulombs = 96,500 pW;
where
p = ionization efficiency
and W = moles of compound
Determination of the ionization efficiency can be made by use of the following
expression which relates p to the signals (X-, and X2) of two identical EC
detectors in series:
X
P = I ~ IT
Xl
X-j^ = 96,500 pW
X2 = p (96,500W - 96,500 pW)
The ionization efficiency should be determined for the operating conditions
and once established, can be used in the coulometric calibration.
3-8
-------
The accuracy of the GOECD approach has been reported by Singh (1977a) to
be better than 10 percent for compounds such as MC that have ambient air
-3 3
mixing ratios of 20 ppt (0.108 x 10 mg/m ) or greater. An overall system
accuracy of ± 10 percent was reported by Singh et al. (1977a) in analyses of
MC in ambient air samples. Two gas chromatographs, each equipped with two EC
detectors, were employed. An ascarite trap was placed between the GC column
and the EC detector to prevent moisture interference. Precision was reported
to be better than 5 percent and instruments were calibrated using multiple
dilutions of pure material. Singh et al., (1981) recently reported linearity
of dual frequency-modulated ECD's over a wide concentration range for MC, down
to low-ppt levels.
3.1.2.3 Standard Methods—The analytical method S335, suggested by NIOSH for
organic solvents in air, utilizes adsorption on charcoal followed by desorp-
tion with carbon disulfide. The resulting effluent is analyzed by gas chroma-
tography. This method was recommended for the range 96 to 405 ppm (518 to
3 •
2,187 mg/m ). Interferences are minimal and those that do occur can be elimi-
nated by altering chromatographic conditions. However, one disadvantage is
that the charcoal may suffer breakthrough, thus limiting the amount of air
that can be sampled. This can be predicted, however, and multiple tubes can
be used. Tubes could also be replaced at predetermined frequencies after
shorter sampling periods to avoid breakthrough.
3.2 PRODUCTION, USE, AND EMISSIONS
Methyl chloroform is principally used for the cold cleaning and vapor
degreasing of fabricated metal parts. Because of its volatility and dis-
persive use pattern, much of the MC produced worldwide is .emitted into the
atmosphere. There are no identified natural sources of emissions. Once in
the atmosphere, MC is subject to atmospheric transport and transformation. To
better assess the effects of present and future emissions of MC on human health
this section profiles MC production, usage, and emissions.
3.2.1 Production
Methyl chloroform is produced by several manufacturing processes (Jordan,
1979; Lowenheim and Moran, 1975; U.S. Environmental Protection Agency, 1979b):
3-9
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1. Hydrochlorination of 1,1-dichloroethylene (vinylidene chloride)
Fed 3
CH£ =
HC1
CH3CC13
2. Hydrochlorination of vinyl chloride
FeCU
CH2 = CHC1 + HC1
CH3CHC12 -ff
CH3CHC12
HC1
3. Chlorination of ethane
CH3CH3 + 3C12 370° to 480°C3 CH3CC13 + 3HC1 + byproducts
In order to prevent catalyst deactivation, hydrochlorination reactions
should be anhydrous (Jorden, 1979). Temperature control of thermal reactors
for Chlorination must be maintained to ensure MC stability (Jordan, 1979). In
reaction (3) above, ethyl chloride and vinylidene chloride are byproducts.
Increased yields of MC can be obtained through recycling of these byproducts.
In 1975, over 60 percent of the U.S. production of MC was derived from
the hydrochlorination of vinyl chloride; derivation from vinylidene chloride
accounted for 30 percent (Lowenheim and Moran, 1975).
In 1977, the U.S. production figure for MC was 288,565 metric tons (U.S.
Environmental Protection Agency, 1979b; U.S. International Trade Commission,
1977). The major producers and their production capacities for 1977 are shown
in Table 3-2. Production of MC in the United States for 1980 was reported as
314,668 metric tons (U.S. International Trade Commission, 1980). Thus, over
the 1977 to 1980 period, production increased at an annual rate of about 3
percent. Quoting production estimates supplied by Dow Chemical U.S.A., Singh
and coworkers (1979a) reported that global production of the chlorocarbon had
been increasing at an annual rate of 12 percent. Estimates did not take into
account production in the Soviet Union and other eastern European countries.
The Dow Chemical estimate for global production in 1977 was 470,000 metric
tons. The annual production growth rate through 1977 was reported to be 7 to
10 percent by trade sources (Chemical and Engineering News, 1979). Future
growth in production depends on the status of other chlorinated solvents under
regulation for which MC may be substituted and upon economic conditions.
3-10
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TABLE 3-2. MAJOR PRODUCERS OF METHYL CHLOROFORM. (Cogswell, 1978)
Organization
Dow Chemical , Freeport, TX
PPG Industries, Inc., Lake Charles, LA
Vulcan Materials Co. , Geismar, LA
1978
204,000
79,000
29,000
Capacity
(Metric Tons)
1982*
204,500
159,000
90,000
*These capacity estimates were provided in the 27 September 1982 Chemical
Marketing Reporter and included production units on standby.
3.2.2 Usage
In 1975, cold cleaning and vapor degreasing operations accounted for 75
percent of the total end use of MC. Approximately 12 percent was used in the
synthesis of vinylidene chloride (Lowenheim and Moran, 1975). Use in metal
cleaning operations was reported to total 67 percent of 1977 production (Cogs-
well, 1978).
MC is also used as a solvent in adhesive formulations, as a spot remover,
as a film cleaner, and as an additive in metal cutting oils (Jordan, 1979;
Keil, 1979; Lowenheim and Moran, 1975). Most commercial formulations of:MC are
stabilized in order to: (1) prevent or retard oxidation, (2) chelate metal
ions, (3) scavenge HC1, and (4) passivate metal surfaces (Jordan, 1979).
Vapor degreasing grades of MC contain 3 to 7 percent (w/w) stabilizers and
additives (Jordan, 1979).
The most commonly used commercial stabilizers are: 1,4 dioxane, 1,3-
dioxolane, butylene oxide, methyl ethylketone, isobutylalcohol, nitromethane,
and nitroethane (Torkelson, 1982).
3.2.3 Emissions
Emissions of MC arise during primary and end product production, during
dispersive use applications, from storage containers, and during disposal of
waste materials.
Singh et al. (1979b) have estimated global emissions in 1977 at 95 percent
of the 300,000 metric tons produced. Lovelock (1977a) estimated that 500,000
metric tons were released globally in 1975. The estimate of the global emis-
sions rate in 1976 by the National Research Council (1979a) was about 439,000
3-11
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metric tons. The WMO (World Meteorological Organization, 1982), using data
reported by Neely and Agin (U.S. E.P.A., 1980), estimated current global
annual releases at 476,000 metric tons.According to McCarthy (1975), almost
all the MC produced is eventually released to the atmosphere.
Recent estimates in a report prepared for the U.S. Environmental Protec-
tion Agency, Office of Toxic Substances, placed 1978 nationwide emission
losses to air at 214,000 metric tons (Katz et al., 1980). A slightly higher
rate (245,000 metric tons) for nationwide emissions from all sources for 1978
was reported by Anderson et al. (1980) in a study prepared for the U.S. Envi-
ronmental Protection Agency, Office of Air Quality Planning and Standards.
Emission estimates by source category are shown in Table 3-3.
TABLE 3-3. 1978 EMISSION LOSSES TO AIR
Source
1978 emission losses to air in
million pounds (kkg) and
percentage of total released to air
Production
1. From Vinyl Chloride
2. From Vinylidene Chloride
3. From Ethane
Metal Cleaning
Aerosols
Adhesives
Texti 1 es
Paints
Inks
Drain Cleaners
Pharmaceuticals
Film Cleaning
Leather Tanning
Catalyst Preparation
Miscellaneous
Total released to the air*
0.21
0.15
0.03
351.70
39.90
38.37
6.44
10.91
6.13
0.61
0.27
0.48
0.23
0.06
14.82
470
(95)
(67)
(14)
(159,500)
(18,100)
(17,400)
(2,920)
(4,950)
(2,780)
(278)
(124)
(218)
(104)
(28)
(16,730)
(213,300)
0.04%
0.03%
0.01%
75%
8%
8%
1%
2%
1%
0.1^
0.06%
0.1%
0.05%
0.01%
3%
(100%)
*An additional 63 million pounds (28,602 kkg) are released to the environment
via solid waste and water.
Adapted from Katz et al. (1980).
Based on annual measurements of global MC since 1975, Rasmussen et al.
(1981a) and Khali 1 and Rasmussen (1981) have determined that emissions of MC
since 1974 have increased at an exponential rate of 8 percent per year. This
3-12
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is somewhat less than half the previous rate. The average rate of accumula-
tion since 1975 was calculated at 12.4 ± 1.5 percent per year.
3.3. ATMOSPHERIC TRANSPORT, TRANSFORMATION AND FATE
The relation of ambient air mixing ratios of MC to the likelihood of
health effects in humans is influenced by many theoretical processes that may
occur in the troposphere and stratosphere. Such processes include: transfor-
mation of MC into other potentially harmful atmospheric components; stability
of such components; urban transport; tropospheric chemical reactivity; and
diffusion into the stratosphere where MC participates in ozone (0~) perturba-
tion reactions.
3.3.1 Tropospheric Removal Mechanisms and Residence Time
Prior to 1977, it was commonly believed that the residence time (i.e. the
average time between release to and removal from the atmosphere) was in the
range of 1 to 2 years (National Research Council, 1976). This residence time
was derived from estimates of globally averaged hydroxyl radical (OH) levels
in the range of 10 X 10 to 30 X 10 molecules cm"3. Reliable estimates of
globally averaged MC levels were not available. It is important to note that
reaction with OH is the principal tropospheric removal mechanism by which many
compounds, including MC, are scavenged from the troposphere.
More recent modeling estimates of the loading of the troposphere by MC
have led to refined estimates of OH mixing ratios and thus, suggest that the
residence time of MC is more likely to be in the range of 5 to 10 years. The
World Meteorological Organization (1982) recently reviewed the data base and
found that, given the uncertainties in release rates and absolute concentra-
tions of MC as discussed by Logan et al. (1981), the current mixing ratios
observed for MC appear to be consistent with a lifetime of 5 to 10 years and
with a globally averaged OH mixing ratio of about 7 X 105 molecules cnf3
This estimate was, in part, based on the redeterminations of the rate constant
for reaction of OH with MC by Kurylo et al. (1979) and Jeong and Kaufman
(1979). The lack of knowledge concerning MC emissions from Eastern Bloc
countries was also a factor that had been considered. Since OH distribution
has only been estimated through the use of models, estimates for the atmos-
pheric lifetime of MC should be viewed cautiously.
3-13
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Support for this estimate by the World Meteorological Organization can be
seen in the data of Rasmussen and Khalil (1981b). These investigators reported
that tropospheric measurements of MC made at six locations in the Northern and
Southern hemispheres, between January 1979 and June 1980, were consistent with
a lifetime in the range of 6 to 10 years. Average concentrations over both
hemispheres were calculated from latitudinal profiles.
Using reaction rates current for 1981, Logan et al. (1981) calculated a
global lifetime of 5 years, by modeling MC mixing ratios over time and latitude
and with altitude profiles of OH averaged over an annual cycle. The model
results agree with those of Campbell (U.S. Environmental Protection Agency,
1980) in that MC removal is particularly sensitive to OH levels in the tropics,
a region that accounts for about 70 percent of the global sink. Approximately
50 percent of the global sink is calculated over the tropical ocean. It was
emphasized that one-dimensional models are inappropriate for calculating the
lifetime of a compound such as MC. The model of Logan et al. (1981) calcu-
fi ~3
lated a 'mean OH level in the northern hemisphere of 1 x 10 molecules cm .
5 3
This is somewhat higher than the earlier estimates (2.5 x 10 per cm ) calcu-
lated by Crutzen and Fishman (1977).
Derwent and Eggleton (1981), using a two-dimensional model, calculated a
global lifetime of 4.8 years. Derwent (1982) has recently evaluated global,
hemispheric, and one-dimensional models in comparison to a two-dimensional
model. This report discusses the limitations and strengths of the various
types of models used to estimate residence time and thus, the inherent dif-
ferences between models in relation to estimates of stratospheric ozone changes.
Methyl chloroform was one of three halocarbons chosen for the evaluation. It
was concluded that the simpler model, with averaged chemical reaction rates
and parameterization of transport processes, tend to under- or overestimate OH
oxidation thus leading to errors in calculation of residence time. With
respect to MC, however, both one- and two-dimensional models are in agreement
with respect to atmospheric lifetime, 4.3 ^nd 4.8 years, respectively. In
contrast, hemispheric and global models yielded a lifetime of about 9 years.
Singh and coworkers (1979b) found that field measurements support a 6 to
8 year global average residence time. Earlier, Singh et al. (1977a, 1977b)
computed a global average residence time between 8 and 11 years, based upon
the observed tropospheric distribution of MC and the rate of reaction with OH
accepted at the time. Estimates from a number of other investigators, using
different approaches, are consistent with a 5 to 12 year range (Altshuller,
1980; Chang and Penner, 1978; Lovelock, 1977b).
3-14
-------
In conclusion, the estimates of the atmospheric lifetime of MC, as deter-
mined by reaction with OH, should be viewed cautiously given the limitations
and uncertainties inherent in the various models. However, unless new infor-
mation is forthcoming, the estimate of 5 to 10 years is consistent with the
state-of-the-art knowledge of the troposphere.
3.3.2 Impact Upon the Ozone Layer
Current understanding of stratospheric science suggests that several
substances produced by human activities may affect stratospheric 0_. A major
O
goal of atmospheric scientists is to determine the net effect of all these
substances on 03 simultaneously.
The National Research Council (1982), drawing upon the data base developed
by the World Meteorological Organization (WHO, 1982), recognizes MC to have
the potential to perturb stratospheric 0, levels. This potential was not
expressed in quantitative terms as had been done for the two principal chloro-
fluorocarbons believed to be the most important, namely, dichlorofluoromethane
and trichlorofluormethane (CF2C12 and CFClp. The Council has stated that
"The abundance of ozone in the stratosphere is determined by a dynamic balance
among processes that produce and destroy it and transport it to the troposphere."
Thus, the actual role of MC in stratospheric processes can best be elucidated
through an examination of multiple perturbation scenarios involving all the
known key contributory factors (National Research Council, 1982). The poten-
tial of MC, however, can be examined in a context apart from other perturba-
tion processes. In this manner, important information concerning the atmos-
pheric chemistry of MC can be used to develop more representative multiple
perturbation simulations. The development of such models has been recommended
by the Council to describe the combined effects of all relevant compounds on
stratospheric 0_.
A tropospheric lifetime of 5 to 10 years for MC, as estimated by the WMO
(World Meteorological Organization, 1982) suggests that a portion of the
amount of MC released to the atmosphere reaches the stratosphere. Singh et
al. (1982) have estimated that as much as 15 percent of the MC released enters
the stratosphere. The National Research Council (1979a), using 1976 global
release rates, estimated that about 12 percent reaches the stratosphere.
Photodestruction of MC in the stratosphere would increase the atomic chlorine
burden, thus accelerating 03 destruction. Preliminary information reported by
Fabian et al. (1981) suggests that MC is rapidly decomposed in the strato-
sphere. This mixing ratio profile awaits verification.
3-15
-------
Reduced 0- concentrations associated with MC photodestruction could
O
result from the following reaction mechanisms:
hv
un~uui~
n .in
Cl- i 03
pin. in
LIU* ' U
nn. -4- Mn
3 nn + n
pi . + n
> m . + NO
(1)
(2)
(3)
(4)
The atomic chlorine produced in reaction (1) would react with 03 to yield
chlorine oxide. The subsequent chain reaction, if not counterbalanced, could
result in a continual depletion of Og.
The modelling results shown in Table 3-4 indicate the efficiency of MC to
reduce 0- relative to other stratospherically important compounds (U.S. Envi-
ronmental Protection Agency, 1980). It must be noted that such results do not
take into account multiple perturbations that occur, some of which could
offset the calculated effects of MC.
TABLE 3-4. RELATIVE EFFICIENCY OF HALOCARBONS IN REDUCING STRATOSPHERIC OZONE
(U.S. Environmental Protection Agency, 1980)
Compound
CFC-11
CFC-12
CFC-113
CFC-114
MC
Percent ozone depletion
after 320 years*
-10.7
-8.5
-8.25
-5.38
-0.933
-1.6b
^production rate assumed, for each species individually, at 1,000 million
pounds annually
abased on 4.5 year lifetime
based on 9 year lifetime
3-16
-------
The World Meteorological Organization (1982), calculated an estimated
steady-state depletion of the total 03 column due to only MC of 0.8 percent.
This estimate included an assumption of continued release at the current
estimated emissions rate, for MC and relied upon the one-dimensional model of
the Lawrence Livermore Laboratory (Wuebbles and Chang, 1981) that incorporated
revised reaction rate kinetics. It should be noted that other scenarios
reported by the WMO, involving other reactive species, did not show a calcu-
lated net reduction in total column 0,.
tj
The recent model calculations of Owens et al. (1982) suggest that natural
and anthropogenic emissions of methane may significantly moderate the 0.,
destroying potential of chlorofluorocarbons. Using a I-D model with chemical
reaction rates and incident solar fluxes recommended by WMO (1982), Owens and
coworkers have calculated that a doubling of methane, if viewed in isolation,
can lead to a total column 03 increase of 3.5 percent. When coupled with
chlorofluorocarbons, the calculation shows an overall total column 0, change
O
of -1.6 percent. Khali! and Rasmussen (1982) also recently suggested that an
increase in the atmospheric levels of methane may serve to protect 0., levels
O
in the stratosphere. After examining measurement data as far back as 1965,
they find the data consistent with a rate of increase in methane ranging from
1.2 to 2 percent per year.
Although a variety of scenarios involving multiple perturbations have
been evaluated by the WMO (1982), it is difficult to assess with confidence
the actual effect of MC in quantitative terms. To date, measurements of
stratospheric 03 have not detected any depletion. The current levels of
calculated 03 depletion are too small to be observed by existing techniques,
which could detect a 2 percent change in total column 0, if it occurred.
«j
Among the confounding factors are the uncertainties and limitations of the
models and the complexity of rapidly-changing knowledge in atmospheric chem-
istry. The NRC (National Research Council, 1982) stated that "These results
should be interpreted in light of the uncertainties and insufficiencies of the
models and observations." An evaluation of the impact of MC upon stratos-
pheric 03 must take into account all factors affecting atmospheric processes
if realistic estimates of 0., perturbation are to be made.
3.3.3 Laboratory Studies
In experiments designed to characterize the UV absorption spectrum for MC
in the wavelength and pressure ranges associated with the stratosphere, Van-
3-17
-------
laethem-Meuree et al. (1979) found that photo-dissociation was the dominant
sink process at altitudes above 25 kilometers.
In Spence and Hanst's (1978) photooxidation studies, when MC was irradi-
o o
ated at maximum intensities of 3100 A and 3650 A, one-fifth of the initial
amount (10 ppm) was consumed after 6 minutes. The principal products observed
were carbon monoxide (1.5 ppm), hydrochloric acid (6 ppm), and phosgene (2
ppm).
The recent reaction rate studies, involving MC and OH, by Jeong and
Kaufman (1979) and by Kurylo et al. (1979), indicate that the rate of this key
reaction is slower and more temperature sensitive than previously had been
indicated. Jeong and Kaufman (1979) measured reaction rates using the dis-
charge-flow method and MC which had been extensively purified. A reaction
-14 3 -1
rate at 293°K was determined at 1.06 ± 0.09 x 10 cm sec . The Arrhenius
expression, within 95 percent confidence limits but not including systematic
errors, was 5.49 ± 1.40 x 10~12 exp [ - (1832 ± 98/T] cm3 sec'1. The authors
indicated that the most recent rate (at 298°K) reported by JPL (Jet Propulsion
Laboratory, 1979), 1.9 x 10 cm sec , is too high by a factor of about 1.7
at this temperature, and by a factor of 1.9 at 265°K, the temperature used by
Singh (1977) in deriving average OH concentrations.
The Arrhenius expression determined by Kurylo et al., (1979) was in close
agreement with that determined by Jeong and Kaufman (1979). Kurylo et al.
(1979) used the flash-photolysis method and also extensively purified the MC
prior to use.
3.4 MIXING RATIOS IN THE ATMOSPHERE
3.4.1 Global Atmospheric Distributions
Before 1977, reliable estimates of globally averaged MC concentrations
were not available. Recent measurements, however, indicate that the global
average concentration is consistent with a long (5 to 10 years) tropospheric
lifetime. Point measurements of MC mixing ratios in the troposphere are shown
-3
in Table 3-5. Typical ambient levels are in the 0.1 to 1 ppb (0.54 x 10
to 5.4 x 10~3 mg/m3) range (Brodzinsky and Singh, 1982). These authors have
evaluated the quality of all ambient data reported for MC between 1970 and
1980 and consider it to range from good to excellent. Compared to the levels
reported in Table 3-5, dispersion models that have been used to estimate popu-
lation exposure to ambient MC predict the maximum annual average to which
3-18
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5 LATITUDE, degrees t
Figure 3-1. Global distribution of methyl chloroform.
Source: Singh et al. (1979a).
people may be exposed is 9.2 ppb (0.050 mg/ms). Exposure to atmospheric
emissions from degreasing operations, the source category of greatest nation-
wide emissions, is not expected to exceed 4.6 to 9.2 ppb (0.025 to 0.050
mg/m3) averaged over a year's time. These predicted values are lower than
those levels reported (for shorter averaging times) at some urban sites in the
United States.
The global average background mixing ratio of MC has been determined by
several laboratories, including SRI International and Washington State Univer-
sity. The field studies of Singh and co-workers at SRI have been reported in
a number of publications (Singh, 1977a; Singh et al. , 1977c; 1978a; 1978b;
1978c; 1979a; 1981; Singh et al., 1982). The global average for late 1977 was
reported as 95 ppt (Singh et al., 1979a). This average was derived from
measurements made in the northern (average = 113 ppt) and southern (average =
77 ppt) hemispheres. Based on data collected at latitudes 30°N to 40°N from
1975 to 1978, MC presence in the atmosphere was increasing at the rate of 15
ppt (or 17 percent) per year, during this time period.
3-24
-------
Field studies by Singh et al. (1979a) were conducted during two oceano-
graphic cruises between latitudes 64°N and 40°S (15 September to 30 October
1977; 20 November to 13 December 1977). Samples were collected in electro-
polished stainless steel and glass vessels and analyzed by GC-ECD, operated
coulometrically. Samples were analyzed i_n situ as well as after storage. The
northern temperate average mixing ratio was reported as 123 ppt. The latitu-
dinal distribution is shown in Figure 3-1.
Rasmussen and Khali1 (1981b) reported that tropospheric levels of MC at
six locations, in the northern and southern hemispheres between January 1979
and June 1980 were consistent with an estimated lifetime in the range of 6 to
10 years. Analysis was made by EC/GC. From these measurements, a global
average concentration of 115 ppt was calculated along with an annual increase
of 6.7 ± 2.0 percent. This global average is consistent with the values
reported by others for previous years. In contrast, however, to the rate of
increase reported by Singh et al. (1979a) for the period 1975 to 1978, the
rate of increase appears to be declining, which Rasmussen and Khalil (1981b)
attribute to an overall reduction in the rate of emissions. The hemispheric
difference in concentration reported by Rasmussen and Khalil (1981b) is con-
sistent with that reported by Singh et al. , (1979b). During the period from
January 1979 to December 1980, the northern hemisphere average was 130 ppt
compared to the southern hemisphere average of 99 ppt (Rasmussen and KhaliT,
1981b).
Rasmussen and Khalil (1982) have obtained new estimates of the average
mixing ratio of MC in both hemispheres, based on tropospheric air samples
collected during the 1978 Project GAMETAG. Based on these samples, the average
hemispheric levels are 117±4 ppt (Northern) and 90±3 (Southern). Data for the
tropical latitudes indicate that MC levels in boundary air (0.12 to 0.36 km)
are 6.4 percent higher than above it. Data limitations precluded a similar
analysis for Northern latitudes.
The vertical profile and latitudinal gradients of MC in the troposphere
and lower stratosphere have been investigated by Cronn and coworkers in a
series of high-altitude studies (Cronn and Robinson, 1979; Cronn et al.; 1976;
1977a; 1977b). Most recently, experiments conducted at the Panama Canal Zone
(9°N latitude) and at 37°N indicated that MC tropospheric abundance was 18
percent lower at 9°N (Cronn and Robinson, 1979). Singh et al. (1979a) re-
corded a 17 percent difference between these latitudes (Figure 3-1). For July
3-25
-------
1977, the average tropospheric background concentration (139 whole air samples
were collected between ground level and 13.7 kilometers) was 97.3 ±4.6 ppt at
9°N. At 37°N (Pacific Ocean west of San Francisco), an average mixing ratio
of 116 ± 14 ppt was measured. Tropopause height at the Canal Zone was 15.7
kilometers. Methyl chloroform tropospheric mixing ratios did not vary signi-
ficantly with increasing altitude at 9°N but there was a precipitous drop in
the mixing ratio across the tropopause. This observation suggests that model-
ing of MC's role in stratospheric processes requires a consideration of ultra-
violet photoreactivity at all altitudes.
Analysis of pressurized tropospheric samples was performed by a dual
GC-ECD. The detection limit for MC was 6 ppt and the precision of analysis
was ±4.2 percent. Analyses were performed on 5-ml aliquots. For the low-
pressure stratospheric air samples, precision was ±7 percent with a detection
limit of 2 ppt. Methyl chloroform was preconcentrated with a freezeout sample
loop. Overall accuracy of analysis was estimated at about 10 percent.
In experiments conducted at 37°N (Cronn et al., 1977a), samples were
collected at altitudes ranging from 6.1 to 14.3 kilometers with the tropopause
height between 11 and 12 kilometers. Samples were analyzed by GC-ECD utiliz-
ing the freezeout concentration method. The average tropospheric background
mixing ratio for MC in April 1977 (excluding samples within 0.8 kilometer of
the tropopause) was 116 ± 14 ppt. This value is consistent with the results
of Singh et al., (1979a), who obtained a value of 117 ppt at this latitude
during the fourth quarter of 1977 (Figure 3-1).
When this observed mixing ratio (116 ppt) was compared with that obtained
in March 1976 at 48°N (Pacific Northwest), an annual atmospheric increase in
MC of 23.7 percent was calculated. Cronn et al. (1976; 1977a) collected whole
air samples at altitudes ranging from 4.6 to 14.6 kilometers (average tropo-
pause height was 10.8 kilometers) during March 1976 over Western Montana and
Idaho. The average tropospheric background mixing ratio for MC was 94.5 ±8.2
ppt. Samples were analyzed by GC-ECD both isothermally with a 5-ml aliquot
and with the freezeout concentration method. Precision of the overall method
was ±4 percent.
The Washington State University (WSU) group has made several studies by
aircraft of the latitudinal distribution of MC (Cronn, 1980a; Robinson, 1978;
Robinson and Harsch, 1978). Interhemispheric differences of MC in the tropo-
sphere in June, 1976, were reported, with northern levels of 96 ppt and south-
ern levels of 88 ppt (Robinson and Harsch, 1978). Lower stratospheric levels
3-26
-------
were 80 and 67 ppt, respectively. Work begun at WSU in 1976, and continuing
at both WSU and the Oregon Graduate Center, has provided annual measurements
of interhemispheric differences of MC of 1.67, 1.72, 1.56, 1.40, and 1.45 for
1975, 1976, 1977, 1978, and 1979, respectively (Khalil and Rasmussen, 1980;
Robinson, 1978). Time-trend monitoring has been conducted nearly continuously
at a ground station in eastern Washington state since July 1977 (Cronn, 1980a).
Increases have exceeded 12 percent per year during that time.
Point measurements made by Lovelock (1977b) during 1972 to 1977 resulted
in a lower global average background mixing ratio (73 ppt). Air samples
collected at rural sites in the British Isles indicated that, during the 5
year period, MC mixing ratios increased from 31 ppt to 97 ppt. In the south-
ern hemisphere (Africa and Antarctica), it increased from 12 ppt to 50 ppt.
Absolute accuracy of the GC-ECD method was reported as ±30 percent.
In field measurements of ambient MC levels over North America (18°N to
65°N) from October 4 to 13, 1976, Pierotti and co-workers (1980) determined an
average tropospheric mixing ratio of 145 ± 25 ppt for this region. The mixing
ratio dropped sharply across the tropopause and in the stratosphere, suggest-
ing a large sink at these altitudes. These investigators suggested that the
data were consistent with a northern hemisphere background mixing ratio of
about 100 ppt.
3.5 LEVELS FOUND IN WATER
Groundwater data available from state sources indicate that 23 percent of
the 1,611 wells tested contained MC (Coniglio, 1981). Concentrations of 344
ppb were measured in discharge waters from a MC manufacturing facility (Battelle,
1977). Chow (1981) reported a level of 700 ppb in testing of residential
wells near a manufacturing site. The highest level reported for drinking
water is 17 ppb (Battelle, 1977). Chi an and Ewing (1977) have examined surface
water at various sites in the United States and found that 11 of 204 sites had
MC levels greater than 6 ppb. The maximum level of MC found was 8 ppb.
Bellar et al. (1974) reported that the influent level of MC to a municipal
sewage treatment plant was 16.5 ppb; upon treatment, the level dropped to
9.0 ppb. Contamination of community drinking water supplies by MC and other
halogenated solvents used as components of cesspool cleaners was reported
(U.S.E.P.A., 1979b). It was cited that levels of MC in observation wells were
3-27
-------
as high as 5,000 ppb. The maximum level of MC found in the contaminated
drinking water wells was 310 ppb. Given the high volatility of MC and the
generally low levels expected in natural aquatic environments, MC is expected
to be nonpersistent.
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3-36
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3-37
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-------
4. METABOLIC FATE AND DISPOSITION
4.1 ABSORPTION, DISTRIBUTION, AND ELIMINATION
Methyl chloroform (MC; 1,1,1-trichloroethane) is currently one of the
most widely used of the chlorinated aliphatic hydrocarbon solvents. Since its
introduction in the mid-1950's as a cold cleaning solvent substitute for
carbon tetrachloride, MC has gained recognition as being among the least toxic
of the chlorinated aliphatic hydrocarbons (Chemical Marketing Reporter, 1982)
and is increasingly replacing other supposedly more toxic chlorinated solvents
such as trichloroethylene (Torkelson et al. , 1958; Prendergast et al. , 1967;
Stewart, 1968). The fact that its isomer, 1,1,2-trichloroethane, is markedly
more toxic than MC (Hardie, 1964; Irish, 1963; Browning, 1965; Carlson, 1973),
is in agreement with the general observation that chlorinated ethanes having
chlorines on both carbon atoms are considerably more soluble in water, blood,
and lipid (Table 4-1). These physical properties could be responsible in part
for the manifestations of differences in chlorinated hydrocarbon toxicity
(Sato and Nakajima, 1979; Clark and Tinston, 1973).
TABLE 4-1. PARTITION COEFFICIENTS OF METHYL CHLOROFORM AND OTHER SOLVENTS
AT 37°C
Compound
1,1,1-Trichloroethane
1 , 1 , 2-Tri chl oroethane
1,1-Dichloroethane
1,2-Di chl oroethane
Trichloroethylene
Tetrachl oroethyl ene
Dichloromethane
Chloroform
Carbon tetrachloride
Vapor Press
torr at 25C
125
25
250
80
436
20
400
250
100
Water
Air
0.93
17.1
2.7
11.3
1.3
0.43
7.6
3.5
0.25
Olive Oil
Air
356
2273
187
447
718
1917
152
401
361
Blood
Air
3.3
38.6
4.7
19.5
9.5 _
13.1
9.7
10.3
2.4
Olive Oil
Blood
108
59
40
23
76
146
16
39
150
Adapted from Sato and Nakajima, 1979.
4-1
-------
4.1.1 Oral and Dermal Absorption
While limited absorption of MC vapor through the lungs is the common
route of entry into the body, MC is also rapidly and completely absorbed from
the gastrointestinal tract (Stewart, 1971). Stewart and Andrews (1966) re-
ported an instance of non-fatal acute intoxication after oral ingestion of a
liquid ounce of MC (0.6 g/kg body weight,BW). The concentration of MC in the
expired air was measured serially and found equivalent to an inhalation exposure
o
of experimental subjects to 500 ppm (2,700 mg/m ). On the other hand, MC
vapor is poorly absorbed through intact skin and, unless it is trapped against
the skin beneath an impermeable barrier, it is unlikely that toxic quantities
can be absorbed (Stewart, 1971; Stewart and Dodd, 1964). Stewart and Dodd
(1964) demonstrated, with continuous immersion for 30 min of the thumbs or
hands of volunteers, that the solvent penetrates the skin, enters the blood
circulation, and is excreted through the lungs in exhaled air. Fukabori and
coworkers (1976, 1977) attempted a quantisation of cutaneous absorption in
humans. Systemic absorption was evaluated by measurement of MC in blood and
exhaled air and its biometabolites [trichloroethanol (TCE) and trichloroacetic
acid (TCA)] in urine. After applications of the solvent to the skin of the
2
forearm in a circumscribed area (12.5 cm ) for 2 hours a day for 5 consecutive
days, or immersion of the hands 11 times a day for 10 minute periods, the MC
concentrations in exhaled air and urinary metabolites corresponded roughly to
3
a 2-hour inhalation exposure to 10-20 ppm (54 to 108 mg/m ) MC in ambient air.
The investigators concluded that absorption through the skin for workers in
direct contact with liquid MC may add to the absorption from vapor exposure.
Additional evidence that MC absorption via skin can be a source of exposure
is provided by the results of Jakobson et al. (1982). Application of a stabilized
formulation of MC to the skin (3.1 cm) of anaesthetized guinea pigs resulted
in peak blood levels (1.9 ug/ml) 30 minutes after exposure. Blood levels then
declined to 0.66 ug/ml 6 hours after exposure MC was applied at two sites on
shaved skin under an occlusive barrier. With elimination studies, using
percutaneous exposure 4 hours in duration, elimination from the blood was
consistent with a two-compartment model exhibiting nonlinear kinetics. During
exposure, no visible skin reactions were noticed at the application sites. In
comparison, Astrand (1975) obtained a level of 4 ug/ml arterial blood after a
3
30-minute exposure of humans to 245 ppm (1,323 mg/m ).
4-2
-------
4.1.2 Pulmonary Uptake and Body Burden
Several studies have been made of the pharmacokinetics of MC pulmonary
uptake, distribution in the body, and metabolism after exposure to low inha-
lation concentrations approximating the accepted TWA concentration. Informa-
tion available from these "studies concerns single exposures under experi-
mentally controlled conditions with animals or human volunteers. Little
information is available regarding long-term exposure, such as may occur in
the workplace or in the environment. Recent studies have greater reliability
than earlier studies because of the availability of gas chromatographic methods
for the analytic determination of MC and its metabolites in alveolar air, body
fluids, and tissues (Schumann et al., 1982a,b; Monster et al. , 1979b; Monster
and Boersma, 1975; Astrand et al., 1973; Eben and Kimmerle, 1974; Briemer et
al., 1974; Humbert and Fernandez, 1976, 1977; Stewart et al. , 1969), although
spectrophotometric methods based on adaptations of the Fujiwara reaction are
still widely used by many investigators for determining urinary excretions of
trichloroethanol (TCE), trichloroacetic acid (TCA), or total urine chloro-
derivatives of MC (Ogata et al., 1974; Imamura and Ikeda, 1973; Tanaka and
Ikeda, 1968; Ikeda and Ohtsuji, 1972).
Inhaled MC rapidly equilibrates with arterial capillary blood across the
lung alveolar endothelium (Astrand, et al., 1973). The rate of pulmonary
uptake or absorption depends largely on the solubility of MC in blood (Ostwald
solubility coefficient) and hence the blood/air partition coefficient (Table
4-1). Vapors with a high partition coefficient are absorbed into the body
readily and may exhibit selective partitioning into various tissues. Con-
versely, vapors with a low partition coefficient are expected to approach
steady-state tissue levels slowly. This is likely since partition occurs more
rapidly with venous blood and alveolar air, and thus vapors are more rapidly
eliminated into expired air following exposure. In comparison with other
common solvents in Table 4-1, MC possesses a relatively low blood/air parti-
tion coefficient of about 3.3 at 37°C.
The magnitude of MC uptake (dose, burden) into the body is related to the
following factors: 1) concentration of MC in the insp-ired air; 2) duration of
exposure until steady-state is reached; 3) pulmonary ventilation during ex-
posure; 4) blood/ air partition coefficient; 5) rates of diffusion into, and
solubility in, the body tissues; 6) total body-lipid repository, and 7) meta-
bolic rate. Consequently, during exposure at a given inspired air concentra-
tion, pulmonary uptake and retention is initially large and gradually decreases
4-3
-------
to a minimum steady-state value as total body equilibrium (and body burden)
with inspired air concentration is reached. Under steady-state conditions,
pulmonary uptake balances the pulmonary and other routes of elimination,
including metabolism. For any given breath cycle during exposure, the pro-
portion of MC absorbed and retained by the body is equal to the inspired air
concentration (C,.) minus the end alveolar air concentration (Cft) and, since
pulmonary uptake is a function of inspired air concentration, the percent
retention is:
% retention of inspired air concentration =
CI-CA
CI
x 100
(1)
This value is large at the beginning of exposure, but gradually decreases as
total body equilibrium is approached (Figure 4-1). The percent retention
value is independent of the inspired air concentration. During experimental
3
exposures of subjects to 70 and 140 ppm (378 and 756 mg/m ) MC for 4 and 8
hours, both Monster et al. (1979b) and Humbert and Fernandez (1977) found
retention to be 30 percent of inspired air concentration at an equilibrium
reached after 4 hours of exposure. In a report submitted for publication,
Nolan et al. (1983) reported that 25% of the MC inhaled by six volunteers at
3
either 35 or 350 ppm (139 or 1,890 mg/m ) during a 6-hour exposure, was
retained.
o
<
cc
Ul O
o ui
z cc
2
100
50
EXPOSURE,
II 111
POST EXPOSURE - MC
BLOOD CONC.
10
TIME, hours
20
Figure 4-1. Absorption and pulmonary elimination of MC, and blood
concentration (see text for explanation).
Source: Davidson (1980).
4-4
-------
The total amount (dose, Q) of MC retained In the body during an inhala-
tion exposure can be estimated by multiplying percent retention by the volume
of air inspired during the exposure period, or:
Q = (C: - CA) V
T
(2)
where V is ventilation rate (£/min) and T is exposure period (min). Since the
retention value decreases exponentially during the exposure until equilibrium
is reached (Figure 4-1), either experimental measurements of retention at
frequent intervals during exposure (Monster et al., 1979b) or integration over
the experimentally determined retention function is required (Humbert and
Fernandez, 1977). The exponential nature of the retention curve is due to
first-order kinetics of saturation of body compartments with MC.
Table 4-2 shows the amounts of MC absorbed into the body during inhala-
tion exposures for volunteers from the studies of Monster et al. (1979b) and
Humbert and Fernandez (1977). Monster and his coworkers estimated pulmonary
uptake of MC by multiplying the minute volume by percent retention during
single 4-hr exposures to 70 ppm and 145 ppm (378 and 783 mg/m ). They ob-
served a direct proportionality between uptake and inspired air concentrations
of MC. Similar results were obtained by Humbert and Fernandez for volunteers
3
exposed for 8 hours to 72 ppm and 213 ppm (389 and 1,150 mg/m ). Comparison
of pulmonary uptake for 4-hr and 8-hr (Table 4-2) exposures indicates that the
amount of MC retained is also proportional to duration of exposure until a
steady-state is reached. Although the values for uptake determined by Humbert
and Fernandez are 40 percent lower than those of Monster et al, this variation
can be ascribed to differences in the experimental methodologies and average
minute volume. Nonetheless, these experimental results indicate that the body
burden resulting from an 8-hr inhalation exposure to 350 ppm (1,890 mg/m )
(TWA) approximates 1.5 to 2 g of MC for a normal 70-kg man.
The body uptake of MC increases with the duration of inhalation exposure
and with physical work or exercise. Monster et al. (1979b) found that during
3
a 4-hr exposure to 142 ppm (767 mg/m ) MC, with physical activity equivalent
to light physical work (100 watts), pulmonary uptake increased from 429 mg to
538 mg MC, an increase of 25 percent (Table 4-2). This increase was primarily
due to an increase of ventilation from 10.7 £min sedentary to 30.6 £min
with work. While it might be expected from equation (2) that the increased
4-5
-------
TABLE 4-2. ESTIMATED UPTAKE OF MC DURING A SINGLE 4-HR INHALATION EXPOSURE
(AVERAGE BODY WEIGHT, 77 kg; 67 kg LEAN BODY MASS), MONSTER ET AL., 1979b
Subject
A
B
C
D
E
F
Av.
Exposure Concentration
70 ppm
140
240
200
185
200
190
193
(at rest*) 145 ppm
Uptake, mg
305
520
465
395
425
465
429
142 ppm*
with work (100
Uptake,
435
610
540
560
575
505
538
watt)
mg
*Venti1ation minute volume increased from average of 10.7 £/min. at rest to
30.6 £/min during light work.
ESTIMATED UPTAKE OF MC DURING A SINGLE 8-HR INHALATION EXPOSURE
(AVERAGE BODY WEIGHT 74 kg), HUMBERT AND FERNANDEZ, 1977
Subject
BH
JC
JO
Av.
Exposure Concentration*
72 ppm
Uptake, mg
293
274
277
281
213 ppm
921
912
—
917
^Average ventilation minute volume 5.7 1/min.
uptake should be directly proportional to the ventilation rate increment, an
increase in ventilation also tends to increase alveolar elimination of MC from
pulmonary venous blood. Similar observations were made by Astrand et al.
(1973) with volunteers exposed to an MC inhalation concentration of 250 and
350 ppm (1,350 and 1,890 mg/m3) for 30 minutes at rest, alternating with
identical exposure plus 50 watts of light work. Table 4-3 shows that their
subjects responded to this work with a 3-fold increase of pulmonary ventila-
tion, nearly a 2-fold increase in cardiac output, and a 50 percent increase in
arterial blood concentration, which is an index of pulmonary uptake. Also,
end alveolar concentration of MC increased while' retention decreased from 50
4-6
-------
TABLE 4-3. MEAN VALUES AND SEN! FOR 12 MALE SUBJECTS
AT REST AND EXERCISE FOR 30 MINUTE PERIODS
MC
At rest
250 ppm
350 ppm
50 Watt
250 ppm
350 ppm
Ventilation
BTPS
£/mi n
6.6 ± 0.4
6.6 ± 0.4
22.5 ±1.0
21.8 ±1.1
Cardiac
output
A/mi n
5.1 ± 0.4
9.7 ±0.6
Alveolar
cone
ppm
125 + 6
179 ± 13
168 ± 7
239 ± 17
Blood
arterial
cone.
Mg/g
3.0 ± 0.
5.0 ± 0.
4.5 ± 0.
7.2 ± 0.
2
5
2
4
From Astrand et al. (1973).
to 33 percent with work. Therefore, physical activity during MC exposure
increases the uptake, but the increase is not directly proportional to in-
creased ventilation and is self-limited by a compensatory increase of pul-
monary elimination. For solvents like MC, with a low blood/air partition
coefficient, physical activity has a smaller effect on net pulmonary uptake
and body retention than those solvents with a higher blood/air partition
coefficient (e.g., trichloroethylene, perchloroethylene, dichloromethane;
Table 4-1; Monster, 1979).
During inhalation of MC, and in the elimination phase after exposure, the
concentration in arterial blood leaving the lung always is directly propor-
tional to the alveolar concentration (Monster et al. , 1979b; Humbert and
Fernandez, 1977; Astrand et al. , 1973; Gamberale and Hultengren, 1973; Eben
and Kimmerle, 1974). Since at equilibrium the end alveolar air concentration
is proportional to inspired air concentration, blood concentration of MC is
also related to inspired air concentration (Monster et al., 1979b; Humbert and
Fernandez, 1977; Astrand et al. , 1973). This fixed relationship between
alveolar air and blood concentration is defined by the blood/air partition
coefficient for MC. Figure 4-2 illustrates this linear relationship for a
volunteer exposed for 30 minutes to increasing increments of MC concentration
4-7
-------
E
Q.
Q.
cc
til
o
Z
O
o
DC
UJ
CC
8
6
4
0
0
100
200
300
400
ALVEOLAR CONCENTRATION, ppm
Figure 4-2. Relationship between methyl chloroform concentration
in alveolar air and arterial blood. Data from one subject. Product
moment correlation: r = 0.985. Data from one subject exposed to
30-min. periods of MC. Alveolar air samples and arterial blood sam-
ples.
Source: Gamberale and Hultengren (1973).
in inhaled air. The relationship was independent of the duration of exposure
over the time period studied. Eben and Kimmerle (1974) exposed rats to 204
o
ppm (1,101 mg/m ) for 8 hours daily, 5 days a week for 14 weeks and found that
the blood concentration of MC determined immediately after daily exposure
remained constant during the entire 3 month period, suggesting that steady-
state was attained.
The blood/air partition coefficient, as determined from j_n vivo measure-
ments of alveolar air concentration and blood concentration of MC, agrees well
with the i_n vitro value of 3.3 at 37°c determined by Sato and Nakajima (1979)
(Table 4-1). Table 4-3 summarizes the data of Astrand et al. (1973), deter-
o
mined for sedentary men exposed to 250 and 350 ppm (1,350 and 1,890 mg/m ) MC
for 30-min periods alternating with 30-min periods of physical activity. The
ratios of arterial blood concentration (ug/g) to alveolar MC concentration
4-8
-------
remain nearly constant over a 2-fold range of alveolar concentrations, with an
average value of 5. Monster et al. (1979b) estimated from their study of men
exposed to 70 and 142 ppm (378 and 767 mg/m ) MC a blood/alveolar air concen-
tration ratio of 6. In comparison to other solvents (Table 4-1), MC has a
relatively small blood/ air partition coefficient and, hence, for equivalent
ambient air exposure concentrations the blood concentration of MC is propor-
tionally lower than for other solvents.
The amount of MC pulmonary uptake is influenced by total body weight and
also by the total fat content of the body (average body fat = 8 percent of
body weight). The capacity of adipose tissue to absorb MC jm vivo is de-
termined by the product of adipose tissue volume and the lipid solubility of
MC. The olive oil/blood partition coefficient for MC (108 at 37°C) is higher
than for most other structurally related solvents (Table 4-1) and, therefore,
the capacity of adipose tissue for MC is relatively high. However, because of
the low rate of perfusion (5 percent of cardiac output), the time needed to
saturate adipose tissue is large in comparison with that for other tissues.
Also, since the blood/air partition coefficient for MC is lower than that of
other solvents, the amount of MC in adipose tissue (concentration) at the end
of an exposure of similar duration will be relatively lower. Monster (1979)
suggests that because of the high solubility of MC in adipose tissue, tissue
storage of MC under non-steady state conditions will vary with repeated daily
exposures, particularly in obese persons. This concept is supported by the
observations of Savolainen et al. (1977) and Vainio and coworkers (1978), who
exposed rats to MC (500 ppm; 2,700 mg/m3) 6 hr daily for 5 days and determined
MC concentrations in perirenal adipose tissue and in other body tissues. Their
data, shown in Table 4-4, indicate that measureable amounts of MC remained in
perirenal fat tissue 18 hours after the previous exposure of day 4, and mark-
edly increased further with a 6-hr exposure on day 5. The adipose tissue/
blood partition coefficient calculated from these data was 21, as compared to
1.6 for brain and liver tissues. On the other hand, Eben and Kimmerle (1974)
exposed rats for 14 weeks (8 hr daily, 5 day per wk) to 204 ppm (1,101 mg/m3)
MC but failed to find MC in adipose or other tissues and concluded that MC did
not accumulate with chronic exposure. It should be noted that low-level, pro-
longed exposures, such as encountered in the ambient environment, would be
expected to result in steady-state tissue concentrations. Under these condi-
tions no further MC would be expected to partition into lipid-rich tissues.
4-9
-------
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4-10
-------
4.1.3 Tissue Distribution
During exposure to MC, distribution of the compound throughout the body
and the amount concentrated by each tissue is primarily governed by the blood
concentration, the blood perfusion rate, and the affinity of MC for respective
tissues, as determined by individual tissue/ blood partition coefficients. In
comparison with other chlorinated hydrocarbon solvents that are known to be
distributed widely in the body, MC has one of the highest lipid/ blood par-
tition coefficients (108 at 37°C, Table 4-1) and would distribute into all
body tissues, particularly those high in lipid content, such as brain and
adipose tissue (Holmberg et al., 1977). Table 4-4 shows the concentrations of
MC in liver, brain, and adipose tissue of rats exposed to 500 ppm (2,700
mg/m ) in their inspired air for 6 hours daily for 5 days (Savolainen et al.,
1977). Adipose tissue appears to have a partition coefficient with blood of
approximately 20, and brain tissue levels are also greater than blood concen-
tration. In man, MC readily passes the blood-brain barrier, as evidenced by
the concentrations in the brain reported by Caplan et al. , (1976) and in the
cerebrospinal fluid (Larsby et al., 1978). While it has not been demonstrated
directly to cross the placenta! barrier into the fetus, it may be expected to
do so (Laseter and Dowty, 1977) like other highly lipid-soluble haloalkanes.
Trichloroethanol (TCE), a major metabolite of MC, is known to cross readily
into the fetus (Bernstine et al., 1954, 1957). Leighty and Fentiman, Jr. (1981)
have recently provided in vitro evidence that TCE, upon incubation in a rat
liver microsomal system fortified with coenzyme A, appears to conjugate with
palmitic acid. While there is no report of MC occurring in colostrum or milk
of nursing mothers, it may possibly distribute into these compartments because
of their high lipid content.
4.1.4 Pulmonary Elimination
Figure 4-1 shows schematically the time-course of pulmonary elimination
of MC after exposure. At termination of exposure, MC immediately begins to be
eliminated from the body into the lungs with blood concentration and alveolar
air concentration describing parallel exponential decay curves with three
major components. These components represent first-order passive diffusions
of MC from three major body compartments: (1) most rapidly from a vessel-rich
group of tissues (VRG) with high blood flow and high diffusion rate constant
(VRG: brain, heart, kidneys, liver, endocrine, and digestive system), (2) more
4-11
-------
slowly from the lean body mass (MG; muscle and skin) and (3) from adipose
tissue (FG) (Fiserova-Bergerova and Holaday, 1979). The rate constants for
the passive diffusion from VRG, MG and FG compartments are dependent on both
the arterial blood flow/tissue mass and the relative solubilities of MC in the
tissues of these compartments (tissue/ blood partition coefficients). How-
ever, the ranking of half-times (t-,/2) of elimination of MC is VRG < MG <
-------
4.1.5 Elimination by Other Routes
There is no report in the literature of significant elimination of MC by
any route other than pulmonary. MC is poorly soluble in water, even when
compared with other chlorinated hydrocarbons (Table 4-1), and with its high
lipid/water partition coefficient, MC is unlikely to be excreted unchanged in
the urine in any significant amounts. Studies of chlorinated compounds in the
urine after exposure of animals and humans to MC have not demonstrated its
appearance in urine (Schumann et al., 1982a; Monster e,t al., 1979b; Eben and
Kimmerle, 1974; Humbert and Fernandez, 1977; Seki et al. , 1975; Stewart et
al., 1961; Hake et al., 1960). Since highly lipid soluble substances like MC
readily cross into the intestinal lumen, some fecal and flatus excretion can
be expected from inhalation exposures. After controlled inhalation exposures,
Humbert and Fernandez (1977) were able to account for 88 to 100 percent of an
estimated retained dose as unchanged MC in postexposure exhaled air and as
metabolites of MC in urine. However, Monster et al. (1979b) could account for
only 60 to 80 percent; furthermore, they noted that the percentage recovered
decreased with higher exposure doses.
4.2 BIOTRANSFORMATION
MC has long been known to be metabolized to only a very limited extent by
mammals. The generally accepted metabolites of MC -- trichloroethanol (TCE),
TCE-glucuronide, and trichloroacetic acid (TCA) -- are excreted primarily by
the kidney, but very small amounts of TCE (<1 percent) are excreted by the
lungs (Monster et al., 1979b). TCE-glucuronide is also excreted to an unknown
extent in bile (Owens and Marshall, 1955a,b).
Filser et al. (1982) evaluated a variety of compounds, including MC, for
their ability to stimulate the endogenous production of acetone. This study
was based on previous observations that certain haloethylenes caused acetonemia
in rats (Filser and Bolt, 1980). Exposure of rats (Filser et al., 1982) to
1,000 ppm (5,400 mg/m3) MC for up to 50 hours did not stimulate acetone production
as measured by gas chromatographic analysis of exhaled air. Furthermore,
these investigators reported that the metabolic rate of MC was below their
limit of detection. Data supporting this latter observation were not provided.
4.2.1 Magnitude of MC Metabolism
More than 20 years ago, Hake and his coworkers (1960), using 14C-labeled
MC, determined that less than 3 percent of MC is metabolized by rats. More
4-13
-------
recently, estimates of the extent of metabolism in man have been made from
controlled inhalation exposures with unlabeled MC (Seki et a!., 1975; Monster
et al., 1979b; Humbert and Fernandez, 1977; Nolan et a!., 1983). From the
experimentally determined retained dose and the amounts of MC metabolites
excreted into the urine, the percent of the dose metabolized in man is esti-
mated to be no more than 6 percent.
Hake et al. (1960) found that 98.7 percent of a dose of MC given to rats
was eliminated unchanged via the lungs. These investigators injected 1,1,1-
trichloroethane-l-14C (700 mg/kg) intraperitoneally into 3 rats (170-183 g),
which were then placed individually in a Roth metabolism unit with air traps
for 14C-MC (cold toluene), 14C02 (NaOH solution) and finally a trap for "meta-
bolites" (quartz-tube furnace and halogen absorber that converted any halo-
genated hydrocarbon to inorganic halogen and CO,,, and absorbed the halogen).
Each rat was kept in the unit for 25 hours, during which time urine and feces
were collected. At necropsy at 25 hours, blood and tissue samples were analyzed
for total 14C-activity. The C-MC used in the experiments was synthesized by
the investigators and was determined by paper chromatography to be 99 percent
pure, with possible contamination by 0.4 percent 1,1,2-trichloroethane and 0.5
percent 1,1-dichloroethane. Their findings, summarized in Table 4-5, indicate
that metabolism of MC in the rat is limited to about 2 percent of a very large
dose. However, only TCE-glucuronide was identified as a metabolite in the
urine, and the source of the C02 in expired air could not be defined because
of the presence of small amounts of C-l,l,2-isomer and C-dichloroethane in
the dose.
Recently, Schumann and coworkers (1982a) evaluated pharmacokinetic para-
meters of 14C-labelled MC to characterize the disposition of the inhaled
compound in male Fischer 344 rats and B6C3F1 mice. The animals, ranging in
age from 2.5 to 3.5 months, were exposed to 150±9 or 1,500±90 ppm [C ] MC (810
O ~\ A
or 8,100 mg/m ) for 6 hours and elimination of c activity was followed for 72
hours. As other investigators have found, Schumann and coworkers also observed
that body burden, end-exposure blood levels, and tissue concentrations of MC
were found to increase in direct proportion with exposure concentration. It
was also observed that, as others have found, that MC was more concentrated in
the lipid stores of both species than in the liver or kidneys immediately
following exposure. However, only 2 percent or less of the initial radio-
activity remained 24 hours after exposure ceased. Thus, it is reasonable to
conclude that MC has little potential for bioaccumulation in these species.
4-14
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TABLE 4-5. HAKE ET AL. (1960) RECOVERY EXPERIMENT WITH RATS (3)
INTRAPERITONEALLY INJECTED WITH 14C-MC (700 mg/kg)
Average % dose
Expired Air
Unchanged MC (by isotopic dilution)
Unknown compound detected by
furnace (assumed to be unchanged MC)
14
C02 (in NaOH) 70% within 4 hr; 100% within 12 hr
Urine
TCE-glucuronide + other volatile compounds but
no detectable TCA
Feces
Uncharacteri zed
Tissues
Uncharacterized except skin (90% unchanged MC)
97.6
1.1
0.5
0.85
0.03
0.18
Schumann et al. observed that between 94 and 98 percent of the total
recovered radioactivity in rats and between 87 and 97 percent of that in mice
was unchanged MC in expired air. These data are in general agreement with the
observations of others reported in this section. The remaining radioactivty
14
consisted of either C02 or nonvolatile radioactivity in urine, feces, and
carcass.
Of particular interest is the observation by Schumann and coworkers that
biotransformation of MC appears to be a saturable and dose-dependent process
in both species. Over the 10-fold exposure range, a 2 to 3-fold increase in
the mean micromole-equivalent of MC metabolized per kilogram of body weight
was observed in both species. Species differences noted were (1) a greater
rate of pulmonary clearance of MC in mice relative to rats, (2) end-exposure
blood levels, (3) and log-linear elimination constants for two-compartment
(rats) and three-compartment (mice) linear models. As the authors concluded,
these differences can reasonably be ascribed to species differences in respi-
ratory minute volume and metabolism. Coupled with the observations of Eben
4-15
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and Kimmerle (1974) the results of Schumann and coworkers suggest that satur-
ation of metabolic pathways occurs somewhere in the range of 500 to 1,500 ppm
o
(2,700 and 8,100 mg/m ) in these rodent species.
The pharmacokinetic description and biochemical basis of saturable met-
abolism in relation to a variety of organic compounds is comprehensively dis-
cussed in a series of reports by Andersen and coworkers (1980) and Andersen
(1981a,b,c).
In a companion study, Schumann and coworkers (1982b) determined that the
fate of inhaled MC is not altered upon repeated exposure of male Fischer 344
rats and B6C3F1 mice. Both rats and mice were exposed to 1,500 ppm (8,100
3 14
mg/m ) unlabeled MC for 16 months and, on the last exposure, to C-MC. The
14
fate of C-MC was followed for 72 hours and compared to age-matched controls
exposed concurrently to chamber air for 16 months prior to receiving the
single C-MC exposure. Results showed that prior repeated exposure did not
significantly alter the quantity of MC excreted via the pulmonary route compared
to those singly exposed, of either species. Although the mean end-exposure
body burden of C-activity was about 2-fold greater in mice than rats, it did
not differ significantly between the singly and repeated exposed groups of
rats or mice. Per kg body weight, mice were observed to metabolize about
5-fold more MC than did rats. Immediately after the end of the final 6-hour
14
exposure, C-activity was found in the liver, kidney, and adipose tissue.
After 72 hours postexposure, levels in all three sites decreased appreciably;
activity was not detectable in adipose tissue of mice nor in liver of rats.
Prior repeated exposure to MC did not significantly alter the concentration of
14
C-activity in any tissue of either species.
When data obtained in the study were compared to similar data obtained
during the course of exposure to younger rodents (Schumann et al., 1982a), an
apparent age-related difference in metabolism was noted. The apparent effect
was greater in mice; there was a 2-3 fold increase in body burden and a 5-6
fold greater extent of metabolism of MC in the 18-month-old mice compared to
the 2.5 to 3.5-month-old mice that had been exposed to 1,500 ppm (8,100 mg/m )
C-MC in only a single 6-hour period. One factor that could account for the
increased body burden and metabolism is a slower rate of pulmonary clearance
14
in the older animals. The older rats cleared about 50 percent less C-MC
during the first three hours after exposure relative to younger rats (Schumann
et al., 1982a); the difference was not as great in mice.
4-16
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As previously discussed man appears to possess a very limited capacity
for metabolism of MC. Table 4-6 summarizes data taken from several investi-
gative studies showing the amount of urinary metabolites excreted after exposure
to various concentrations (4.3 to 213 ppm; 23.2 to 1,150 mg/m ) of MC in
inspired air. Seki et al. (1975) surveyed workers in 4 printing plants where
MC was the sole organic solvent in use. The workers were exposed 8 hours
daily, 5 1/2 days per week, over a period of at least 5 years. Urine samples
were collected in the latter half of the work week. Their data in Table 4-6
show a proportional increase of both TCE (as glucuronide) and TCA, and also
total trichlorinated compounds (TTC) with increased air concentrations of MC.
Seki et al. expressed their data as a linear relationship between inspired MC
concentration and urinary metabolite excretion, with an added constant increment
presumably due to continued excretion from the previous day's exposure. From
this observation and from evidence of continued excretion during exposure-free
weekends, they suggested that MC accumulates in the body up to a steady-state
body burden defined by the air concentration of each daily exposure. Seki et
al. also noted that the amounts of metabolites excreted daily in the urine of
these workers were less than 5 percent of that observed with comparable ex-
posures to trichloroethylene, a compound that Monster (1979) has shown to be
metabolized from 60 to 80 percent to TCE and TCA. This comparison suggests
that about 5 percent or less MC is metabolized.
The data (Table 4-6) of Monster et al. (1979b) and of Humbert and Fernandez
(1977) were obtained from subjects given single 4-hr and 8-hr exposures to 70
and 145 ppm (378 and 783 mg/m3), and 72 and 213 ppm (389 and 1,150 mg/m3) MC,
respectively. Total urinary excretion of TCE (glucuronide) and TCA is observed
to be proportional to the inspired air concentration of MC and to the expected
body burden from these exposures (with duration of exposure also taken into
account). Both these research groups attempted a balance study by estimating
the retained body dose of MC either by measuring lung clearance during exposure
(Monster et al., 1979b), or by integrating over the MC alveolar air decay
curve to infinite time postexposure (Humbert and Fernandez, 1977). The per-
centages of the retained dose metabolized to TCE and TCA were 2.5 and 6.3
percent, respectively. The difference can be ascribed to the different metho-
dologies used for estimating body dose and their inherent imprecisions. In
both studies, the percentages of MC metabolized were found to be independent
of the retained dose (Table 4-6). A similar observation with rats was made by
4-17
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TABLE 4-6. RELATION BETWEEN INHALATION EXPOSURE AND URINARY METABOLITES OF MC
Seki et al., 1975
Average concentration of metabolites in urine samples from workers daily
exposed to MC.
Plant
Plant
A
B
C
D
Air cone.
MC, ppm
0
4.3
24.6
53.4
No.
Subjects
30
10
26
10
TCE*
TCA
0
1.2
5.5
9.9
0
0.6
2.4
3.6
TTC/g
creatim'ne
0
2.1
6.8
15.0
Monster et al., 1979b
Averaged amounts of metabolites in total urine collected 70-hr post single
exposure (4 hr).
Air Cone.
MC, ppm
No.
Subjects
72 6
145 6
Estimated as % retained dose
Humbert and Fernandez. 1977
TCE*
mg
5.5
11.5
2%
TCA
mg
1.5
2.8
0.5%
Averaged amounts of metabolites in total urine collected for 12 days post
single exposure (8 hr).
Air
MC,
72
213
Estimated as
cond.
ppm
% retained
No.
Subjects
3
2
dose
TCE*
mg
15.2
30.7
4.6%
TCA
mg
5.2
13.0
1.7%
*TCE found as glucuronide.
Eben and Kimmerle (1974), who measured urinary excretion of TCE and TCA for 3
days after a 4-hr inhalation of MC at 221 and 443 ppm (1,193 and 2,392 mg/m ).
The amounts of the metabolites excreted were proportional to the inspired
4-18
-------
concentration of MC. A 1.7-fold increase in the quantity of urinary metabo-
lites was measured with the 2-fold range of exposure levels. In rats, the
urinary ratio of TCE/TCA was 20/1 rather than the 3/1 observed in humans
(Table 4-6).
Nolan et al. (1983) in a report submitted for publication found that 5 to
6% of the amount of MC inhaled by volunteers during a 6-hour exposure was
excreted in urine as TCE and TCA. The amounts were reported to be extremely
variable, thus indicating that such measurements provide at best only a rough
estimate of exposure.
Caperos et al., (1982) have developed a kinetic model simulating occupat-
ional exposures and one which may be suitable for biologic monitering. This
model was reported to be consistent with the experimental data of Humbert and
Fernandez (1977). The results of the simulation of varying workday exposures
suggested that TCE is the most sensitive and representative indicator of
exposure; fluctuations in exposure concentration had a marked influence upon
urinary TCE concentrations, but no effect upon urinary TCA.
A controlled human exposure study designed to determine if simultaneous
measurement of MC and Pco2 in alveolar air represents a useful technique by
which MC exposures can be monitored was conducted by Guillemin and Guberan
(1982). A linear relationship between MC levels and Pco2 in alveolar air
samples was demonstrated. Both MC and Pco2 levels varied, depending on the
technique used to collect alveolar air samples and the extent to which the
eight volunteers hyper- or hypoventilated. Analysis of variance for those
tests that used a "standard" sampling technique showed an average coefficient
of variation of 5.6 percent. During this series, the sample was collected
during the last part of a prolonged (but not forced) expiration following a
normal inspiration. All 8 volunteers were exposed, at rest, 4 hours both in
the morning and afternoon to MC ranging in concentration from 203 to 236 ppm
o
(1,096 to 1,274 mg/m ). Alveolar sampling was performed on the first post-
exposure day between 8 and 12 a.m. The results demonstrated that MC and Pco2
should be corrected for hyper- and hypoventilation and for dilution of alveolar
air with dead space air by a proportional adjustment of MC concentration at
the mean normal alveolar Pco2 or by disregarding the samples with a Pco2
outside the normal range.
A reasonable conclusion from these studies is that MC is minimally meta-
bolized by man on the order of 3 to 6 percent of the inhaled dose. The per-
centage of the dose metabolized is constant. Due to the limited extent of
4-19
-------
metabolism of MC and the observations that metabolism in rats and mice is
saturated at high concentrations, saturation of metabolism does not appear to
play a significant role in the pharmacokinetics of MC during likely human
exposure situations.
4.2.2 Kinetics of Blood and Urine Metabolites
The blood and urinary metabolites of MC (total amount, excretion ratios,
and excretion time-course) are of interest as quantitative indices of exposure
and body burden. However, the known metabolites of MC—TCE, TCE-glucuronide,
and TCA — are not pathognomonic of MC, but are also metabolites of other
chlorinated hydrocarbons, e.g., trichloroethylene.
3
From studies of men exposed to 70 and 145 ppm (378 and 783 mg/m ) MC
inhaled for 4 hours, Monster et al. (1979b) found that the TCE concentration
in blood was proportional to both the inspired concentration and the blood
concentration of MC. TCE blood concentration was about 4 percent that of MC
-i -i 3
[0.2 mg H and 0.09 mg 8, TCE for 145 and 70 ppm (783 and 378 mg/m ) MC
inspired, respectively]. After termination of exposure, blood TCE concentra-
tion declined exponentially with a half-life of 10 to 12 hours. Urinary
appearance of TCE and TCE-glucuronide paralleled the disappearance of blood
TCE, and daily excretion decreased with a half-time of renal elimination of 10
to 12 hours. This value is in agreement with that observed after ingestion of
TCE itself (Briemer et al. , 1974; Muller et al. , 1974). In contrast to the
first-order blood decay kinetics of TCE, blood concentrations of TCA progres-
sively increased after the end of MC exposure for about 40 hours before declin-
ing exponentially with a half-life of 70 to 85 hours. Consequently, TCA
appeared in the urine in almost equal daily amounts for 3 days before decreas-
ing. Exogenous TCA administered to men has a similarly long half-time of
renal elimination of 50 to 82 hours, presumably because of very tight non-
covalent binding to plasma proteins (Paykoc and Powell, 1945; Muller et al. ,
1974). Therefore, the rise in plasma concentration of TCA during the 24 to 48
hours period following exposure is due to a rate of formation of TCA from TCE
greater than the rate of renal elimination of TCA. Similar observations on
daily urinary excretion of TCE, TCE-glucuronide, and TCA following acute MC
inhalation exposure have been made in man by Humbert and Fernandez (1977), and
in rats by Eben and Kimmerle (1974).
Stewart et al. (1969) investigated urinary metabolite excretion in men
3
repeatedly exposed to MC (500 ppm; 2,700 mg/m ), 7 hours per day for 5 days.
4-20
-------
The daily ratio TCE/TCA during exposure remained relatively constant (about
2.8), but rapidly decreased within several days after exposure (5th day, 0.4),
indicating that the daily urinary TCE excretion decreased while the TCA excre-
tion decreased at a lesser rate or may have gradually increased. Eben and
Kimmerle (1974) also chronically exposed rats for 14 weeks (8 hours daily, 5
days per week) and measured blood levels of MC, TCE, and TCA, as well as daily
urinary excretion. Blood concentrations of MC and TCE (determined immediately
after daily exposure) remained essentially constant during the entire 14 week
period, with a concentration ratio of MC:TCE of 10:1. Weekly urinary excre-
tion of TCE reached a plateau within 3 to 4 wk, whereas TCA weekly excretion
was constant throughout the entire 14-wk period. The urinary excretion ratio
TCE/TCA was thus initially low, but after 3 to 4 weeks exposure reached a
constant value of approximately 18.
These findings show that the daily urinary ratio of TCE/TCA (mg/day)
during chronic MC exposure of both man and rats is determined by the relative
rates of metabolic formation and by the differences in renal elimination of
TCE (rapid, half-time 10 to 12 hours) and TCA (slow, half-time 50 to 70 hours).
In blood, both metabolites achieve steady-state plateau concentrations (TCE
more rapidly than TCA) related to the inspired air concentration of MC and its
metabolites. In urine, the TCE/TCA ratio, initially low, rises to a constant
daily value. After termination of either acute or chronic exposure, the ratio
of the amounts of daily urinary metabolites, TCE/TCA (mg/day), is highest 24
hours after exposure. Thereafter, it progressively decreases daily to less
than unity by the 5th or 6th day postexposure because of the relatively rapid
renal elimination of TCE and the slow renal elimination of TCA (Stewart et al. ,
1969; Eben and Kimmerle, 1974; Monster, 1979).
The profile of TCE and TCA urinary excretion during and after MC exposure
is similar to that observed for trichloroethylene (Nomiyama and Nomiyama,
1971; Muller et al. , 1974; Sato et al. , 1977; Monster et al. , 1976, 1979a;
Fernandez et al. , 1977). However, the ratio of TCE/TCA excreted is 2-fold
greater for MC than for trichloroethylene, suggesting differences in the
pathways and rates of metabolism to TCE and TCA for these two chlorinated
hydrocarbons. Trichloroethylene, a chlorinated olefin, is thought to be
metabolized by hepatic microsomes to an epoxide, then to chloral hydrate, and
thence to TCE and TCA (Liebman and Ortiz, 1977; Henschler, 1977; Van Duuren,
1977). However, neither epoxide nor chloral hydrate have been identified as
4-21
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metabolites of MC. Comparison of the percentage of the body dose metabolized
to TCE and TCA (MC, 3-6 percent vs trichloroethylene, 80-90 percent) explains
the 25- to 30-fold difference in the total amounts of these metabolites (mg/day)
excreted into urine for comparable body doses retained after inhalation exposure
(Seki et al., 1975; Stewart, 1968; Stewart et al., 1969; Monster, 1979; Ikeda
and Ohtsuji, 1972; Ikeda et al., 1972). In a comparative study of rats exposed
to 200 ppm (1,080 mg/m3) MC or its 1,1,2-isomer, Ikeda and Ohtsuji (1972)
reported that the urinary excretion of total chlorinated metabolites was less
for the 1,1,2-isomer, indicating that the 1,1,2-isomer is metabolized to an
even smaller extent than MC. These workers determined the urinary metabolites
of the 1,1,2-isomer as TCE and TCA by a spectrophotometric method based on the
Fujiwara reaction. However, the MC isomer requires a shift of a chlorine atom
from one carbon to the other in order to form TCA or TCE, an unlikely reaction
in vivo for a saturated aliphatic, and it is probable that dichlorometabolites
14
were actually measured. Yllner (1971a) injected C-labeled 1,1,2-trichloro-
ethane, i.p., into mice. Over a 3-day period, 73 to 87 percent of the activity
was recovered in the urine, less than 2 percent in the feces. Expired air
14
contained 16 to 22 percent of the radioactivity (60 percent C02 and 40
percent unchanged parent compound). From 1 to 3 percent remained in the
animal. This indicates that the metabolism of 1,1,2-trichloroethane is sig-
nificantly greater than MC.
The interactions of MC and tetrachloroethylene upon their metabolism is a
focus of the research investigations of Ikeda and coworkers. An 8-hour exposure
of male Wistar rats to a mixture of these compounds at their TLV® levels was
reported to cause a statistically significant (p < 0.01) decrease in TCE, the
principal urinary metabolite of MC. In the opinion of the authors, this
apparent suppression of MC metabolism by tetrachloroethylene is most likely a
result of suppression of oxidation of MC to TCE (Koizumi et al., 1982). Ln
vitro kinetic studies are in progress.
4.2.3 Enzyme Pathways of Methyl Chloroform Metabolism
The metabolic pathways and enzyme mechanisms for the metabolism of halo-
genated hydrocarbons assume considerable importance for understanding and
assessing cellular toxicity. Compounds that in the course of their metabolism
form intermediates reactive with cellular macromolecules, e.g., epoxides or
free radicals, are associated with enhanced cellular toxicity and carcinogenic
4-22
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potential (Van Duuren, 1977). In comparison with other halogenated hydro-
carbons, MC is not extensively metabolized by mammalian systems; this may
explain, in part, its lower toxicity and carcinogenic potential (Weisburger,
1977).
Figure 4-3 summarizes the presently postulated enzyme steps in the bio-
transformation of MC to TCE and TCA, the only known metabolites appearing in
the plasma and urine of animals and man (Table 4-6). It is assumed that
metabolism occurs principally in the liver, although jm vitro experiments that
directly demonstrate and evaluate MC metabolism by the liver have only been
reported for TCE production (Ivanetich and Van Den Honert, 1981).
TCA
CI3C - C
Cl - C - CHo
MICROSOMAL
P450 SYSTEM
NADPH, 02
PEROXISOME \^\ TCE
OXIDASE,
CATALASE CI3C ~ CH2OH
OH
SYSTEM
"CHLORAL HYDRATE
DEHYDROGENASE
MICROSOMAL
DEHYDROGENASE
NADP
MICROSOMAL
ETHANOL
OXYGENASE
NADPH, O2
ALCOHOL
DEHYDROGENASE?
GLUCURONYL
TRANSFERASE
V ,
TCE-GLUCURONIDE
CI3C - C
H
CHLORAL
Figure 4-3. Postulated pathways of hepatic biotransformation of MC.
Source: Davidson (1980) and Ivanetich and Van Den Honert (1981).
4-23
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Schumann et al. (1982a), using
in both rats and mice.
14
C-MC studied the routes of elimination
Hake et al. (1960), found that about 0.5 percent of
14
the dose (of the 2 percent metabolized; see Table 4-7) was converted to CO,,,
following 1,1,1-trichloroethane-l- C administration (intraperitoneal) to
14
rats, suggesting carbon-carbon cleavage. The origin of the CO^ is an open
question since both Schumann et al. (1982a) and Hake et al. did not identify
14
the source. The C-MC used by Hake et al. was contaminated with trace amounts
14
of the MC isomer 1,1,2-trichloroethane-l- C (0.4 percent) and 1,1-dichloro-
ethane-1- C (0.5 percent). Both of these compounds were reported by Van Dyke
and Wineman (1971) and by Van Dyke (1977) to be readily dechlorinated by the
rat liver microsomal P450 system. The optimal configuration for dechlorination
was a dichloromethyl group. Hence, 1,1,2-trichloroethane, the more toxic isomer,
was readily dechlorinated but 1,1,1-trichloroethane was not. For the isomer
1,1,2-trichloroethane, the products were identified as mono-and dichloroethanol
and mono- and dichloroacetic acid (Van Dyke and Wineman, 1971), which are known
to be metabolized to C0? or to form glutathione conjugates (Yllner, 1971a,b).
Carlson (1973) found that pretreatment of rats with phenobarbital, but not
methylcholanthrene, potentiated hepatotoxicity (measured as serum SCOT and
SGPT) of both inhaled MC and its 1,1,2-isomer, but th,e toxicity of the, isomer
was increased to a far greater extent. These several observations suggest
that 1,1,2-trichloroethane, but not MC, may be extensively metabolized by the
oxidative dechlorination system of Van Dyke and Wineman (1971).
Figure 4-3 proposes that the initial pathway of MC transformation is hy-
droxylation of MC to TCE by the microsomal P450 mixed function oxidase system
(Ivanetich and Van Den Honert, 1981).
This view is supported by the observations of Cox et al. (1976) and
Pelkonen and Vainio (1975) in which a P450 type I binding spectrum was demon-
strated upon aerobic incubation of MC with rat liver microsomes plus NADPH.
Apparent induction of the P450 drug metabolizing system was observed in the
experiments of Fuller et al. (1970) and Lai and Shah (1970). Lai and Shah
(1970) exposed Swiss albino, random-bred male mice to 3,000 ppm (16,200 mg/m3)
MC for either 24 hours or for 4 or 8 hours/day for several days. Hexobarbital
(80 mg/kg), sodium barbital (275 mg/kg) or chloral hydrate (350 mg/kg) induced
sleeping time was measured at predetermined times after exposure. The 24-hour
exposure produced a maximum reduction in the duration of hexobarbital sleeping
time but had no effect when either barbital or chloral hydrate was used.
4-24
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The significance of the difference of means between control and experimental
groups was determined by the Wilcoxon Rank Sum Test. Where the 24-hour expo-
sure was completed in 3 to 6 exposure periods, each separated by 18 to 21
hours, NIC inhalation showed a statistically significant cumulative effect. A
single 8-hour exposure was ineffective. •
Fuller et al. (1970) extended these observations by investigating the
inductive effect of 1C on hepatic drug metabolism. Male Sprague-Dawley rats
and Swiss albino, random-bred mice were exposed to MC (2,500 to 3,000 ppm;
13,500 to 16, 200 mg/m3) in an air flow-regulated chamber for 24 hours. Pre-
treatment with inhibitors of protein synthesis (cyclohexamide and actinomycin
D) were used in an attempt to block the effect of MC on the hepatic drug-
metabolizing system. Inhalation of MC by untreated rats decreased the dura-
tion of action of hexobarbital, meprobamate, and zoxazolamine. This was
accompanied by an increase in the metabolism of hexobarbital, zoxazolamine and
aminopyrine in vitro by hepatic microsomal enzymes. Cytochrome P-450 and
cytochrome c reductase were also increased. Pretreatment with cyclohexamide
and actinomycin D prevented the MC-induced decrease in hexobarbital sleeping
time and the increase in drug metabolism. The authors suggested that the
results indicate that the increase in drug metabolism after MC inhalation is
related to induction of new enzyme protein. It was further suggested that
this offers a sensitive prepathologic measure of MC toxicity since induction
occurs after exposure .to concentrations of MC that do not produce histologi-
cally detectable liver lesions.
These observations raised the possibility that MC might induce its own
metabolism with repeated daily exposure. However, a number of observations by
others indicate that this is not the case. Platt and Cockrill (1969) reported
MC to be essentially without effect in enhancing the hepatic MFO system of
mice orally dosed with 1,650 mg/kg/day for 7 days. Savolainen et al. (1977)
did observe increased P-450 content above controls at 3 hours of exposure of
male Sprague-Dawley rats to 500 ppm (2,700 mg/m3.) but upon cumulative exposure
of rats for 5 days, P-450 decreased below control values. This decrease is
consistent with the observations of Vainio et al. (1976) who treated animals
intraperitoneally. As previously discussed in this chapter, Schumann et al.
(1982b) found that repeated exposure of rats and mice to 1,500 ppm (8,100
mg/m3) for 16 months did not significantly alter the disposition of MC com-
pared to singly-exposed rats and mice.
4-25
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In contrast to the results observed earlier by Lai and Shah (1970), Shah
and Lai (1976) found that when MC was administered to Swiss-albino, random-
bred mice by i.p. injection, a potentiation of pentobarbital sleeping time and
a reduction in the metabolism of hexobarbital by liver microsomes were observed.
When MC was topically applied, pentobarbital sleeping time was reduced by 36
percent, similar in action to that observed previously by inhalation. Similar
results had been reported earlier by Plaa et al. (1958) in male albino mice of
the Princeton strain when MC was administered subcutaneously. MC signifi-
cantly (p = 0.01) potentiated sleeping time induced by pentobarbital. These
observations may be explained on the basis of different amounts of MC reaching
the liver by various routes and methods of administration.
The origin of TCA as a plasma and urinary metabolite of MC is postulated
to occur from enzymic oxidation of TCE. TCE-glucuronide, also present in
plasma and urine, is presumed to be formed by glucuronyl transferase. TCE,
exogenously administered to man, yields TCA as a metabolite (Marshall and
Owens, 1954; Owens and Marshall, 1955a,b; Muller et al., 1974). Marshall and
Owens (1954) also observed TCA formation during TCE incubation with rat or dog
liver slices. The most likely reaction for the oxidation of-TCE to TCA would
involve the enzyme alcohol dehydrogenase (Figure 4-3). However, i_n vitro
studies with alcohol dehydrogenase purified from horse liver, and with rat
liver cytosol fractions, have shown that TCE is a poor substrate for alcohol
dehydrogenase and that significant conversion to trichloroacetaldehyde (chloral)
does not occur (Sellers et al., 1972; Friedman and Cooper, 1960; Marshall and
Owens, 1954). The reverse reaction —reduction of chloral to TCE -- proceeds
rapidly with a Km of 2.7 x 10~3M for horse liver enzyme (Butler, 1948, 1949;
Marshall and Owens, 1954; Owens and Marshall, 1955a,b; Friedman and Cooper,
1960; Sellers et al., 1972). Also, chloral hydrate has been sought, but not
detected, as an intermediate metabolite in plasma of rats and man exposed by
inhalation to MC (Monster et al., 1979a; Eben and Kimmerle, 1974). On the
other hand, chloral hydrate exogenously administered is very rapidly metabolized
in vivo with a half-life of only a few minutes, yielding both TCE and TCA as
metabolites in plasma and urine (Butler, 1948; Marshall and Owens, 1954; Owens
and Marshall, 1955a,b; Breimer et al., 1974; Muller et al., 1974; Cole et al.,
1975).
The conversion of chloral to TCA is usually ascribed to so-called "chloral
hydrate dehydrogenase," a substrate-specific NAD-dependent enzyme described by
Cooper and Friedman (1958). This enzyme was obtained from rabbit liver acetone
4-26
-------
powders. Cooper and Friedman reported that acetaldehyde was not a substrate,
but rather a markedly effective inhibitor; they did not demonstrate a normal
endogenous substrate. Human cytosolic acetaldehyde dehydrogenase does not
convert chloral hydrate to TCA (Kraemer and Deitrich, 1968; Blair and Bodley,
1969; Sellers et al. , 1972). Grunnet (1973) reported that chloral hydrate is
not a substrate for mitochondria! NAD-dependent acetaldehyde dehydrogenase.
Microsomal NADP-dependent acetaldehyde dehydrogenases with broad substrate
specificity have been described, but whether chloral hydrate is a substrate
has not been determined (Tottmar et al., 1973).
There is little evidence which precisely defines the enzymatic pathways
for the metabolism of MC to TCE by P450 mixed function oxidase system, and TCE
to TCA by classical alcohol dehydrogenase and acetaldehyde dehydrogenase
enzymes. Although as yet uninvestigated, it is possible that TCE may be a
substrate for the microsomal P450 ethanol oxidative system (MEOS) described by
Lieber and his coworkers for ethanol and other alcohols (Lieber and De Carli,
1968; Teschke et al. , 1977). TCE has been shown by Uehleke et al. (1976) to
give a binding spectrum with rat microsomes. An alternative pathway also
uninvestigated is afforded by the peroxisoma! oxidase-catalase system (Figure
4-3), which is known to readily oxidize ethanol and a broad spectrum of other
substrates (Masters and Holmes, 1979; Chance et al., 1977). Indeed, peroxi-
somes may perhaps be involved in the initial oxidation of MC to TCE and other
halogenated hydrocarbons as well.
4.3 SUMMARY AND CONCLUSIONS
Like other solvents of this group, inhalation and lung absorption of MC
vapor in the air is the most important and rapid route of absorption into the
body. Absorption through the skin by direct liquid contact is sloWv At the
3
accepted TWA value (350 ppm; 1,890 mg/m ) for an 8-hr exposure, less than 2 g
may be expected to be absorbed into the body of a normal 70 kg man. This is
because pulmonary absorption is directly related to the blood/air partition
coefficient, which for MC is less than that of most other structurally related
solvents. MC total body dose increases in direct proportion to inspired air
concentration until steady-state is reached; it is also increased by physical
activity during exposure. MC distributes throughout the body, readily cross-
ing the blood-brain barrier and possibly the placenta! barrier as well. It
4-27
-------
can be assumed that NIC may also distribute into the colostrum or milk of
nursing mothers, although no specific data are available. Relative body
tissue concentrations are not known, but there is a high affinity for adipose
tissue due to the higher lipid/ blood partition coefficient of NIC compared
with that of other related solvents. Blood and tissue concentrations achieved
at equilibrium are directly proportional to inspired air concentration.
MC is metabolized in man to a very limited extent—no more than 6 percent
of the total body dose. Metabolism appears to occur principally in the liver,
and to an unknown extent in other tissues, yielding only trichloroethanol
(TCE) and trichloroacetic acid (TCA) as identified metabolites. Urinary
excretion of these metabolites is proportional to inspired air concentration
and the total body dose of MC at low exposure concentrations. At high concen-
trations (1500 ppm), the biotransformation of MC appears to be a saturable,
dose-dependent process. However, saturability is of little practical signifi-
cance since metabolism occurs to a limited extent and ambient air concentra-
tions are orders of magnitude lower than those associated with metabolic
saturation. Detailed information concerning the enzymatic pathways of meta-
bolism is lacking. The mechanism(s) of the initial biotransformation of MC to
TCE are speculative.
During post-exposure, between 80 to 90 percent of MC is excreted un-
changed by the lungs. Alveolar air concentration and blood concentration
decline in a parallel exponential fashion exhibiting three major components of
elimination with half-times of approximately 1, 9, and 30 hours. The long
half-time of elimination (30 hours) is related to elimination from adipose
tissue.
4-28
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Sato, A., T. Nakajima, Y. Fugiwara, and N. Murayama. A pharmacokinetic model
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Savolainen, H., Pfaffli, P., Tengen, M.
1,1,1-trichloroethane: effects on
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and Vainio, H. Trichloroethylene and
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Schumann, A. M., T. R. Fox, and P. G. Watanabe.
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[14C] Methyl
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14
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5. TOXIC EFFECTS
Since its commercial introduction in 1954, methyl chloroform has been
used increasingly as an industrial solvent and in consumer products. Usually
the most important path of entry into the body is by inhalation. The prin-
cipal health effect of over-exposure involves the central nervous system.
While thresholds for such effects are difficult to characterize, levels around
1,000 ppm (5,400 pg/m ) may result in coordination problems. At much higher
levels, anesthesia becomes apparent and death may occur at 1 to 3 percent due
to anesthesia and/or cardiac toxicity. Unlike other chlorinated hydrocarbons,
MC has not been associated with clearly evident liver or kidney damage. The
available literature on the possible toxic effects of MC is reviewed in this
chapter.
5.1 HEALTH EFFECTS IN HUMANS
5.1.1 Experimental Studies
•Experimental studies in humans have centered around three general areas:
(1) clinical experiences with MC as an anesthetic; (2) the kinetics of MC
absorption and excretion after exposure via the inhalation and cutaneous
routes; and (3) the impairment of psychophysiological functions in humans
exposed to MC.
Dornette and Jones (1960) administered MC to 50 patients undergoing
elective surgery. Nitrous oxide-oxygen (4:1) was used as the vehicle and as a
supplemental anesthetic agent. The concentration of MC required for induction
of surgical plane anesthesia varied from 10,000 to 26,000 ppm (54,000 to
140,400 mg/m ), and for maintenance of light anesthesia from 6,000 to 22,500
O
ppm (32,400 to 121,500 mg/m ). Rapid induction and recovery, analgesia, and
the absence of disagreeable odor, respiratory depression, postoperative de-
pression, nausea and vomiting were listed as advantages in the use of MC. A
definite disadvantage was depression of blood pressure during anesthesia of
moderate depth. The tendency to develop ventricular arrhythmias during hypoxia
was also observed but this effect was reversed when effective oxygenation was
reestablished.
Cardiac sensitization from exposure to high concentrations of halogenated
hydrocarbons, with resultant increased susceptibility of the heart to cate-
5-1
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cholamine-produced arrhythmias, e.g., ventricular fibrillation or ventricular
tachycardia, is a well-known phenomenon. This topic has been comprehensively
reviewed by Aviado et al (1976). Clark and Tinston (1973) believe that cardiac
sensitization caused by these relatively inert, lipid soluble hydrocarbons is
very likely to be a structurally non-specific action on myocardial membranes
by solution and distribution within these membranes, and is, therefore, an
example of "physical toxicity" (see Sect. 5.2.3). From an evaluation of 14
halogenated hydrocarbons in dogs, Clark and Tinston (1973) found that the
cardiac sensitizing potencies, as determined by the partial pressure in in-
haled air needed to sensitize the heart to epinephrine, was directly related
to their saturation vapor pressures. In a similar way, the anesthetic potency
or narcotic action of the halogenated hydrocarbons, a structurally nonspecific
action, is directly related to the blood/air or lipid/air partition coeffi-
cient for these compounds (Table 4-1) (Miller et al., 1972; Eger et al., 1965;
Sato and Nakajima, 1979).
TABLE 5-1. SUBJECTIVE AND PHYSIOLOGICAL RESPONSES TO A CONSTANTLY INCREASING
METHYL CHLOROFORM VAPOR CONCENTRATION OVER A PERIOD OF 15 MINUTES
Concentration
(ppm)
Responses to Exposure
0 to 1000
1000 to 1100
1900 to 2000
2600
2650
Increasing awareness of a slightly sweet, not unpleasant
odor.
Mild eye irritation noted in 6 of 7 subjects.
6 of 7 subjects aware of throat irritation.
1 subject very lightheaded.
2 subjects unable to stand. 3 subjects very lightheaded,
but able to stand. 2 subjects were not lightheaded, and
one of these was able to demonstrate a normal Romberg
test.
Source: Stewart et al. (1961).
Siebecker et al. (1960) reported that electroencephalographic patterns
during MC anesthesia showed little change before circulatory depression and
that the changes were similar to those during halothane anesthesia. The
investigators found MC to be less potent clinically than either chloroform or
halothane in supplementing nitrous oxide-oxygen for anesthesia.
5-2
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Stewart et al. (1961) evaluated the acute effects of increasing concen-
trations (0 to 2,600 ppm; 0 to 14,040 mg/ms) of MC over a 15-minute exposure
period. A commercial grade of MC was used. Table 5-1 shows the subjective
and physiological responses during exposure to MC. In another experiment in
which 6 subjects were exposed to MC at 500 ppm (2,700 mg/ms) for 78 minutes or
186 minutes, no eye irritations or dizziness occurred, nor were balance or
coordination affected. Exposure at 900 to 955 ppm (4,860 and 5,157 mg/m3) for
73, 35, and 20 minutes produced a number of psychophysiological effects which
are listed in Table 5-2.
TABLE 5-2. SUBJECTIVE AND PHYSIOLOGICAL RESPONSES TO METHYL CHLOROFORM
VAPOR CONCENTRATIONS OF 900 TO 1000 PPM
Average
Concentration
Responses to Exposure'
900 ppm for 20
minutes
(3 subjects)
910 ppm for 35
minutes
(2 subjects)
951 ppm for 73
minutes
(3 subjects)
Positive Romberg in one subject. Greater effort re-
quired to perform a normal Romberg in two subjects
. after 10 minutes of exposure. Heel-to-toe walking
normal. Two subjects experienced lightheadedness
above 900 ppm.
Greater mental efforts required to perform a normal
Romberg test after 10 minutes of exposure. All
heel-to-toe walking performed well. One subject
experienced persistent lightheadedness above
900 ppm.
Greater mental effort required to perform a normal
Romberg test after 10 minutes of exposure. After
15 minutes of exposure one subject had consistently
positive Romberg. Heel-to-toe walking performed
well by all during exposure. No lightheadedness.
Mild eye irritation was noted by all subjects when vapor concentrations rose
above 1000 ppm.
Source: Stewart et al. (1961).
In another study, Torkelson et al. (1958) exposed humans to MC. Exposure
o
to 550 ppm (2,970 mg/m ) for 90 minutes had no measureable effect on the vital
signs being monitored. Exposure to 500 ppm (2,700 mg/m^) for 450 minutes
produced no significant changes in pulse, respiration, blood pressure, reflexes,
or equilibrium; liver function tests were also negative. Exposure to 1,000
ppm (5,400 mg/m^) for 30 minutes was also without effect. However, exposure
5-3
-------
to 900 to 1,000 ppm (4,860 to 5,400 mg/m3) for 75 minutes resulted in slight
eye irritation and a feeling of lightheadedness, and the Flanagan and Romberg
tests revealed a slight but definite loss of coordination and equilibrium.
ECG and liver function tests were normal. Exposure to 1,900 ppm (10,260
mg/m3) for 5 minutes resulted in an obvious disturbance of equilibrium and an
abnormal Romberg test.
In 1969, Stewart et al. reported that exposure to 500 ppm (2,700 mg/m3)
for periods of 6.5 to 7 hours/day for 5 consecutive days resulted in mild
subjective responses (sleepiness, eye irritation, and mild headache). The
only untoward physiological response was an abnormal Romberg test. However,
the two individuals who had the abnormal response had, according to the authors,
demonstrated difficulty performing the test before any exposure. None of the
clinical tests performed during or following the exposure was abnormal. "
Twelve subjects were exposed to 250, 350, 450, and 550 ppm (1,350; 1,890;
3
2,430; and 2,970 mg/m ) of MC in inspired air during four continuous 30 minute
periods in an experiment reported by Gamberale and Hultengren (1973). The
air-gas mixture was supplied via a breathing valve and a mouthpiece with very
low resistance. The effects of the introduction of the breathing tube were
not assessed. The presence or absence of MC was reportedly disguised by the
use of menthol crystals. In the final 20 minutes of each exposure period,
five performance tests were made. Two of the tests were of perceptual speed
and the others were tests of simple reaction time, choice reaction time, and
manual dexterity. The same subjects were also studied under control condi-
tions in which inspired air contained no MC but in which all operations and
measurements were the same as during exposure to the solvent. To balance the
training effects between experimental and control conditions, the order of
conditions was reversed for half the subjects.
The change in mean performance level during exposure to the increasing
concentrations of MC differed systematically from the change in performance
under control conditions. The level of performance in the manual dexterity
test and two perceptual tests were affected by training; however, the training
effect was less pronounced during exposure to MC. The tests of reaction time
were less sensitive to training and, with these, there was an absolute decline
in performance capability as the exposure concentration increased. Statis-
tically significant performance differences between experimental and control
conditions were reported for all tests with exposure levels of 350 ppm (1,890
3
mg/m ) or greater, but the subjects were tested repeatedly. Such testing usually
5-4
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yields more significant results than warranted (Benignus and Muller, 1982).
This study contained several .drawbacks: e.g., the substance used to disguise
the MC odor may itself have had a toxic effect, and the introduction of a
breathing tube may have induced stress in the subjects.
Salvini et al. (1971) evaluated psychophysiological effects after ex-
posing six male university students in two groups of three to an average vapor
concentration of 450 ppm (2,430 mg/m ) MC for two periods of 4 hours, separated
by a 1.5 hour interval. During exposure they alternated their activities with
a one-hour study period followed by 20 minutes of physical excercise. Each
subject was examined on two different days, four days apart. . Each group
alternated in the order in which they were exposed to the control and experi-
mental atmospheres. The psychophysiological tests included a perception test
with tachistoscopic (brief exposure to visual stimuli) presentation, the
Wechsler Memory Scale test, a complex reaction time test, and a manual dexterity
test. After two exposures, no disturbances in motor function, coordination,
equilibrium, or behavior patterns were observed in any of the subjects.
However, there were some complaints about eye irritation at peak concentrations
when values ranged up to 500 ppm (2,700 pg/m3). Under mental stress conditions,
exposure to 450 ppm (2,430 mg/m3) MC was reported to have "decreased perceptive
capabilities". Mental fatigue was determined by comparing perception test
results between the morning and afternoon tests. A small reduction in perfor-
mance was also observed but was not statistically significant.
In 1975, Stewart et al. reported an experiment in which 20 individuals
were exposed to 500 ppm (2,700 (jg/m3) or less for 7.5 hours per day, 5 days
per week, for 3 weeks. No serious deleterious effects upon health or per-
formance were detected and the health of the exposed individuals remained
unimpaired during the inhalation studies. The blood chemistries, hematolo-
gies, urinalysis, electrocardiograms, and pulmonary function tests remained
normal. Of the ten females exposed, nine reported that the odor of 350 ppm
(1,890 (jg/m3) was objectionable. In contrast, none of the males objected to
odor at any of the levels used. The authors stated that there was a lack of
sleepiness and fatigue which had been reported by the subjects in the first
repetitive study by Stewart et al. (1969). The authors suggested that monotony
and boredom may have been the responsible factors in the first study. The
authors stated further that "This study failed to corroborate the findings, of
Gamberale and Hultengren (1973) who reported that exposures to 1,1,1-trichloro-
ethane at 350 ppm for 30 minutes impaired reaction time, perceptual speed and
. 5-5
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manual dexterity In our study, these cognitive tasks were not performed
during the first 30 minutes but after several hours when the blood 1,1,1-tri-
chloroethane was higher and when the decrement in performance should have been
more pronounced ... our findings are in agreement with those reported by
Salvini et al. (1971), who observed that exposure to 450 ppm for 4 hours
failed to impair performance of a series of different psychophysiologic tests."
The effect of exposure to MC alone, and in combination with m-xylene,
upon psychophysiological functions in humans was evaluated by Savolainen et
al. (1981). Exposure of nine male volunteers to 200 and 400 ppm (1,080 and
2,160 mg/m3) MC and to 400 ppm (2,160 mg/ms) MC and 200 ppm m-xylene resulted
in no marked effect upon reaction time, critical flicker fusion thresholds, or
body balance. However, MC appeared to have a biphasic effect upon body balance.
Exposure to MC at 200 ppm (1,080 mg/ms) tended to decrease body sway whereas
the higher exposure level had the opposite effect. No kinetic interactions
between MC and m-xylene were observed. Savolainen and coworkers (1982) recently
reported confirmation of the biphasic effect, using an identical protocol with
the same subjects.
5.1.2 Occupational Studies
Chronic occupational exposure to other chlorinated solvents has occasion-
ally been reported to cause adverse neurological and behavioral effects.
Based on the absence of effects at 500 ppm (2,700 mg/ms) in the better con-
ducted human studies, relatively low level ambient exposures to MC would not
be associated with adverse neurological and behavioral effects.
A recent attempt to assess the central and peripheral nervous system
effects of MC in occupational situations was undertaken by Maroni and co-
workers (1977). They studied a very small group (22 subjects) of female
workers exposed to MC vapors at concentrations ranging from 110 to 990 ppm
(594 to 5,346 mg/m3). When compared to workers who were reportedly unexposed,
no differences were found in clinical symptoms or measures of nerve conduction
velocity and psychometric function.
In another study, Seki et al. (1975) surveyed 196 male workers employed
in four Japanese printing factories where MC was the sole organic solvent in
use. The four groups of workers were exposed to average concentrations of
4, 25, 28, and 53 ppm (22, 135, 151, and 286 mg/m3). The workers participated
in a medical interview coupled with a test for sense of vibration (studied at
5-6
-------
the distal joints of the thumbs and great toes using a 128 Hz tuning fork) as
well as routine laboratory examinations, including peripheral hemograms,
determination of blood specific gravity, and urinalysis for urobilinogen and
protein. These examinations revealed no consistent dose-related adverse
effects among the four groups of workers.
In the most recent study, Kramer et al. (1978) measured numerous physio-
logical parameters of workers in two adjacent textile plants. Detailed blood
chemistry and hematology studies were conducted for 151 matched pairs of
employees to compare the exposed and unexposed partners. All employees in the
exposed group had been exposed to MC (and other solvents), in varying concen-
trations, for up to 6 years. The concentration range was 11 to 838 ppm (59 to
3 3
4,525 mg/m ), with a mean of 115 ppm (621 mg/m ) MC. Of the 151 employees,
149 had been exposed for 12 to 60+ months. 135 out of 151 had TWA exposures
of 50 to 149 ppm while 116 of 151 had a TWA exposure of 100 to 249 ppm. The
control group was not exposed to chlorinated solvents.
Pairs were matched with regard to age, race, sex, work shift, job des-
cription and socioeconomic status, and examined within a 10 week period.
Subject height, weight, blood pressure and pulse were obtained, and electro-
cardiograms were recorded. Laboratory blood determinations included hema-
tocrit, hemoglobin, red blood cell .count (RBC), white blood cell count (WBC),
mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) alkaline
phosphatase, SCOT, SGPT, gamma glutamyl transpeptidase, total bilirubin, urea
nitrogen, LDH, uric acid, total protein, A/G ratio, albumin, calcium, and
phosphorus. For quantitative variables, t tests and tests of homogeneity of
variance were made. Multiple regression analysis was performed on paired
differences with respect to environmental variables and on the combined matched
exposed and control populations with respect to demographic variables.
Breathing zone samples were collected in charcoal tubes, except in a few
locations where area sampling was more practical, and analyzed on a portable
gas chromatograph equipped with a flame ionization detector. Samples of
expired air were analyzed immediately after collection by gas chromatography.
After explaining that some data were eliminated on the basis of subjects'
smoking habits, high blood pressure, or prior illness, the authors presented
statistical findings but no individual data. MC concentrations in the breath
ranged from "less than 5 ppm" to "greater than 30 ppm," with the majority
(127/151) between 5 and 29 ppm (27 and 157 mg/nr*). Comparison of the health
5-7
-------
test data between exposed and control subjects revealed no statistically
significant differences except in a decrease in SGPT and a slight increase in
albumin in the exposed group. The authors concluded that no health impairment
was suffered by workers exposed to an average daily concentration of 115 ppm
(621 mg/ma) MC.
5.1.3 Accidental Exposure
Accidental exposure to excessive concentrations MC can lead to death.
Table 5-3 lists the signs and symptoms of a number of cases in which the
patients survived. These results suggest that MC has only a minimal potential
*or producing liver or kidney injury in man upon acute exposures. The primary
toxic effect appears to be a reversible depression of the CNS, typical of an
anesthetic agent. While abuse or misuse of any organic solvent has been
Difficult to control, the problem has diminished in recent years. The follow-
ing is a review of some of the acute exposure incidents related to MC but
v/here no exposure measurements were possible.
Two fatal cases in which the subjects intentionally inhaled cleaning
fluids containing MC were reported by Hall and Hine (1966). A 19-year-old
woman who was observed sniffing cleaning fluid over several'days and acting
irrationally was later found dead. Pathologic findings on autopsy were con-
fined to the respiratory system, stomach and brain.
There were indications of chronic, intentional inhalation of a cleaning
fluid containing MC in the other fatal case studied by Hall and Hine (1966).
On autopsy, pathologic findings were confined to the respiratory system and
the ki''ne.'s. In neither of these cases were drugs or solvents detected in the
stomach contents, and no barbiturates were found in the blood. Blood levels of
MC were 72.0 and 13.0 mg percent, respectively.
Tventy-nine cases of presided sudden death from sniffing MC during 1964
to 1969 were reported by Bas ,1970). These were among 110 cases of sudden
death attributed to sniffing volatile hydrocarbons and halocarbons summarized
by the author. In 18 of the 110 cases, death followed sniffing coupled with
some form of exercise. No anatomical abnormalities were found from gross or
microscopic postmortem examinations that could explain the sudden deaths.
The author discussed the possibility that these deaths resulted from cardiac
sensitization to endogenous catecholamines.
Six fatal cases were reported by Stahl et al. (1969). In the first case,
a 20-year-old man was found dead in a closed space in which he had been working
5-8
-------
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5-9
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with a "paint remover." Gas chromatographic analysis revealed MC in all
tissues. Upon autopsy, the lungs were found to be congested and moderately
edamatous; the liver, spleen, kidneys, and brain were also congested. Micro-
scopic examination of the brain suggested anoxia as the cause of death.
In the second case, a 17-year-old man was found dead in a room in which
he was cleaning an air vent with MC. Gas chromatographic analysis revealed
the presence of MC although blood levels were only 0.15 mg percent. The
authors concluded that death was probably due to 0? deprivation.
In the third case, an obese 24-year-old man was found dead in bed after
having cleaned electrical equipment with MC. In the fourth, fifth, and sixth
cases, three males were found dead in an unventilated 4 x 5 ft. compartment
where they had been cleaning electrical equipment with MC. Blood levels of MC
were 12.0 mg percent, 6.2 mg percent, and 6.0 mg percent in these victims.
Hatfield and Maykoski (1970) reported on a 27-year-old who was found dead
in an aircraft tank that he had been cleaning with MC. The diagnosis was
acute passive congestion of the viscera with petechial hemorrhages in the lung
and brain. Hatfield and Maykoski estimated the concentration level of MC to
which this worker was exposed at 62,000 ppm (334,800 mg/ms).
Caplan et al. (1976) reported a fatal intoxication in which a 40-year-old
female was apparently overcome while painting a bathroom. On external examin-
ation, the only significant abnormality was in the respiratory system. Histo-
logically, the lungs showed acute edema and congestion, and the liver showed a
mild fatty change. MC was identified in tissue samples.
Twenty-one deaths assumed to be related to the abuse or gross misuse of
decongestant aerosol sprays containing MC in the solvent resulted in removal
of several such products from the market (Federal Register 38:21935-36, 1973).
As a followup, a Federal Register notice appeared in 1977 (Federal Register,
1977) and discussed this matter. It was stated: "The action concerning
trichloroethane for inhalation is being taken because of the lack of safety
and effectiveness data available on trichloroethane when it is used as a
component of an aerosolized drug product intended to be inhaled by a sick
person. The drug products containing trichloroethane implicated with 21
deaths were used as cough suppressants, primarily in infants."
5-10
-------
5.2 EFFECTS ON ANIMALS
5.2.1 Acute and Subacute Effects
The LD5Q's of 1C for various species are found in Table 5-4. Admini-
stration of single oral doses yielded LD5Q's for laboratory animals ranging
from 8.6 gm/kg for guinea pigs to 14.3 gm/kg for rats (Torkelson et al. ,
1958). The solvent caused slight transitory irritation in the eyes. The
toxicity from skin absorption was found to be low, as doses of 4 gm/kg failed
to kill any rabbits exposed for 24 hours. Even 15.8 gm/kg failed to kill all
rabbits tested with undiluted liquid MC, applied under a cuff for 24 hours.
Repeated multiple daily application of 500 mg/kg to the skin of rabbits caused
no effect other than reversible local irritation. In other experiments,
laboratory animals were exposed repeatedly to 500, 1000, 2000, and 10,000 ppm
(2,700; 5,400; 10,800; and 54,000 mg/m3) in order to establish conditions safe
for repeated exposure. Rats, guinea pigs, rabbits, and monkeys were unaffected
after 6 months of repeated 7 hour exposures 5 days/week to 500 ppm (2,700
mg/ms). Female guinea pigs, which were found to be the most sensitive in
previous experiments, were able to._tolerate 1000 ppm (5,400 mg/m3) for 0.6 hour/
day for 3 months and 2000 ppm (10,800 mg/m^) for 0.1 hour/day with no detect-
able adverse effects. Male rats tolerated exposure to 10,000 ppm (54,000
mg/m^) for 0.5 hour per day with no organic injury. The effects of MC were
shown to be primarily anesthetic, with only a slight capacity to cause revers-
ible injury to the lungs, and liver. Based on this work, Torkelson et al. '
(1958) suggested that the maximum allowable concentration (ceiling) for MC be
500 ppm (2,700 mg/m§) to avoid anesthetic effects.
In another study, Eben and Kimmerle (1974) exposed rats acutely (4 hours)
and subchronically (8 hours/day, 5 times/week for 3 months) to 220 and 440 ppm
(1,188 and 2,376 mg/ms) and 200 ppm (1,080 mg/m^), respectively. During the
periods of exposure, the authors reported that "the animals did not differ in
any way from their controls in behavior, appearance, or body weight gain.
Hematologic examination performed at the end of the exposure period did not
reveal any pathologic abnormalities".; Liver and renal function tests and
blood glucose were normal. At autopsy, the organs of the exposed rats were
normal; organ weights did not deviate significantly from those of the control
animals.
Prendergast et al. (1967) repeatedly exposed 15 Sprague-Dawley rats, 15
Hartley guinea pigs, 3 squirrel monkeys, 3 New Zealand albino rabbits, and 2
5-11
-------
TABLE 5-4. ACUTE TOXICITY OF METHYL CHLOROFORM
Reference
Torkelson et al. ,
1958
Torkelson et al. ,
1958
Torkelson et al. ,
1958
Torkelson et al. ,
1958
Torkelson et al. ,
1958
Takeuchi
1966
Priestly and Plaa
1976
Klaassen and Plaa
1966
Torkelson et al. ,
1958
Adams et al . ,
1950
Plaa et al. ,
1958
Klaassen and Plaa
1967
Klaassen and Plaa
1967
Gehring
1968
Woolverton and
Balster
Species
rats
rats
mice
albino
rabbits
guinea
pigs
SM mice
CF-1 Swiss
mice
Sprague-Dawley
rats
albino
rabbits
rats
Princeton
mice
Swiss-Webster
mice
dog
Swiss-Webster
mice
CD-I mice
Route
oral
oral
oral
oral
oral
i.p.
i.p.
i.p.
dermal
inhalation
i.p.
i.p.
i.p.
i.p.
inhalation
Sex
•M
F
F
mixed
mixed
?
M
M
mixed
mixed
M
M
M
F
M
LD50
mg/kg
14,300
11,000
9,700
10,500
8,600
2,568
4,008
5,054
15,800
18.0001
16,000
5,080
4,140
4,700
22.2402
95% confidence
limits
12,100 to 17,000
9,500 to 13,000
9,700 to 11,300
6,100 to 12,200
3,558 to 4,522
4,389 to 5,586
12,700 to 21,400
4,140 to 6,010
4,320 to 5,110
1LC50 in ppm for 3 hr exposure; 7 hr exposure resulted in a LCSO of 14,250 ppm (12,950 to
15,675).
2LC5o in ppm for 30 min.
5-12
-------
beagle dogs (8 hour/day, 5 days/week for 6 weeks) to 2200 ppm (11,880 mg/m3)
MC. The same number of animals were exposed continuously for 90 days to 135
ppm and 370 ppm (729 and 1,998 mg/m3). The repeated exposure to 2200 ppm
(11,880 mg/m3) did not result in any deaths or visible signs of toxicity,
although weight loss was observed in rabbits and dogs. The continuous expo-
sure at 370 ppm (1,998 mg/m3) did not cause any deaths, visible toxic signs,
significant growth depression or biochemical, hematologic or pathologic changes.
Continuous exposure to 135 ppm (729 mg/m3) resulted in three deaths (2 rats
and one rabbit) but no visible toxic signs or impaired growth in any of the
survivors. Autopsy and subsequent histopathblogic examination of the experi-
mental animals revealed lung congestion and pneumonitis which may have been
severe enough to have caused the deaths of the two rats and the rabbit, but
which were not attributed to the exposure.
The acute behavioral and toxic effects of ethanol and inhaled MC alone
and in combination have been examined by Woolverton and Balster (1981) on CD-I
albino mice. The animals were exposed to doses of MC ranging to-20,000 ppm
(108,000 mg/m3) for 30 minutes and then tested for behavioral decrements using
the inverted screen test, a test that requires mice to climb to the top of an
inverted screen. Lethality was measured at 24 hours. The inverted screen
test was scored by counting the number of animals to fall off plus the number
on the underside of the screen, expressing these numbers as a percentage. To
test the effect of ethanol, animals were first intubated with varying doses of
ethanol and then exposed to MC.
At the highest concentration of MC tested for behavioral effects (7,000
3
ppm; 37,800 mg/m ), half of the affected mice recovered within 5 minutes after
removal from the chamber and all recovered within 60 minutes. In contrast, at
the highest dose of ethanol tested (4 g/kg), half of the animals recovered
within 180 minutes and 80 percent recovered within 360 minutes. Twelve mice
were used for each data point.
The ECj-n for MC at 1 minute following a 30 minute exposure was in the
tDU -3
range of 4,644 to 5,778 ppm (24,077 to 31,201 mg/m ). The 24-hour LC5Q was in
the range of 22,080 to 22,404 ppm (119,232 to 120,981 mg/m3). For ethanol,
the ED™ was in the range of 1.7 to 2.7 g/kg and the 24-hour LD5Q was in the
range of 7.5 to 8.0 g/kg.
When the combined effect was evaluated, the investigators observed that
low doses of ethanol and MC resulted in a greater-than-additive response both
5-13
-------
on behavioral and lethal endpoints; higher doses of ethanol were additive or
less-than-additive with MC. In general, the LC50/EC5Q ratio of MC increased
as the dose of ethanol increased (from 0 to 2.0 g/kg). One anomalous finding
was at the ethanol dose level of 0.25 g/kg. In contrast to the potentiating
effect of ethanol at other dose levels, this dosage increased the EC™ for MC,
in comparison and reduced the LC50/EC5Q ratio for MC. This dosage of ethanol
was not reported to have effects of its own on the parameters measured but
apparently decreased the effects of MC. The combined effects data are shown
in Table 5-5. Additional information is discussed in section 5.2.2.
TABLE 5-5. COMPARISON OF LETHAL AND BEHAVIORAL EFFECTS.OF MC
IN COMBINATION WITH ETHANOL
Ethanol dose
(g/kg)
0
0.125
0.25
0.5
1.0
2.0
MC
LC56
(ppm)
22,241
20,273
19,852
19,067
17,924
16,834
MC
ECso
(ppm)
5,173
3,834
6,006
3,157
2,200
618
LC -/EC •
4.3
5.3
3.3
6.0
8.1
26.5
Source: Woolverton and Balster, 1981
Because the liver is a target organ for many chlorinated hydrocarbons,
the effects of MC on hepatic function have been studied by a number of investi-
gators.
McNutt et al. (1975) reported hepatic lesions in mice after continuous
inhalation exposure to MC. CF-1 mice were exposed to determine the nature of
pathologic alterations and to obtain data that would be useful in establishing
acceptable levels for continuous exposures. Two levels were chosen: 1,000
ppm (5,400 mg/ms) and 250 ppm (1,350 mg/ms), the first in order to produce a
definite, mild toxic effect and the other as an estimate of a concentration
that might give a threshold response or possibly, no detectable effect. Mice
were continuously exposed for up to 14 weeks with a weekly serial sacrifice.
In the high exposure group (from 1 to 14 weeks of exposure), cytoplasmic
alterations were observed in the centrilobular hepacytes upon electron micro-
scopic evaluation. Alterations consisted of vesiculation of the rough endo-
5-14
-------
plasmic reticulum with loss of attached polyribosomes and increased smooth
endoplasmic reticulum, microbodies, and triglyceride droplets. Necrosis of
individual hepatocytes occurred in 40 percent of the mice in the high exposure
group, exposed for 12 weeks. The necrosis was associated,with an acute inflam-
matory infiltrate and hypertrophy of Kupffer cells. Moderate liver triglycer-
ide accumulation was evident and peaked at 40 mg/gm of tissue after 7 weeks of
exposure; by 14 weeks the triglyceride level had decreased to 16 mg/gm.
In contrast, cytoplasmic alterations were described as mild to minimal in
the 250 ppm (1,350 mg/m3) group. Necrosis was not evident nor was fat accumu-
lation elevated above control values. The authors concluded that the observed
effects of MC were of a type similar to those produced by carbon tetrachloride
but appeared to be much less severe.
As part of the same study described above, rats, dogs, and monkeys were
also exposed continuously to 250 and 1,000 ppm (1,350 and 5,400 mg/m^) MC.
The most prominent observation in the rats was the presence of chronic Tespira-
tory disease which was found in 12 of 40 controls, 28 of 40 rats in the low
exposure.group and in 17 of 40 rats exposed to 1,000 ppm (5,400 mg/m3). Other
observations included focal areas of tubular dilation in the kidney. Since
the incidence in the treated and control animals was similar, the observation
was interpreted as being unrelated to the exposure. No evidence of fatty
infiltration of the liver was observed in the treated animals. No adverse
health responses were observed in dogs or monkeys that were related to expo-
sure.
The combined effect of nominal concentrations of methylene chloride (100
ppm) and MC (1,000 ppm) in rats, mice, dogs, and monkeys was investigated by
the Aerospace Medical Research Laboratory (1975). Animals were exposed con-
tinuously for 13 weeks. Though most animals were sacrificed, subgroups of
each species were held for an additional period of time to determine the
reversibility of any alterations that might occur. In rats, dogs, and monkeys,
there were no significant differences from the control animals. In mice,
however, there was a consistent finding in liver tissue of multifocal peri-
acinal areas in which there was vacuolization of surrounding hepatocytes.
Increased amounts of fat were observed in these periacinal areas. The effect
was reversible and ameliorated within 14 days post-exposure.
Table 5-6 shows the relative hepatotoxic effect of MC in comparison to
other chlorinated solvents. MC, according to Plaa et al., (1958) was judged
to be the least hepatotoxic of the seven solvents investigated.
5-15
-------
TABLE 5-6. THE RELATIVE HEPATOTOXIC EFFICACY OF CHLORINATED SOLVENTS
Compound
Relative hepatotoxic
efficacy
1,1,1-tri ch1oroethane
Tetrachloroethylene
Tri chloroethylene
Sym-tetrachloroethane
1,1,2-trichloroethane
Chloroform
Carbon tetrachloride
1
3
8
12
40
60
190
Adapted from Plaa et a!., (1958).
5.2.2 Nervous System and Behavior
There have been few animal studies of the effects of MC on the nervous
system and behavior. In the animal studies that have been performed, effects
of MC were either not observed or were observed at exposure levels of 4,000 ppm
(21,600 mg/m3) or greater (Table 5-7). The effects were upon motor abilities
(Woolverton and Balster, 1981), vestibular control (Larsby et al., 1978) or on
fixed-interval response rate (Moser and Balster, 1982). Studies apparently
have not been performed upon such behaviors as learning or extinction, accuracy
or efficiency of complex schedule-controlled behaviors or sensory discrimina-
tions. The endpoints that have been evaluated were studied in only a pre-
liminary manner. Nervous system electrophysiology, neurochemistry, and neuro-
pathology are other areas that warrant attention.
Exposure of 10 Sprague-Dawley male rats to 500 ppm (2,700 mg/m3) was not
found to significantly affect activity level. Rats were exposed by inhalation
for 4 days, 6 hours/day. They were evaluated one and 17 hours after the last
exposure in an open field setting. Ambulation, grooming, rearing, defecation,
and urination were measured (Savolainen et al. , 1977).
York et al. (1982) reported that exposure of female Long-Evans rats by
3
inhalation before and/or during pregnancy to 2,100 ppm (11,340 mg/m ) did not
affect mating (anecdotal data) nor were open field activity, running wheel
5-16
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rates affected in the offspring. Open field tests were done on post-partum
day 21 and running wheel tests from day 21 through day 110. Amphetamine
administered to all groups before testing on days 110 through 120 did not
alter running wheel rates with respect to controls.
Woolverton and Balster (1981) showed a concentration-related increase in
the percent of CD-I mice affected by MC inhalation on an inverted-screen
climbing test. The EC™ for the 12 mice evaluated was about 5,200 ppm (28,080
mg/m3). At a level of 7,000 ppm (37,800 mg/m?), about 80 percent of the mice
were affected. Tests were run beginning one minute after the termination of
exposure. Ethanol doses, given prior to MC exposure, shifted the concentration-
effects curve to the left by amounts depending upon MC concentration. It is
quite possible that the observed decrements were due to vestibular rather than
motor deficits.
The fixed interval response rate in adult male CD-I mice was measured
during inhalation exposure to 1,000, 2,000, 4,000, and 8,000 ppm (5,400,
110,800, 21,600, and 43,200 mg/m-) MC (Moser and Balster, 1982). Response
rate was reduced proportional to concentration in about 5 minutes after the
initiation of exposure, beginning at 4,000 ppm (21,600 mg/m ). It was unclear
whether mice were trained in unexposed conditions.
An apparent disturbance in vestibular function due to MC was reported in
rabbits by Larsby et al. (1978). MC was infused intravenously and the blood
level of MC was monitored. At 75 ppm, rabbits began showing nystagmus during
axial body tilts. A crude estimate made from the data reported by Astrand et
al. (1973) indicated that 75 ppm MC in human arterial blood would result upon
inhalation of air containing about 4,000 ppm (21,600 mg/m ).
Protein and RNA content in the brains of Sprague-Dawley male rats at 0,
3
2, 3, 4, and 6 hours after the last exposure to 500 ppm (2,700 mg/m ) MC for
4 days, 6 hours/day was measured by Savolainen et al. (1977). No effects were
significant after Bonferroni correction for repeated t-tests (Benignus and
Muller, 1982).
The available information indicates that, at high levels, MC acts as a
CNS anesthetic and at somewhat lower levels it has CNS depressant effects.
Until more pertinent information is available, very few conclusions about
threshold effects are warranted.
5-18
-------
5.2.3 Cardiovascular Effects ,
The cardiovascular effects produced by exposure to MC have been exten-
sively studied in recent years. Halogenated alkanes, like many organic solvents,
have been shown to sensitize the heart to catecholamines and at the same time
produce cardiac depression.
Krantz et al. (1959) were the first to observe the cardiac depression
produced by MC. Rats were deeply anesthetized with MC for 1 hour, at which
time the hearts were immediately removed. Cardiac ventricular slices were
promptly prepared and the oxygen uptake over the 1 hour period was measured.
MC anesthesia was associated with a significant diminution (33.3 percent) in
oxygen uptake of the myocardium. Blood pressure studies in dogs also revealed
that anesthesia with MC elicited a depressed response. At the point of respi-
ratory arrest, the blood pressure was reduced to approximately one-half its
normal value. Ether anesthesia under similar conditions results in only a
slightly depressed blood pressure. Six dogs and two rhesus monkeys were
anesthetized and electrocardiograms recorded. The pattern of the electro-
cardiogram was essentially unaltered; the heart rate was increased and the
T-wave was either flattened or inverted. At the point of respiratory arrest,
a depressed S-T segment was observed; however, tachycardia was absent.
Herd and co-workers (1973, 1974) confirmed the initial observations of
Krantz et al. (1959). They exposed dogs to 10,000 to 40,000 ppm (54,000 to,
216,000 mg/m3) MC per minute and observed a dose-dependent biphasic decline in
arterial blood pressure. Since cardiac output increased initially, the initial
decline in pressure (within 10 to 15 seconds after introduction of MC) was due
to a decrease in total peripheral resistance (TPR). Injection of phenyle-
phrine (pure alpha-agonist) reversed the peripheral vascular effects, indica-
ting that MC does not act directly on the vascular musculature. The second
phase of blood pressure decline was found to be associated with a decrease in
myocardial contractility, reflected by a decline in both heart rate and stroke
volume. Exogenous Ca reversed the MC-induced decline in myocardial con-
tractility, but had no effect on the initial phase of peripheral vasodilation.
Taylor et al. (1976) exposed New Zealand white rabbits to a series of
haloalkane chemicals. The animals were first anesthetized with sodium pento-
barbital, then fitted with various cannulas allowing for the measurement of
mean arterial pressure, left ventricular pressure, left ventricle dP/dt,
cardiac output, stroke volume, heart rate, left ventricular end-diastolic
5-19
-------
pressure, central venous pressure, and peripheral vascular resistance. Table 5-£
lists the left ventricular and hemodynamic effects of 50,000 ppm (270,000 mg/nr)
MC. There was no effect on heart rate, left ventricular end-diastolic pressure,
central venous pressure, or peripheral vascular resistance. During exposure,
no cardiac arrhythmias were observed nor was there a significant change in pH,
oxygen, or carbon dioxide tensions. While this level of exposure would probably
be lethal in humans, it is many orders of magnitude greater than levels found
in ambient air.
Numerous hydrocarbon compounds have been shown to sensitize the myo-
cardium to catecholamines. In humans, for example, several unexplained deaths
have been associated with solvent abuse or overexposure; ventricular fibrilla-
tion due to cardiac sensitization has been suggested as the underlying mechanism.
The cardiac sensitization potential of MC has been investigated under various
circumstances. In an early study, Rennick et al. (1949) reported MC and
epinephrine induction of idioventricular rhythms in dogs. Attempts to induce
MC anesthesia resulted in the sudden death, presumably of cardiac origin, of
two animals. Ventricular extrasystoles and ventricular tachycardia were seen
in five dogs under barbital anesthesia when epinephrine was injected after
"repeated small doses" of MC. Maximum sensitization of the heart to epin-
ephrine occurred after the administration of 0.25 to 0.4 ml/kg of MC. Further
administration of this compound produced severe hypotension. Somani and Lum
(1965), Lucchesi (1965), and Hermansen (1970) administered MC to dogs or mice
as a method for induction of cardiac arrhythmias in studying and characteriz-
ing the sensitivity and specificity of adrenergic blocking agents.
Clark and Tinston (1973) evaluated the cardiac sensitizing potencies of
14 halogenated hydrocarbons in conscious beagle dogs. During the last 10
seconds of a 5 minute exposure, epinephrine (5 mg/kg) was injected intraven-
ously (bolus). As a control, epinephrine was given prior to exposure and also
10 minute after the end of exposure. The electrocardiogram (lead II) was
monitored continuously during this procedure. Several dose levels of the
chemical being tested were used, each differing by a factor of two. The
concentration at which 50% (EC™) of the animals could be sensitized was
calculated by a moving average interpolation. The mean concentration of MC
which produced cardiac sensitization in 50% of the animals tested was 7,500
ppm (40,500 mg/m3). The authors found that they could directly predict the
EC50 for cardiac sensitization by knowing the vapor pressure at 37 C and the
5-20
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5-21
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partial pressure at the EC5Q. Table 5-9 lists the EC™ for a number of dif-
ferent halogenated hydrocarbons. MC appears to be a highly potent sensitizing
agent at near anesthetic levels, with tetrachlorodifluoromethane being the
most potent in the series. The authors concluded that cardiac sensitization
was probably a structurally nonspecific action and that it was physically
toxic.
TABLE 5-9. CONCENTRATION OF CHEMICALS CAUSING CARDIAC SENSITIZATION
AND THEIR PHYSICAL PROPERTIES (IN PPM)
Chemical
Tetrachl orodi f 1 uoromethane
Carbon tetrachloride
Trichloroethane (MC)
Halothane
Trichlorotrifl uoroethane
Methyl ene chloride
Tri chl orof 1 uoromethane
Di chl orof 1 uoromethane
Di chl orotetraf 1 uoroethane
Vinyl chloride
Propane
Bromotri f 1 uoromethane
Chl orotri f 1 uoromethane
EC50
1,200
5,000
7,500
20,000
io",ooo
24,000
12,500
25,000
100,000
50,000
200,000
200,000
800,000
Vapor pressure
at 37 C (mmHg)
Ps
99
190
210
480
524
661
1,186 ,
2,052
2,310
4,218
9,538
15,276
40,698
Partial
pressure at
EC5Q(mmHg)
Pcs
2
4
6
15
8
18
10
19
76
38
153
153
610
Relative
saturation
for cardiac
sensitization
Pcs/Ps
0.02
0.02
0.03
0.03
0.02
0.03
0.01
0.01
0.03
0.01
0.02
0.01
0.02
Source: Clark and Tinston (1973).
5-22
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Reinhardt et al. (1973) investigated five commonly used industrial and
household solvents, including MC, in order to rank their relative cardiac-
sensitizatibn potentials. In this screening test, the investigators con-
ducted experiments on unanesthetized, healthy male beagle dogs that had been
trained to breathe through a one-way face mask while supported in a standing
position by a sling. The dog inhaled house air for 7 minutes and the test
compound for 10 minutes. Epinephrine (8 ug/kg in 1 ml of normal saline) was
injected i.v. in 9 seconds as a control dose after 2 minutes of breathing air,
and as a challenge dose after 5 minutes of breathing the test compound. The
10-minute interval between the two doses was found to be adequate to prevent
additive effects when tested in 13 control dogs subjected to the same proce-
dures, but with air substituted for the test compound. A positive response
was considered to be either cessation of cardiac output (ventricular fibril-
lation) or the development of an arrhythmia that was not observed following
the control dose and that was considered to pose a serious threat to life
(multiple, consecutive beats of ventricular origin). The concentration of the
test compound being delivered to the animal was determined by gas chromato-
graphy at 2-minute intervals.
The results obtained for MC indicated no response in 12 dogs at a nominal
concentration of 0.25 percent (2,500 ppm) (V/V) and a marked response in 3 of
18 dogs exposed at a nominal concentration of 0.5 percent (5,000 ppm) (V/V);
12 of 12 responded at a nominal concentration of 1.0 percent (10,000 ppm)
(V/V). However, unlike the other substances, MC-induced ventricular fibril-
lation in the animals from the 1.0 percent group reverted to multiple consecu-
tive ventricular beats within a matter of seconds and eventually recovered to
a normal cardiac rhythm. The results confirm earlier reports that MC is
capable of sensitizing the dog heart to exogenous epinephrine although none of
the dogs died.
Trochimowicz and co-workers (1976) were concerned with the problem of
haloalkane-induced cardiac sensitization in patients who had survived myo-
cardial infarction. They induced myocardial infarction in beagle dogs and
tested for cardiac sensitization to epinephrine after exposure to MC at levels
of 2500, 3700, and 5000 ppm (13,500; 19,980; and 27,000 mg/m3). Myocardial
infarction did not significantly alter the threshold for cardiac sensitiza-
tion. There was no greater potential for cardiac sensitization among dogs
having recovered from myocardial infarction compared to normal healthy animals.
5-23
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Rabbits have recently been shown, by Carlson (1981), to respond to a
combined inhalation exposure to MC and a challenge dose of epinephrine, result-
ing in spontaneous arrhythmias (premature ventricular contractions). Controls
did not exhibit arrhythmias at an exposure level of 5,600 ppm (30,240 mg/nv?)
up to 60 minutes, in the absence of a challenge dose of epinephrine. Induc-
tion of arrhythmia was examined also by first administering phenobarbital (40
mg/kg) i.p. daily for 4 days prior to exposure or administering microsomal
mixed function oxidase inhibitors. Although none of the results reported by
Carlson (1981) were statistically significant, both inhibitors appeared to
increase the responsiveness of the heart to arrhythmias when compared to
controls. Fifty percent of the rabbits administered SKF-525A (50 mg/kg) 30
minutes prior to exposure exhibited arrhythmias after 7.5 minutes of exposure
to MC, following a challenge dose of 2 ug/kg epinephrine. In both inhibitor
groups there was a rapid return to normal cardiac rhythm during the recovery
period.
Blood analysis revealed trichloroethanol (TCE) as the principal metabolic
product. Phenobarbital resulted in an increase in blood TCE but it was not
statistically significant. The inhibitors resulted in an increased amount of
blood MC. This observation and the tendency of the inhibitor-treated animals
to show a higher incidence of arrhythmias led the investigators to conclude
that MC, not the metabolites, was responsible for the arrhythmias.
At sufficiently high levels (in the thousands of ppm, for durations of
exposure on the order of minutes), MC has been reported to cause significant
alterations in cardiovascular function in a variety of experimental animals,
including mice, rabbits, dogs, and possibly monkeys. The concentration levels
reported for these effects are generally more than an order of magnitude
3
greater than the current TWA for humans of 350 ppm (1,890 mg/m ) and are
likely to be lethal to the experimental animals if the exposure is continued
for more than several minutes. Indeed, some experimenters appeared to find it
necessary to increase the oxygen content of the inspired gas/air mixture to 40
percent in order to prevent asphyxia of the exposed animals. That they appeared
unable to find effects at lower exposure levels is noteworthy. Also noteworthy
is the fact that many of the investigators reported complete recovery of the
animals if the exposure was stopped. This was the case, for example, with the
dog experiments of Reinhardt et al. (1973) and Trochimowicz et al. (1976).
At necropsy following sacrifice of several of the animals several months after
exposure, there was no pathology attributable to the exposure per se. Delayed
cardiotoxicity from acute exposure has not been demonstrated.
5-24
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5.2.4 Dermal Effects
MC was one of seven solvents tested for dermal effects by Kronevi et a'l.
(1981). Guinea pigs (17) were anesthetized with pentobarbital, i.p., and one
ml of an inhibited formulation of MC was applied to an area (3.1 cm2) on the
clipped back skin, through a hole in the cover glass. The hole was closed
with a small piece of glass glued with orcyanoacrylate. At 15 minutes, 1.4,
and 16 hours, glass rings were removed and whole skin specimens from exposed
sites were excised and fixed in 10% formalin. Samples from adjacent non-
exposed sites were taken as controls.
Pyknotic nuclei were seen in all layers of epidermis, beginning with the
15 minute sample. It was reported that degeneration of nuclei progressed in
proportion to increased exposure times involving1pyknosis and karyolsis. A
marked intercellular edema was observed after 15 minutes; edema was hardly
recognizable at 1 hour and not at all after 4 and 16 hours; at this time, it
was totally replaced by junctional separation. A slight, diffuse pseudo-
eosinophilic infiltration appeared in the upper part of the dermis after 4
hours and became severe after 16 hours.
Karyopyknosis was a finding common to all the solvents evaluated. Kary-
olysis, intercellular edema and junctional separation was common to most of
the solvents.
The results of this one study suggest that skin contact with MC be mini-
mized.
5.3 TERATOGENICITY, MUTAGENICITY, AND CARCINOGENICITY
It has been proposed that the susceptibility of individuals to cancer is
related to their ability to metabolize carcinogenic compounds to benign mole-
cules and, conversely, to /their capacity to metabolize benign compounds to
carcinogens (Kellerman et al. , 1973). Susceptibility may be partly explained
by genetic differences involving enzymatic pathways, repair mechanisms, and
immune response mechanisms. An assessment of the carcinogenic potential of MC
must therefore take into account tissue concentrations of MC during typical
exposures as well as the extent and nature of MC metabolism. Both these
aspects have been extensively reviewed in Chapter 4. It can be concluded that
MC is metabolized in humans to a very small extent; about 6 percent or less of
the body dose is converted to carbon dioxide, trichloroethanol, and trichloro-
5-25
-------
acetic acid. The mechanism of this overall reaction is unknown. The avail-
able studies on carcinogenicity and teratogenicity are summarized in Table
5-10.
5.3.1 Teratogenicity, Embryotoxicity, and Reproductive Effects '
5.3.1.1 Overview—The basic viewpoints and definitions of the terms "terato-
genic" and "fetotoxic" were summarized by The Office of Pesticides and Toxic
Substances (U.S. EPA, 1980c) as follows:
Generally, the term "teratogenic" is defined as the tendency to
produce physical and/or functional defects in offspring iji utero.
The term "fetotoxic" has traditionally been used to describe a wide
variety of embryonic and/or fetal divergences from the normal which
cannot be classified as gross terata (birth defects) — or which are
of unknown or doubtful significance. Types of effects which fall
under the very broad category of fetotoxic effects are death, reduc-
tions in fetal weight, enlarged renal pelvis edema, and increased
incidence of supernumary ribs. It should be emphasized, however,
that the phenomena of terata and fetal toxicity as currently defined
are not separable into precise categories. Rather, the spectrum of
adverse embryonic/fetal effects is continuous, and all deviations
from the normal must be considered as examples of developmental
toxicity. Gross morphological terata represent but one aspect of
this spectrum, and while the significance of such structural changes
is more readily evaluated, such effects are not necessarily more
serious than certain effects which are ordinarily classified as
fetotoxic—fetal death being the most obvious example.
In view of the spectrum of effects at issue, the Agency suggests
that it might be useful to consider developmental toxicity in terms
of three basic subcategories. The first subcategory would be embryo
or fetal lethality. This is, of course, an irreversible effect and
may occur with or without the occurrence of gross terata. The
second subcategory would be teratogenesis and would encompass those
changes (structural and/or functional) which are induced prenatally,
and which are irreversible. Teratogenesis includes structural
defects apparent in the fetus, functional deficits which may become
apparent only after birth, and any other long-term effects (such as
carcinogenicity) which are attributable to jri utero exposure. The
third category would be embryo or fetal toxicity as comprised of
those effects which are potentially reversible. This subcategory
would therefore include such effects as weight reductions, reduction
in the degree of skeletal ossification, and delays in organ matura-
tion.
Two major problems with a definitional scheme of this nature must be
pointed out, however. The first is that the reversibility of any phenomenon
is extremely difficult to prove. An organ such as the kidney, for example,
may be delayed in development and then appear to "catch up". Unless a
5-26
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5-27
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series of specific kidney function tests are performed on the neonate,
however, no conclusion may be drawn concerning permanent organ function
changes. This same uncertainty as to possible long-lasting after effects
from developmental deviations is true for all examples of fetotoxicity.
The second problem is that the reversible nature of an embryonic/fetal
effect is one species might, under a given agent, react in another species
in a more serious and irreversible manner.
It is not possible, on the basis of limited available data, to define the
full potential of MC to produce adverse teratogenic or reproductive effects.
Human epidemiology studies are difficult to conduct in order to evaluate the
effects of MC on the exposed population. Each of the available mammalian
studies had methodological drawbacks that do not allow for conclusive evalua-
tion of the ability of MC to produce a teratogenic response over a wide range
of doses, which should include doses high enough to produce signs of maternal
toxicity and lower doses which do not produce this effect. Some of the tera-
tology studies used rats and mice and only single doses of MC which produced
signs of maternal toxicity. Other studies in chicken embryos have indicated
that MC disrupts embryogenesis in a dose-related manner (Elovaara et al., 1979).
However, since administration of MC directly into the air space of chicken
embryo is not comparable to administration of a dose to animals with a placenta,
it is not possible to interpret this result in relationship to the potential
of MC to cause adverse human reproductive effects.
5.3.1.2 Human Studies—No clinical reports associate maternal exposure to
methyl chloroform with congenital malformations in offspring. No epidemic-
logical studies have been performed.
5.3.1.3 Animal Studies—All studies performed to date in mammals have been
done in rats and mice. On the basis of these studies, it does not appear that
short- or long-term exposure to MC results in teratogenic effects in rats or
mice. Delays in fetal development have been observed in both species, but
these are believed to be reversible effects. The inhalation studies indivi-
dually evaluated dosages twice the maximum excursion limit for short-term
exposures of rats and mice and six times the maximum excursion limit for
long-term exposures of rats. In addition, no multigenerational studies of
mammalian reproductive performance have been performed under conditions of
inhalation exposure.
5.3.1.3.1 Rats--Schwetz et al. (1975) report results from Sprague-Dawley rats
exposed via inhalation to 875 ppm (4,725 mg/m3) of MC for 7 hours daily on
5-28
-------
days 6 through 15 of gestation (Day 0 '= the day sperm were observed in smears
of vaginal contents). Control rats were exposed to filtered air. Dams were
evaluated for body weight gain, food consumption and various organ weights.
Maternal carboxyhemoglobin level determinations were performed on blood samples
collected via orbital sinus puncture immediately following the third and tenth
(last) exposure. One-half of the fetuses in each litter were examined for
soft-tissue malformations (free-hand sectioning), and one-half were stained
and examined for skeletal malformations. One fetus in each litter was randomly
selected and evaluated using histological techniques following serial section-
ing.
Twenty-three litters from dams exposed to 875 ppm (4,725 mg/m3) of methyl
chloroform were evaluated. No effect was observed on maternal body weight or
food consumption. The mean absolute liver weight was increased as compared
with control, however the mean relative liver weight was unchanged. No embryo-
toxic or teratogenic effects were observed which were attributable to maternal
MC exposure.
York et al. (1982) exposed female Long-Evans rats by inhalation to dosages
of 2100 ± 200 ppm (11,340 ± 1,080 mg/m3) MC for 6 hours daily, 5 days per
week, in the following regime: (1) two weeks prior to mating through day 20
of gestation; (2) two weeks prior to mating only; (3) throughout gestation
only; (4) controls which were exposed to filtered air before and during preg-
nancy. Day 1 of pregnancy was designated as the day spermatozoa were observed
in smears of vaginal contents. One-half of each group was sacrificed on
day 21 and assessed for signs of maternal toxicity, embryotoxicity or terato-
genicity. The other one-half were allowed to deliver young naturally and the
young were later evaluated for behavioral alterations and for observation of
gross lesions. When possible, litters were culled to four pups of each sex on
day 4 postparturition and to two pups of each sex at 21 days postpartum. Pups
were evaluated for behavioral effects (see 5.2.2) and carcinogenicity. Surviv-
ing rats were sacrificed and necropsied at 12 months of age.
York et al. (1982) reported no teratogenic effects due to exposure but
both total fetal body weight and male fetal body weight were"significantly
depressed in litters when dams were exposed during pregnancy. Delayed ossifi-
cation was more frequent in fetuses which had been exposed before and during
pregnancy, and a delay in the development of the kidney was a more frequent
observation in fetuses in the group exposed prior and during gestation.
5-29
-------
These effects are thought to be indicative of a slight delay in development
since they occurred in more than 5 percent of the population, including controls
as well as exposed. No treatment-related behavioral effects were observed in
the pups, nor were significant signs of maternal toxicity observed.
5.3.1.3.2 Mice—Schwetz et al. (1975) exposed Swiss-Webster mice to 875 ppm
(4,725 mg/m3) of MC, twice the maximum excursion limit, for 7 hours daily, on
days 6 through 15 of gestation (day 0 = day a vaginal plug was observed).
Dams were Caesarean sectioned on day 18 of gestation and thirteen litters were
evaluated. Methodology similar to that described previously (Schwetz et al.
1975) in rats was used, with the exception that food consumption was not
monitored.
Mean and relative maternal liver weights in the exposed mice were slightly
but not statistically decreased. Also, fetuses from the exposed group were
slightly but not statistically smaller (both the crown-rump length and the
body weight). This type of observation is thought to indicate a slight, but
perhaps reversible delay in fetal development. No increased soft tissue or
skeletal variations of the fetuses were attributable to maternal exposure to
MC.
Lane et al. (1982) investigated the effects of MC on the reproductive
capability of ICR Swiss mice using a multigenerational reproductive protocol
modified to include screening for teratogenic and dominant lethal effects.
Animals were administered MC in drinking water for 35 days, then 10 male and
30 female mice (parental generation F/0) were mated to produce the first set
of offspring (FIA). After the F/1A were weaned, the F/0 adults were remated
to produce the second set of offspring (F/1B). A parental stock was chosen
from the F/1B litter (30 females, 10 males) to produce a second generation of
offspring (F/2A). After weaning the F/2A, the F/1B adults were remated to
produce offspring (F/2B) for use in teratology and dominant lethal screening
tests.
The MC (Aldrich Chemical Co. 95% pure with 3% dioxane) was dissolved in a
solution of Emulphor EL-620 (GAF Corp. Kinden, New Jersey) (1% Emulphor dis-
solved in deionized water) then further diluted with deionized water to con-
centrations of 0.58, 1.75, and 5.83 mg/ml or a nominal dose of approximately
100, 300, and 1000 mg/kg/day (assuming a 35 g mouse consumes 6 ml/day). The
test animals were continuously maintained on MC solutions or control solution
(deionized water, or water containing 0.17 mg/ml p-dioxane dissolved in 1.0%
5-30
-------
Emulphor solution). 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 decreases in the amount of fluid consumed in
either MC or the solvent control groups.
In this study MC 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 F/1A, F/1B, or F/2A generations. There were sporadic inci-
dences of increased mortality throughout the generations but these were not
dose-related. There was no difference in the litter sizes at birth, pup body
weights, survival of pups at days 4 and 21 of birth. There was a decrease in
the survival indices for the F/2A generation as compared to values for the
F/1A and F/B generation but these decreases were not dose related. In addi-
tion, in the dominant lethal screening there were statistically significant
differences; however, both increases as well as decreases were observed in the
ratio of dead to live fetuses. In the teratology screening, the continuous
administration of MC produced no apparent adverse reproductive effects, nor
visceral or skeletal abnormalities. In the lowest dosage groups, none of the
females had copulating plugs which made it impossible to time the pregnancies;
therefore, no teratological examinations were performed. The reason for this
effect was not disclosed in the paper.
In conclusion, this study is inadequate from the standpoint of deter-
mining whether MC has the potential to cause adverse reproductive or tera-
togenic effects because animals were not given doses high enough to produce
overt signs of toxicity. Another confounding factor is that a determination
of the amount of MC that may have partitioned into the head space of the
inverted drinking solution bottles was not made (Lane, personal communication,
1982). Thus, it is not clear how much MC each animal received between solu-
tion changes. It had been determined, however, that MC was stable in solu-
tion, based upon measurements of stored drinking solutions. It appears that
under the conditions of the experiment, mice (Swiss ICR) administered MC in
drinking water (nominal doses of 100, 300, and 1000 mg/kg/day) did not exhibit
any observable adverse reproductive effects.
5.3.2 Mutagenicity
Methyl chloroform (MC) has been tested for its ability to cause gene
mutations in bacteria, Drosophila, and yeast; chromosome aberrations in rats;
5-31
-------
micronuclei in mice; and unscheduled DNA synthesis in mouse and rat liver
cells iji vitro. Studies have also been reported on the ability of MC to reach
germinal tissue and cause adverse effects there. These studies are evaluated
and discussed below.
5.3.2.1 Gene Mutations in Bacteria--Several reports have been prepared about
the mutagenicity of MC in bacteria; all were conducted using the Salmonella/
mammalian microsome system (Table 5-11).
Because of MC's volatility and low solubility in water, special precautions
should be taken to ensure adequate exposure of test organisms. No such precau-
tions were taken in the tests conducted by Litton Bionetics (1975) for Dow
Chemical Company. In this study various formulations of MC (i.e. 99+%, 96%,
95.65%, and 93.75%) were assayed for mutagenicity in Salmonella strains TA1535,
TA1537, and TA1538, both with and without metabolic activation. A number of
separate experiments (both liquid suspension tests and spot tests) were conducted
each with and without an S9 mix prepared from various tissues (liver, lung,
testes) of PCB-induced mice, rats, or monkeys. No information was provided
concerning the stabilizers or other components present in the formulated
samples tested. Preliminary toxicity testing was conducted in association
with the liquid suspension assays to determine the appropriate test doses; the
test doses used were different for each formulation and are presented in Table
5-11. The contractor reported that the formulated samples were not soluble in
the aqueous testing environment and stated, for the 99+% formulation, that
"the toxicity from test to test was quite variable depending upon the ability
to effectively disperse the compound in the testing medium."
The low solubility of MC coupled with its high volatility raise the
concern that exposure of the test organisms may have been minimal in the tests
conducted by Litton Bionetics. Salmonella tester strain TA1535 was reported
to exhibit a reproducible mutagenic response to the 99+% formulation in suspen-
sion tests, but the response was considered by Litton Bionetics as equivocal
for the following reasons:
A. "The positive response is only evident at high dose levels
which generally result in low population survivals (high
toxicity). Thus, one cannot exclude some type of selection.
B. The data from activation plate tests does not indicate any
activity."
5-32
-------
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in most of the samples. The substances identified included vinylidene chloride,
1,1-dichloroethane, trichloroethylene, 1,1,2-trichloroethane, 1,2-epoxybutane,
and others. Experiments are needed to resolve whether the mutagenicity of
commercial MC is due to MC ger se or to stabilizer.components. Perhaps.fraction-
ation of a mutagenic commercial sample and subsequent demonstration that the
activity is due to substances in peaks other than the one containing MC would
resolve this issue.
The tests performed by Simmon and co-workers (1977) were conducted using
the standard battery of Salmonella typhimurium strains .TA1535, TA1537, TA1538,
TA98, and TA100, both with and without PCB-induced rat liver.S9 mix for meta-
bolic activation. The concentrations used for testing were 0, 100, 200, 300,
400, 500, 750, and 1000 ul/9-liter desiccator. A weak dose-related response
was observed for TA100 both with and without metabolic activation, showing
that commercially available MC (from Aldrich Chemical Company) possesses
mutagenic activity in Salmonella. When the study was repeated with the same
sample of MC, no significant mutagenicity was observed (Dr. Yi Wang, SRI
International, personal communication, 1982). Subsequently, a sample from
Matheson, Coleman, and Bell (MCB) was tested by Simmon and colleagues, and
weakly positive dose-related increases in mutant frequency were obtained at
doses ranging from 100 to 500 ul/dl desiccator. This sample was analyzed (Dr.
Ronald Spanggord, SRI International, personal communication, 1982) and found
to be 84% pure and to contain several contaminants, including p-dioxane;
1,1-dichloroethylene; nitromethane; 1,1-dichloroethane; chloroform, and the
reported mutagens trichloroethylene and 1,2-epoxybutane. After distillation
to increase the purity to 98% and acid hydrolysis to remove the 1,2-epoxybutane
contaminant, the material was reanalyzed and tested for mutagenicity. The MCB
samples showed nearly the same mutagenic responses before and after purifica-
tion (Dr. Kristien Mortelmans and Dr. ,Yi Wang, SRI International, personal
communication, 1982). These results are consistent with the hypothesis that
the response is due to MC itself and not to mutagenic stabilizers or contami-
nants. However, unidentified impurities (1-2% of the sample) remained in the
purified MCB sample; thus, it is not possible to ascribe the mutagenic activity
of this sample to MC unequivocally.
In the studies of Snow et al. (1979), two samples of MC were tested in
Salmonella strain TA100 both with and without an S9 metabolic activation mix-
ture prepared from the livers of MC-induced Syrian golden hamsters. Precau-
tions were taken to prevent evaporation by exposing the bacteria to doses of
5-38
-------
0, 500, 750, 1000, and 1500 pi in 5.6-liter Billups-Rothenberg modular incuba-
tor chambers. Similar linear dose-responses were observed (see Table 5-11)
for each of two MC samples. One of the samples was from Aldrich Chemical
Company (97% MC stabilized with 3% p-dioxane) and the other was from PPG
Industries (also estimated to be 97% pure, but the stabilizers were not report-
ed). These samples were analyzed by mass spectrometry three years after the
testing was conducted (Dr. Stephen Nesnow, U.S. EPA, personal communication).
The Aldrich sample was found to contain dioxane; 1,1- or 1,2-dichloroethylene;
nitromethane; trichloroethylene; methylisobutyl ketone; 1,1,2-trichloroethane;
and toluene. „ Acetone; nitromethane; methyl ethylketone, an unidentified
substance tentatively identified as dimethyl formamide; toluene; trichloroethy-
lene, and 1,1- or 1,2-dichloroethylene were found as contaminants in the PPG
sample. Of these impurities, only 1,2-dichloroethylene (vinylidene chloride)
and trichloroethylene have been reported in the 1iterature, to be mutagenic.
It is not known whether these chemical compositions differ from those of the
original samples tested for mutagenicity.
Butylene oxide was not identified in either sample tested by Snow et al.
(1979), yet this mutagen was present in 19 of 22 samples analyzed by Henschler
et al. (1980). It is possible that this substance was present in the original
samples tested by Snow et al. (1979) but has since degraded. However, if the
chemical analyses reflect the original composition of the material tested for
mutagenicity and butylene oxide was not present, it is likely that the positive
responses reported by Snow et al. (1979) were due to MC itself. Vinylidene
chloride and trichloroethylene were present only as minor components, and most
of the impurities were accounted for by acetone, methyl ethylketone, toluene,
and dioxane. Even,if one were to assume that the 3% impurities consisted ex-
clusively of vinylidene chloride or trichloroethylene, it is not likely that
the mutagenic responses can be accounted for by these substances. Both sub-
stances require the presence of an exogenously supplied metabolic activation
system to yield a positive response in bacteria, yet linear dose-related posi-
tive responses were obtained for the methyl chloroform samples without meta-
bolic activation. Thus, the positive responses obtained by Snow et al. (1979)
must have been due to MC per se or to a mutagenic stabilizer that degraded
between the time of testing and that of chemical analysis.
Nestmann et al. (1980) used Salmonella strains TA98, TA100, and TA1535
both with and without PCB-induced rat liver S9 mix in their tests of MC.
5-39
-------
Aliquots of 100, 500, and 1000 ul (Fisher Scientific; purity not given) were
placed in open glass dishes inside a dessicator. The plates were exposed to
MC vapors for 16 hours and incubated for a total of 72 hours before scoring.
The authors report that under the conditions of the test, MC was not detected
as mutagenic by strain TA98 but induced up to a 2.5-fold increase in numbers
of revertants in strain TA100 and up to a 6.5-fold increase in revertants in
strain TA1535. However, the authors' conclusions cannot be independently
assessed, because experimental values were not presented.
Gocke et al. (1981) used Salmonella strains TA100 and TA1535 both with
and without PCB-induced rat liver S9 mix for metabolic activation to assess
the mutagenic potential of MC (Merck Darmstadt, purity not given). Aliquots
of 500, 1000, 1500, and 2000 ul (values extrapolated from a figure in the
paper) were placed in airtight desiccators, and the bacteria were exposed for
8 hours. After incubation and scoring, a 1.3-fold increase in revertants was
recorded for TA100 and a 10-fold increase in revertants was recorded for
TA1535 at the highest dosage.
The results of Snow et al. (1979), Nestmann et al. (1980), and Gocke et
al. (1981) strengthen the claim by Simmon et al. (1977) that commercial samples
of MC are weakly mutagenic in Salmonella when exposure occurs in sealed chambers.
The nearly identical responses obtained with samples of MC obtained from dif-
ferent sources is consistent with the hypothesis that MC itself is a weak
mutagen in Salmonella, causing primarily base-pair substitution mutations.
However, firm conclusions cannot be reached because of the possibility that
the mutagenic activity is ascribable to chemical substances added as stabili-
zers or to impurities.
For proprietary reasons, manufacturers of MC are reluctant to divulge the
identity and amount of chemical substances they use as stabilizers in their
commercial products. Without this knowledge, however, it is not possible to
determine whether the mutagenicity of these products is due to MC or to its
stabilizers.
Commercially available samples of MC are clearly weakly mutagenic in
Salmonella. Two studies, both unpublished, have been conducted using MC
samples with known levels of identified mutagenic stabilizers (Domoradzki,
1980 and Williams and Shijnada, 1983). The argument is made that the activity
of commercial grade MC is due to the presence of highly mutagenic materials,
such as butylene oxide, which are present as stabilizers.
5-40
-------
In the study by Domoradzki (1980) CHLOROTHENE VG Solvent* (i.e., 93.33%
MC, 0.73% 1,2-butylene oxide, and 5.94% other stabilizer components), low
stabilized MC (i.e., 99.9%+ MC and 0.038% 1,2-butylene oxide), and 1,2-butylene
oxide (i.e., 99+% 1,2-butylene oxide, < 0.2% butyraldehyde, and < 1% iso-buty-
lene oxide) were evaluated for mutagenic activity in the Salmonel1 a/mammalian
microsome mutagenicity assay. Salmonella strains TA98, TA100, TA1535, TA1537,
and TA1538 were exposed to the test substances in airtight desiccator jars
with and without Aroclor 1254-induced rat liver S9 mix. The vapor pressures of
the test chemicals at 30°C were determined from Antoine constants, and the
concentrations of the test materials were calculated using the ideal gas law.
Test plates containing the indicator bacteria were placed in the desiccators,
exposed to the test material for 60 hours, and subsequently scored. Positive
and negative controls were performed concurrently with the test chemicals. No
monitoring of the actual exposure concentrations was performed.
Positive responses (approximately tenfold increases over the spontaneous
level) were obtained in strain TA1535 for CHLOROTHENE VG Solvent (at 20,664
and 41,328 ppm methyl chloroform, or 2.1 and 4.1%, respectively) and 1,2-buty-
lene oxide (from 39 to 1198 ppm or 0.001 to 0.12%) and in strain TA100 for
1,2-butylene oxide (from 599 to 1198 ppm or 0.06 to 0.12%) both with and
without metabolic activation. No increase in revertants over the spontaneous
level was observed when low stabilized methyl chloroform was tested in these
strains (Domoradzki, 1980).
Williams and Shimada (1983) also tested fully stabilized (Triethane 324A)
and low stabilized (Triethane 321, 99.80% MC) samples of MC for mutagenicity
in the Salmonella/mammalian microsome assay. The substances were obtained
from PPG Industries' Lake Charles Plant. Purity analyses were performed by
the Barberton Technical Center, but the percent composition of MC in the fully
stabilized material was not given, and the identities and amounts of stabiliz-
ing materials were not reported for proprietary reasons. Salmonella strains
TA98, TA100, TA1535, TA1537, and TA1538 were exposed to 0, 2.5, 5.0, 7.5, or
10% MC for 18 hours in sealed chambers and then incubated for an additional
30-54 hours. Gas chromatographic analysis of MC in the exposure chamber was
performed for one dose and showed that the desired air concentration of the
test material for the dose was reached within the limits of sampling error
^Trademark of Dow Chemical Company.
5-41
-------
(i.e., for the 5% dose level, 5% MC was measured at 0 hours, and 4.2% was
measured at 17 hours).
Treatment with low stabilized MC led to revertant counts twofold above
background for strains TA100 and TA1535 with and without metabolic activation
(i.e., Aroclor-induced rat liver S9 mix), but only at the 10% concentration
level (100,000 ppm) of MC. This concentration resulted in greater than 95%
toxicity. Fully stabilized MC caused increases in numbers of revertant colo-
nies in TA1535 at concentrations of 2.5% (25,000 ppm) and higher with and
without metabolic activation; increased numbers of revertants in TA100 were
detected at 2.5% (25,000 ppm) with activation and at 5% (50,000 ppm) without
activation (Williams and Shimada, 1983).
The results of Domoradzki (1980) and Williams and Shimada (1983) show
that commercially available samples of MC are mutagenic. They are consistent
with the hypothesis that the mutagenic activity is due to the presence of
mutagenic stabilizers, but they do not prove the hypothesis. In summary,
commercial samples of MC are weakly mutagenic to Salmonella TA1535 and TA100.
It may be that the mutagenic response is caused by chemical substances added
as stabilizers or to contaminants of manufacture.
5.3.2.2 Gene Mutations in Eukaryotes—In addition to testing MC in Salmonella,
Gocke et al. (1981) used the Drosophila sex-linked recessive lethal test
(Table 5-12). A 25 mM solution of MC in 5% saccharose was fed to wild-type
Berlin K male flies for an unreported period of time (25 mM is reportedly
close to the LD,-n). These males were then mated to Base females. Three
broods were scored (i.e., offspring from virgin females mated to treated males
on days 1-3, 4-6, and 7-10 after exposure). Two lethals were observed in 1226
cultures screened in the first brood; the values for broods 2 and 3 were 0/557
and 0/76, respectively. These values do not demonstrate an increased incidence
in sex-linked recessive lethals. However, the experiment is an inadequate
test, because the small number of chromosomes scored would preclude the detec-
tion of a moderate or weak mutagen. At least 7000 chromosomes must be scored
in order to make the determination that exposure to a chemical does not increase
the mutation frequency twofold (Lee et al., in press). In addition, the
actual amount of MC ingested by the exposed males in the tests by Gocke et al.
(1981) may have been less than predicted, because of the volatility of the
substance.
Loprieno et al. (1979) reported that pure, unstabilized MC (Dr. Silvio
Paglialunga, Coordinamento Medicina Ambiente Tossicologia Industriale, personal
5-42
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communication, 1981), administered to B6C3F1 hybrid mice by gavage (5000 mg/kg),
did not increase the incidence of forward mutations in Schizosaccharomyces
pombe in a host-mediated assay. The yeasts were injected intraperitoneally,
and measurements were made after treatment times of 3, 6, and 16 hours. The
control values (mutations/cells scored) for the 3 time points were 9/118,386,
8/80,552, and 11/87,183, respectively. The corresponding values for the
treated cells were 2/82,814, 9/115,831, and 16/165,588. In this forward
mutation assay, which is based on the detection of altered colony color, there
is no selection technique for detecting mutants. Therefore, the most important
consideration in the analysis of this test is the number of colonies scored.
No more than 1.65 x 105 cells were scored for any exposure period. Additional-
ly, no information was provided concerning the ability of MC to be absorbed
and transported to the peritoneum, thereby adequately exposing the yeast
cells. Thus, the possibility of weak mutagenicity cannot be excluded because
of the limited sample size, coupled with the possibility of limited exposure
to the test organisms. The report also does not provide adequate information
concerning the design of the tests, and this makes it difficult to assess the
significance of the results.
5.3.2.3 Chromosomal Aberrations—Two studies have been published on the
ability of MC to cause chromosomal aberrations (Table 5-13). In the test by
Quast et al. (1978) Sprague-Dawley rats were exposed to 0, 875, and 1750 ppm
MC for 6 hours/day, 5 days/week, for 52 weeks. The highest dose was about 1/8
the reported LD5Q. Three males and three females per group were sacrified,
and bone marrow cells were analyzed for aberrations. Fifty cells per male and
fewer than 50 cells per female were scored. Although no increased incidence
of aberrations was observed, the small number of cells analyzed makes the
study inadequate. Also there was no concurrent positive control.
Gocke et al. (1981) assessed the ability of MC (Merck, Darmstadt; purity
not given) to cause micronuclei in polychromatic erythrocytes (PCE). Two male
and two female NMRI mice were used for each of 3 dose levels (266, 1000, and
2000 mg/kg). The highest dose approximates the LD5Q for mice. Two intraperi-
toneal injections of each dose were given at 0 and 24 hours, and the animals
were sacrificed at 30 hours; bone marrow smears were made and 1000 PCE per
animal were scored for the presence of micronuclei. An apparent dose-related
response was observed. The untreated controls had 1.3% micronuclei compared
to 2.9% in the animals receiving 2 injections of 2000 mg/kg. Although these
values represent a twofold increase in micronucleus formation, the control
5-44
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value was low compared to the other controls reported in the paper (about 2%
PCE with micronuclei). Thus, the response is considered to be merely sugges-
tive of a positive effect.
5.3.2.4 Other Indicators of DNA Damage—Litton Bionetics (1975) tested MC for
its recombinogenicity, as indicated by mitotic gene conversion in the yeast
Saccharomyces cerevisiae (Table 5-14). The study was conducted at the same
time as the testing of MC in bacteria, and many of the deficiencies noted
above concerning the bacterial tests apply to the yeast tests as well (e.g.,
no special precautions were taken to prevent evaporation of the test compound;
the compound was not soluble under the conditions of the test; and the toxicity
results were highly variable). The results were negative, but the tests are
judged to be inadequate. The volatility and insolubility of MC and the absence
of reproducible, dose-dependent toxicity suggest that the cells may not have
been adequately exposed.
In unpublished papers, Williams and Shimada (1983) and Williams (1983)
tested samples of MC samples for their ability to cause unscheduled DNA synthe-
sis in rat and mouse primary hepatocyte cultures (HPC DNA repair assay). In
the Williams and Shimada (1983) report two samples were tested using rat
primary hepatocytes (Table 5-14). One was fully stabilized and contained
0.02% vinylidene chloride, 0.35% butylene oxide, 5.3% unidentified materials,
and 94.1£ MC. The other sample, referred to as "low-stabilized," contained
0.02% vinylidene chloride, 0.1-0.2% unidentified materials, and 99.80% MC.
Two types of test were conducted. One was a conventional HPC assay, in which
the hepatocytes were exposed to MC added to the culture medium. The other
test was a modified HPC assay, and the cells were placed in small glass dishes
3
in a sealed incubator and exposed to MC gas. Tritiated thymidine ( H-TdR) was
added to the culture medium in both types of tests. If MC caused DNA damage
3
repaired by the excision repair pathway, H-TdR would be incorporated into the
repaired DNA. To detect such incorporation, the cells were fixed on glass
slides and subsequently coated with radiotrack emulsion. After an incubation
period, the slides were developed and examined microscopically to count devel-
oped silver grains in the radiotrack emulsion over the cell nuclei.
Williams and Shimada (1983), used 2-acetylaminofluorene and vinyl chloride
as positive controls for the conventional assay and the modified gaseous expo-
sure assay, respectively. 2-acetylaminofluorene was highly active (170 grains/
nucleus at 10 M) and vinyl chloride was weakly active at 2.5 and 5% (5 and 11
5-46
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5-48,
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grains/nucleus, respectively) in increasing unscheduled DNA synthesis compared
to the negative controls (3 grains/nucleus).
In the modified HPC DNA repair assay, cells were exposed to dosages up to
5% MC in the air for 3 or 18 hours. No increase in the grain count was observed
(the number of grains/nucleus ranged from 0.6 ± 17 to 4.6 ± 3.8 in treated
cells compared to 2.75 ± 2.76 in the untreated controls). Similarly, negative
results were reported in the conventional assay where the cells were exposed
to concentrations as high as 1% MC in the medium.
There are several factors to consider in the analysis of this assay.
(1) The system is designed to detect the occurrence of a specific type of
repair (i.e. "long-patch" excision repair). Xenobiotics, which cause damage
that is not repaired or that is repaired by another mechanism, will not be
detected as possessing genotoxic activity in the system. (2) The endpoint
measured may not actually reflect repair synthesis of chromosomal DNA in the
nucleus (Lonati-Galligani et al. , 1983). Enhanced incorporation of 3H~TdR
Q
into mitochondria! DNA or suppressed H-TdR incorporation into mitochondria!
DNA without a concomitant increased or decreased incorporation into nuclear
DNA can lead to false negative or positive results, respectively. Thus, both
nuclear incorporation and cytoplasmic incorporation should be measured and the
dose-responses plotted for each to serve as a basis for deciding whether or
not the compound has induced UDS. (3) Nuclear grain counts vary with nuclear
size (Lonati-Galligani et a!., 1983); thus, it may be appropriate to express
grain counts as a percentage of the measured nuclear area. In the studies by
Williams and Shimada (1983) the values were transformed by subtracting cyto-
plasmic grain counts from total nuclear grain counts to give net grain count;
as a result, the data are difficult to evaluate, and the" way they are presented
may preclude the detection of weakly active agents. Thus, although the results
of this study are negative, they do not provide convincing evidence that MC
does not induce UDS.
In the Williams (1983) study, MC (source and purity not reported) was
assayed in the HPC DNA repair test using a conventional liquid exposure pro-
tocol. Primary hepatocytes from BgC^ mice and Osborne Mendel rats were
tested in two separate test series. A positive response, apparently inversely
proportional to dose, was obtained in the mouse cells between 10 M and 10 M
(a greater than 30-fold increase in grain count was noted at 10 M MC compared
to the negative controls). The inverted shape of the dose-response curve may
5-49
-------
be due to toxicity. MC was reported to be nontoxic between 10 M and 10 M,
but the criteria for measuring toxicity (i.e., absence of S-phase cells and
general morphology) is subjective and perhaps not sensitive. It would have
been preferable if lower doses had been used to determine the minimal effec-
tive dose and to determine a range of concentrations yielding positive dose-
response. A negative response was obtained in testing conducted with rat
cells (see Table 5-14) but the lowest dose tested was 10 M MC. At this dose,
the response in the mouse cells was suboptimal. It would be appropriate to
test lower doses using rat hepatocytes to rule out the possibility that a
positive response would have been observed had a lower dose been used. Positive
and negative controls were employed for both sets of experiments and responded
appropriately, but the concerns regarding the presentation of data in the
Williams and Shimada (1983) paper apply here as well. In spite of this, the
positive response in the mouse cells shows that a commercially available sam-
ple of MC is genotoxic in mammalian cells. It should be noted that the posi-
tive response may be due to stabilizer materials present in the sample tested.
It is noteworthy that MC was one of the compounds tested in the collabo-
rative study entitled the International Program for the Evaluation of Short-
Term Tests for Carcinogenicity (IPESTTC) (de Serres and Ashby 1981), which was
sponsored by Imperial Chemical Industries and the Medical Research Council in
the United Kingdom and the National Institute of Environmental Health Sciences
in the U.S.A. The study assessed the ability of short-term tests to predict
the carcinogenicity of 42 coded chemical substances of high purity in 23
different assays (e.g., gene mutation tests in bacteria, yeast, and mammalian
cells in culture; SCE formation iji vitro and i_n vivo; chromosome aberrations
TT\ vitro, etc.). Overall, the results were negative. Although a battery of
tests was conducted in this study, it is judged not to provide an adequate
assessment of the mutagenicity of MC. The intent of the program was to deter-
mine how well standard assays compared when testing coded samples. Many of
the chemical substances selected for testing were chosen because of the diffi-
culty of detecting them in short-term assays. Preliminary toxicity and range-
finding tests were limited, because only small samples of the test materials
were sent to the investigators. Furthermore, no information was provided to
the investigators about the solubility and volatility of the materials, and
the investigators were encouraged to use standard protocols. Thus, volatile
and insoluble compounds like MC would not be likely to give positive responses,
especially if they are weak mutagens.
5-50
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5.3.2.5 Gonadal Effects—There are two studies that bear on the ability of MG
to reach germinal tissue and cause adverse effects. In the study by Riddle et
al. (1981), male and female ICR Swiss mice were given daily doses of 0, 99.4,
2,640, or 8,250 mg/kg in their drinking water. It was reported that the oral
LD™ was about 10,000 mg/kg. No dose-dependent effects on fertility, gestation,
O U . .
viability, or lactation indices were observed, and no dose-related effects
were detected in the F/0 generation. These conclusions cannot be evaluated,
however, because they were presented in abstract form without supportive data.
Topham (1980) gave five daily i.p. injections of MC (purified by J. Ashby
of Central Toxicology Laboratory of ICI Ltd., Alderly Park, U.K.) to groups of
five (CBA x BALB/c) F male mice. The doses were 0.1, 0.25, 0.5, 1.0, and
2.0 mg/kg. The highest dose was reported to be about half the LD5Q; however,
this is not consistent with other investigators who reported the LD™ for i.p.
injection of MC in mice to be from 2 to 16 g/kg (e.g., Gocke et al. , 1981).
Five weeks after the last dose, smears of caudal sperm were coded and examined
for head abnormalities. Cyclophosphamide monohydrate (20 mg/kg/day/5 days) and
injections of either saline, corn oil, or-0.5% Tween 80 in water were used as
concurrent positive and vehicle controls, respectively. MC was reported to be
negative in this study, but the conclusions cannot be confirmed because no
data were presented. Therefore, conclusions cannot be drawn concerning the
ability of methyl chloroform to reach germ tissue because the information is
too limited.
5.3.2.6 Summary and Conclusions—Methyl chloroform has been tested for its
ability to cause gene mutations in Salmonella and DrosophiTa, chromosomal
mutations in rats and mice, and unscheduled DNA synthesis in rat hepatocytes.
It has also been tested for its ability to cause adverse effects in mouse
testes, as indicated by the dominant lethal test and the sperm abnormality
test. In addition, it was one of 42 chemicals tested in a battery of mutageni-
city assays (e.g., gene mutation tests in bacteria, yeast, mammalian cells in
culture; SCE formation i_n vitro and in vivo; chromosome aberrations j_n vitro,
etc;) as part of the International Program for the Evaluation of Short-Term
Tests for Carcinogenicity (IPESTTC). However, these tests and most of the
other experiments are judged to be inadequate because conventional protocols
were used; MC is difficult to test under such conditions because of its volatil-
ity and relatively low solubility in water. Care must be taken to ensure that
the indicator organisms (or appropriate cells in whole animal tests) are
5-51
-------
exposed to the materials in sufficient concentrations to provide an adequate
test.
Commercially available MC has been shown to be weakly mutagenic in
Salmonella, under special treatment conditions, by several different labora-
tories. Negative responses have been reported in gene mutation tests in yeast
and in the Drosophila sex-linked recessive lethal test. However, these negative
results cannot be interpreted as indicating a lack of mutagenic potential of
MC, because no precautions were taken to ensure adequate exposure of the test
organisms or because of such factors as small test populations or uncertainties
about the chemical composition of the material tested. Chromosomal aberration
tests have been reported to be negative, but these tests are also inadequate
because of inadequacies in experimental design, such as the failure to score a
sufficiently large experimental population. It should be noted that a micro-
nucleus test yielded an equivocal weak positive response, but this apparent
effect may be due to the low concurrent negative control values. Although MC
has not been shown to be mutagenic in a higher eukaryote, a positive response
was obtained in an unscheduled DNA synthesis assay using cultured primary
hepatocytes from mice. This test indicates the potential of MC for causing
genetic damage in mammalian cells.
The ability of MC to reach the gonads and cause genetic damage has not
been systematically studied. Only two studies were identified—a dominant
lethal assay and a test for morphologically abnormal sperm heads. Both re-
ported negative results but do not provide convincing evidence that MC has no
potential to cause heritable effects. The dominant lethal assay is not a sen-
sitive test and the assay for morphologically abnormal sperm heads is not
characterized genetically.
Based on the weight of available evidence, it is concluded that commer-
cially available samples of MC are genotoxic to mouse hepatocytes and are
weakly mutagenic in Salmonella under treatment conditions where sufficient
exposure is ensured. The available data are inadequate, however, for reaching
firm conclusions regarding the ability of MC to cause gene mutations in other,
organisms; however, the possibility that this substance, its associated sta-
bilizing materials, or its metabolites may have mutagenic effects in humans
has not been eliminated.
In view of the deficiencies in the available information, additional
testing is advisable. At least three additional assays should be conducted to
5-52
-------
resolve whether commercially available samples of MC are mutagenic in organisms
other than bacteria. These assays should include a test for gene mutations in
a nonbacterial system (e.g., mammalian cells in culture), a test for chromoso-
mal aberrations (e.g. , an in vivo or in vitro mammalian test), and a test for
other indications of DNA damage (e.g., sister chromatid exchange or DNA bind-
ing). Experiments are needed to determine whether methyl chloroform is mutagenic
in the absence of stabilizing materials or contaminants. An approach with the
potential to resolve this issue is to fractionate a mutagenic sample of MC and
subsequently demonstrate whether the mutagenic activity is associated with the
MC peak. Of course, in order to resolve the issue clearly, the fractional on
would have to be performed in such a manner as to separate the components of
the commercial sample without chemical modification. Even if it is shown that
the mutagenic activity of MC is due to one or more of the stabilizers, it
would be appropriate to ensure that commercial formulations of MC are also
adequately tested, because human exposures generally involve the commercial
formulations rather than purified samples.
Because of MC's physical properties, all additional studies should be
designed to ensure appropriate exposure of the indicator organisms; specifi-
cally, precautions should be taken to prevent excessive evaporation and to
overcome the low solubility of the compound in water.
5.3.3 Evaluation of the Carcinogenicity of Methyl Chloroform
The purpose of this section is to provide an evaluation of the likelihood
that MC is a human carcinogen. The evalution of carcinogenicity depends
heavily on animal bioassays and epidemiologic studies when available. However,
information on mutagenicity, pharmacokinetic behavior, and metabolism, particu-
larly in relation to interaction with DNA, have an important bearing on the
qualitative assessment of carcinogenicity. The available information on these
subjects is reviewed in the other sections of this document. This section
presents an evaluation of animal bioassays dealing with the carcinogenicity of
MC and its contaminant, 1,4-dioxane, two cell transformation studies, and
finally, a summary and conclusions dealing with all relevant aspects of carcino-
genicity.
5.3.3.1 Epidemioloqic Studies—No epidemiologic studies relating to the
carcinogenicity of MC are available.
5.3.3.2 Animal Bioassays - Rats—Three carcinogen bioassays have been completed
in rats: (1) a National Cancer Institute gavage study in Osborne-Mendel rats
5-53
-------
published in 1977; (2) a Dow Chemical Company inhalation study in Sprague-Dawley
rats published in 1978; and (3) a National Toxicology Program (NTP) gavage
study in F344/N rats described in a peer-reviewed draft report in 1983 (it has
since been withheld for re-review by NTP for possible serious data discrepancies
and, as such, is not further discussed here).
5.3.3.2.1 National Cancer Institute (1977) Rat Study—The National Cancer
Institute (NCI) bioassay (1977) was done with technical grade MC purchased
from Aldrich Chemical Company, Inc., Milwaukee, Wisconsin. The purity was
checked by Hazelton Laboratories of America, Inc., Vienna, Virginia, using
gas-liquid chromatography (GLC) and infrared spectrophotometry. Analyses by
GLC showed that it contained 95% MC and 3% 1,4-dioxane, an inhibitor routinely
added to commercial preparations of MC. The remaining 2% of the GLC peak area
contained several minor impurities, two of which may have been 1,1-dichloro-
ethane and 1,1-dichloroethylene. In this study, Osborne-Mendel rats were
treated with 750 mg/kg and 1500 mg/kg MC in corn oil five times a week for 78
weeks by gavage (NCI 1977). The rats were observed an additional 32 weeks
with the experiment ending at 110 weeks. Both males and females were used,
with 20 of each sex being untreated controls and 50 of each sex being tested
at each MC dose level.
The study was inadequate because only 3% of the treated rats survived to
the end of the experiment. The survival of both sexes of dosed rats was less
than that of the matched control groups, which was also inadequate in the
males. In male rats, 6/20 (30%) of the controls, 32/50 (64%) of the low-dose
group, and 36/50 (72%) of the high-dose group died within a year of the start
of the study. The Tarone statistical test of survival showed a dose-related
positive trend (P < 0.001) in the proportions of deaths over the period of the
experiment, although this differential mortality is not reflected in the
survival at 78 weeks. In female rats, 1/20 (5%) of the matched controls,
24/50 (48%) of the low-dose group, and 21/50 (42%) of the high-dose group died
in the first year. As in male rats, the statistical test for a positive
dose-related trend was significant (P < 0.04). In both sexes, the early
mortality in the treated rats may have reduced the incidence of late-appearing
tumors; this is especially true in the males, since none survived to the
scheduled termination of the study.
Fewer of the rats receiving MC survived at both 78 and 110 weeks than did
the positive control rats receiving the known carcinogen carbon tetrachloride
5-54
-------
(Table 5-15). A variety of neoplasms (Table 5-16) were observed in the MC-
treated groups, but with incidences not statistically different from matched
controls. Because of the high mortality in rats, this negative result cannot
be used to draw any conclusions regarding the carcinogenicity of MC. The NCI
Clearing House on Environmental Carcinogens concluded that the carcinogenicity
could not be determined from this study (NCI Clearing House 1977). It should
be noted that the isomer, 1,1,2-trichloroethane, is carcinogenic in mice,
inducing liver cancer and pheochromocytomas in both sexes. Dichloroethanes,
tetrachloroethanes, and hexachloroethane also produce liver cancer in mice and
other types of neoplasms in rats (Table 5-17).
5.3.3.2.2 The Dow Chemical Company (1978) Rat Study—The Dow Chemical Company
study (Quast et al., 1978) treated groups of Sprague-Dawley rats by inhalation
under conditions that were similar to those experienced by workers (6 hours/day,
5 days/week, over one-half of a lifetime). Rats were treated 12 months and
observed until death or until they reached the age of 31 months. Doses of 875
and 1750 ppm were 2.5 and 5 times the threshold limit value of 350 ppm. The
composition of the formulation of the MC is given in Table 5-18. The average
frequency of tumor occurrence in the treated animals was similar to that of
controls (Table 5-19).
When tumors at each site by tumor type, both benign and malignant, were
examined, there were eight differences between control and treated animals at
the P < 0.05 level (Fisher Exact Probability Test). Seven of these were
decreased tumor incidence; one was an increase in ovarian granulosa cell
tumors in females at the 875 ppm dose. There were no ovarian tumors in 189
controls; however, there were three in 33 female rats treated at 875 ppm (P =
0.003), and two in 82 female rats treated at 1750 ppm (P = 0.14). Since there
was a smaller response at this site in the high-dose group than in the low-dose
group, the positive findings may not be related to the administration of MC.
The Dow study suffers from two drawbacks: (a) the animals were treated
for only 12 months rather than a lifetime, but they were observed for another
12 months, and (b) it is not evident that the maximum tolerated dose was used
during the treatment period. There is no evidence that a range-finding study
(subchronic) had been done before the start of the experiment. The treated
animals in the Dow study did not differ from the untreated animals in body
weight, terminal organ weight, or mortality. The only sign of toxicity was an
increased incidence of focal hepatocellular alterations in female rats at the
highest dosage.
5-55
-------
TABLE 5-15. COMPARISON OF SURVIVAL OF CONTROL GROUPS, METHYL CHLOROFORM-TREATED
AND CARBON TETRACHLORIDE-TREATED (POSITIVE CONTROL) RATS
(NCI Bioassay 1977)
control
low dose
high dose
Females
Methyl Chloroform
Carbon Tetrachloride
Group
Initial
No. of
Animals
Number
Alive at
78 Weeks
Number
Alive at
110 Weeks*
Initial
No. of
Animals
Number
Alive at
78 Weeks
Number
Alive at
110 Weeks*
Males
20
50
50
7
1
4
0
0
0
20
50
50
20
34
35
12
15
8
control
low dose
high dose
20
50
50
14
9
12
3
2
1
20
50
50
18
38
21
14
20
14
*Time in study at last weighing.
5-56
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TABLE 5-18. COMPOSITION OF THE FORMULATION OF METHYL CHLOROFORM
USED IN CHRONIC INHALATION STUDIES IN RATS
(Quast et al. 1978)
Compounds1*
Liquid volume%
Calculated weight%
Methyl chloroform
Nitromethane
Butyl ene oxide
1,4-Dioxane
94.71
0.44
0.74
3.93
95.88
0.38
0.46
3.11
^Analysis of methyl chloroform Lot TA02013B by gas chromatography.
TABLE 5-19. AVERAGE FREQUENCY OF TUMOR OCCURRENCES IN RATS TREATED WITH
METHYL CHLOROFORM
(Quast et al. 1978)
Control
875 ppm
1750 ppm
Number
Male
189
91
93
of Animals
Female
189
92
93
Total
Male
1.06
0.85
1.11
Neoplasms/Animal
Female
2.97
2.67
3.23
5-3.3.3 Animal Bioassays - Mice—Two carcinogen bioassays have been completed
in B6C3F1 mice: (1) the National Cancer Institute (NCI) study in 1977 and (2)
the study of the National Toxicology Program (NTP), released as a preliminary
report in 1983. However, since the NTP study is undergoing re-review by NTP
for possible serious data discrepancies, only the NCI study will be discussed
here.
5-3.3.3.1 NCI (1977) Mouse Study—In the NCI (1977) bioassay, B6C3F1 hybrid
mice were used with 20 animals of each sex in the control group and 50 animals
of each sex at each treatment dose. The time-weighted average doses were 2807
mg/kg and 5615 mg/kg. The mice were treated by gavage 5 days a week for 78
weeks and observed for another 12 weeks for a total of 90 weeks in the experi-
ment.
5-59
-------
Table 5-20 summarizes the survival of the animals at 78 and 90 weeks. It
shows that only 25 to 40% of those treated with MC survived until the time of
terminal sacrifice. The NCI concluded that this early mortality in mice
receiving MC may have lowered the incidence of late-appearing tumors. In
•addition, the treated animals gained less weight than the controls.
TABLE 5-20. COMPARISON OF SURVIVAL OF CONTROL GROUPS,
METHYL CHLOROFORM-TREATED, AND CARBON TETRACHLORIDE-TREATED
(POSITIVE CONTROL) MICE
(NCI Bioassay 1977)
Methyl Chloroform
Group
Males
control
low dose
high dose
Femal es
control
low dose
high dose
Initial
No. of
Animals
20
50
50
20
50
50
Number
Alive at
78 Weeks
6
21
14
12
28
14
Number
Alive at
90 Weeks
2
15
11
11
23
13
Carbon
Initial
No. of
Animals
20
50
50
20
50
50
Tetrachloride
Number
Alive at
78 Weeks
13
11
2
18
10
3
Number
Alive at
90 Weeks
7
0
1
17
0
1
A variety of neoplasms (Table 5-21) were observed in treated groups but
with incidence not statistically different from matched controls. Because of
the high mortality in mice, this negative result cannot be used to draw any
conclusions regarding the carcinogenicity of MC.
5.3.3.4 Cell Transformation Studies—Price et al. (1978) exposed Fischer rat
embryo cell cultures (F1706, subculture 108) to MC liquid at concentrations of
9.9 x 10+1 and 9.9 x 102 pM for 48 hours. MC was diluted with growth medium
to yield the appropriate doses. The MC sample obtained from the Fisher
Scientific Company was purportedly > 99.9% pure, but a personal communication
from Mr. Carlson of the Fisher Scientific Company revealed that the MC (which
was supplied by Fisher to Dr. Price) was really the product of the Dow Chemical
Company and was actually about 95% pure. The chemical composition of Dow's MC
as reported by Quast et al. (1978) of the Dow Chemical Company is given in
5-60
-------
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5-61
-------
Table 5-18. The cells were grown in Eagles minimum essential medium in Earle's
salts supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-
essential ami no acids, 100 ug penicillin, and 100 (jg streptomycin per ml.
Quadruplicate cultures of Fischer rat embryo cells (F 1706, subculture 108) at
the stage of 50% confluence were treated for 48 hours with low (9.9 x 10 |jM)
2
and high (9.9 x 10 uM) doses of MC. These doses were determined in previous
experiments to be only minimally toxic. After treatment, cells were cultured
in growth medium alone at 37°C and observed for successive subcultures.
Transformation of cells was characterized by progressively growing foci
composed of cells lacking contact inhibition and orientation. In the low- and
high-dose cultures, transformation was first observed at 23 days (two subcul-
tures after treatment) and 44 days (five subcultures after treatment). There
was no transformation of cells grown in medium alone or in the presence of
1:1000 acetone concentration.
At the fourth subculture after treatment, the cells were plated on soft
agar (50,000 cells per dish) and held for 4 weeks at 37°C in a humidified C02
incubator prior to staining. In the low- and high-dose cells, 52 and 55
macroscopic foci were observed, respectively.
At the fifth subculture, cells from the low-dose treatment were injected
subcutaneously into newborn Fischer 344 rats. Sixty-eight days after injection,
fibrosarcomas were observed in all of the eight animals treated. Cells grown
in growth medium alone were not injected, but cells treated with 1:1000 acetone
induced no local fibrosarcomas in 13 animals injected.
Hatch et al. (1983) exposed Syrian hamster embryo cells to gaseous MC and
measured the chemically induced enhancement of cell transformation by adeno-
virus. The agent used, obtained from Aldrich Chemical Company, was said to be
97% pure and to have 3% 1,4-dioxane added as a stabilizer (manufacturer's
specifications). A special gas flow chamber was constructed to ensure exposure
to the agent. They found that a statistically significant (P < 0.05) enhance-
ment of activity occurred at three of four dose levels tested. They also
mentioned that MC from other sources, which had no 1,4-dioxane, was just as
active, but did not publish data for these other preparations.
5.3.3.5 Carcinogenicity of l,4-Dioxane--Methyl chloroform contains a small
amount of stabilizing substances. The concentration of specific stabilizers
that had been identified in various commercial MC products is shown below
(Aviado 1977 and Detrex 1976, cited by Mazaleski 1979).
5-62
-------
Volume %
0.4 -
0.4 -
2.5 -
1.0 -
1.0 -
1.0 -
0.2 -
1.0 -
1.8
0.8
3.5
1.4
1.4
1.4
0.3
1.4
Nitromethane
Butylene oxide
1,4-Dioxane
Dioxolane
Methyl ethyl ketone
Toluene
2-Butyl alcohol
Isobutyl alcohol
Not all of these stabilizers are in every product, but the maximum total
inhibitor package (combinations of stabilizers) appears to be between 7 and 8%
by volume (Aviado 1977, cited by Mazaleski 1979).
Since 1,4-dioxane is a contaminant in MC (about 3%), the carcinogenicity
of 1,4-dioxane has been studied extensively. The results of these studies are
summarized in Table 5-22.
It should be noted that 1,4-dioxane causes liver and nasal tumors in more
than one strain of rats and hepatocellular carcinomas in mice. Liver tumors
have been induced by dioxane in both male and female rats, as well as in mice.
These animal results, coupled with a reported finding of nasal carcinomas in
furniture workers exposed to 1,4-dioxane (NCI 1978, p. 108), suggest that
1,4-dioxane is a potential human carcinogen. A detailed evaluation of the
carcinogenicity of 1,4-dioxane is currently being prepared by the CAG.
5.3.3.6 Summary and Conclusions—No epidemiologic studies are available
investigating the carcinogenicity of MC.
In animal tests, two rat studies have failed to produce positive results
for carcinogenicity. The NCI (1977) gavage study in Osborne-Mendel rats could
not be interpreted because of extremely poor survival. The Quast et al.
(1978) inhalation study in Sprague-Dawley rats showed no carcinogenic response
except for ovarian granulosa cell tumors in the low-dose group. This site was
not affected in the other rat studies. However, the lack of toxic effects in
the Quast et al. (1978) study indicates that the dose was too small for maximum
sensitivity.
In B6C3F1 mice, only one study is available. The NCI (1977) study showed
no response, although relatively high mortality limited its sensitivity.
Technical grade MC has been shown to be weakly mutagenic in Salmonella
and to transform rat and Syrian hamster animal cells in vitro. Technical
5-63
-------
1
4-DIOXANE
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grade MC contains about 3% of the stabilizing compound 1,4-dioxane, which
shows evidence of being an animal carcinogen. The possibility that this
contaminant is responsible for the responses observed has apparently been
ruled out in the case of hamster embryo cells, but remains an open question
for the Salmonella findings and the rat embryo cell responses.
In conclusion, there are negative carcinogenic results in rats and mice
and the possibility of contamination as a cause of the weak response in the
mutagem'city of MC.
If the criteria of the International Agency for Research on Cancer (IARC)
were used, the overall weight of evidence would indicate that the overall
evaluation of MC would be equivalent to the IARC group 3 category of chemicals,
in which a chemical cannot be classified as to human carcinogenicity.
5.4 SUMMARY OF ADVERSE HEALTH EFFECTS AND LOWEST OBSERVED EFFECTS LEVELS
5.4.1 Inhalation Exposure
The high volatility of methyl chloroform (MC), the extensive use of this
compound as an industrial solvent, and the early interest in MC as an anesthetic
has resulted in many reports in the scientific literature of human exposure to
MC. Human experimental studies using acute and subchronic exposures have been
performed using a few clinical-level tests of motor and cognitive performance.
In many cases, these studies were poorly documented and studied only a few
subjects using non-conservative statistics, where statistics were used at all.
No studies using sensitive tests of cognitive performance or tests of vigilance
or sensory function were found. While information on human chronic inhalation
exposure to MC is available from studies of occupationally exposed groups,
there is uncertainty in these studies as to the exact extent of exposure; some
studies are complicated by simultaneous exposure to other chemicals used in
the workplace. Although these human studies have deficiencies which preclude
any individual study from being used for human risk assessment, the combined
information presented in these studies can be used to estimate approximate
dose-response relationships which provide a relatively clear description of
the toxic effects of MC. However, because no quantitive studies or sensitive
dependent variables were studied, no definitive conclusions about the effects
of human exposure of MC can be drawn at this time.
Studies in experimental animals are available for acutn and subchronic
exposure to MC, but the uncertainty of extrapolation from animals to humans
5-65
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makes this animal data useful only in a supportive role for human risk assess-
ment.
5.4.1.1 Effects of Single Exposures—Lethal effects of acute exposure to
MC have been reported after accidental exposure and abuse, with death result-
ing from CMS depression followed by respiratory and cardiac failure (Hall and
Hine, 1966; Bas, 1970; Stahl et al. , 1969; Bonventre et al., 1977; Klienfeld
and Feiner, 1966; Hatfield and Maykoski, 1970; Caplan et al., 1976). From the
levels of MC in the blood and brain of cadavers, estimates have been made of
the concentration of MC present in the air during the time the victim was
3
exposed. These estimates range from 200 to 102,900 ppm (1,080 to 555,660 mg/m ),
with the majority of values being between 6,700 and 18,600 ppm (36,180 and
o
100,440 mg/m ) (Table 5-23). However, these estimates are extremely crude and
probably low since it is not known to what degree the MC had dissipated from
the victims' bodies prior to obtaining tissue samples. Also, some of the
observed deaths which occurred at relatively low MC concentrations may have
resulted from aspiration pneumonia prior to extensive involvement of the CNS.
A clearer indication of the levels of MC needed to produce narcosis is available
in the study of Dornette and Jones (1960) investigating the use of MC as an
anesthetic in humans. Although this study gives an indication of the levels
of MC which cause CNS depression, presumably a higher concentration would be
needed to cause depression severe enough to result in respiratory or cardiac
failure. From these data and the LC5Q concentrations of 14,250 ppm (75,950
mg/m ) obtained by Adams et al. (1950) for rats, it can be estimated that a
3
single short-term exposure to as much as 5,000 ppm (27,000 mg/m ) would not be
expected to be lethal; exposure to higher concentrations and longer duration
of exposure, however, could produce narcosis and possibly death.
The effects of exposure of human volunteers to MC for single short-term
periods were reported for MC levels far below the levels which cause narcosis
(Table 5-24). Stewart et al. (1961) exposed 7 subjects to vapors of MC which
3
were increased from 0 to 2,600 ppm (0 to 14,040 mg/m ) in increments of 15
minutes each. The progress of observed effects is presented in Table 5-1,
3
with 6 of 7 subjects complaining of throat irritation at 1,900 ppm (10,260 mg/m ).
The progressive exposure method used in this study and the small number of
subjects makes the results difficult to interpret. However, in a second
3
experiment in which subjects were exposed to 900 ppm (4,860 mg/m ) MC for
periods of 20, 35, or 73 minutes, difficulty was observed in the performance
5-67
-------
TABLE 5-24. MOM-LETHAL EFFECTS OF METHYL CHLOROFORM OM HUMANS
Study
Dose/Species
Effect
Oornette & Jones, 1960
Siebecker et al. , 1960
Stewart et al. , 1961
Torkelson et al., 1958
Torkelson et al., 1958
Stewart, 1968; 1971
Am. Ind. Hyg. Assoc.
Stewart et al., 1969
Stewart et al., 1975
Gamborale and Hulten-
gren, 1973
Salvini et al., 1971
Maroni et al., 1977
Seki et al., 1975
Kramer et al., 1978
Stewart and Andrews,
1966
Stewart, 1971
Litt & Cohen, 1969
10,000-26,000 ppm
6,000-22,500
Anesthesia
0-2,650 ppm
for 15 min
500 ppm
900-1000 ppm
900-1000 ppm
for 75 min
1,900 ppm for
5 min
Results of single
exposures
500 ppm
6.5-7 hr/day for
5 days
500 ppm
250, 350, 450, 550
ppm for 4-30 min
periods
450 ppm for 2-4 hr
periods
110-990 ppm
4, 25, 28, 53 ppm
11-838 ppm -* x = 115
Induction of anesthesia
Maintain light anesthesia
Depressed BP during mod. an-
esthesia
Tendency to develop ventri-
cular arrhythmias during
hypoxia (reversed by oxy-
genation)
Little change in EEC-similar
to halothane anesthesia
See Table 5-1
No effects
See Table 5-2
Slight eye irritation
and lightheadedness;
Flanagan and Rombert test
showed slight loss of
coordination and equili-
brium
Equilibrium disturbed,
positive Romberg test
See Table 5-7
Mild subjective
complaints; abnormal
Romberg
See Table 5-3
Change in manual dexterity
and perceptual tests in
exp. vs. control groups
Decreased perceptive
capability under stress,
eye irritation
No effects
No effects
No effects, although SGPT
and albumin were different
in exposed vs. control
group.
See Table 5-4
5-68
-------
of the Romberg tests, and some lightheadedness was experienced. No effects
o
were observed in subjects exposed to 500 ppm (2,700 mg/m ) for 1C for 78 or
186 minutes. In both of these experiments, commercial grade MC was used which
contained the inhibitors dioxane (2.4-3.0%), butanol (0.12-0.3%) and small
amounts of 1,2-dichloroethane. These compounds may have also had some effect
on the subjects. In support of the above findings, Torkelson et al. (1958)
obtained similar results with 2 groups of 4 subjects each exposed to 500 or
a
900 ppm (2,700 or 4,860 mg/m ) of pure MC for 75 to 90 minutes. Subtle changes
in perceptive capability were observed following exposure to 350 and 450 ppm
3
(1,890 and 2,430 mg/m ) MC by Gamborale and Hultengren (1973) and Salvini et
al. (1971). However, these last two observations are questionable since the
effects of menthol used to mask the odor of MC in the first study were not
evaluated, and in the second study the control subjects were not matched for
all intervening variables such as food and drinking habits. Since the above-
mentioned studies were largely qualitative and since only gross dependent
variables were observed, conclusions are tentative at best.
Although a precise dose-response gradient of effects produced by MC
exposure cannot be derived, it is possible to establish a crude relationship
between exposure and effects from a compilation of the human data available.
A summary of the estimated dose-response relationships for acute effects of
single short-term exposures is presented below:
>5,000 ppm
1,900-2,650 ppm
1,000 ppm
350-500 ppm
100 ppm
Onset of narcosis
Lightheadedness, irritation of the throat
Disturbance of equilibrium
Slight changes in perception, obvious odor
Apparent odor threshold
The approximate concentrations of MC which elicit a particular adverse
effect have greater uncertainty at the two extremes, that is, at levels greater
3
than 10,000 ppm (270,000 mg/m ), and the no-observed-adverse-effect level
5-69
-------
* 3
(NOEL) , 350-500 ppm (1,890 to 2,700 mg/m ). This results from the inherent
difficulties in obtaining exact exposure values in the cases of accidental
overexposure to MC, and the difficulty in the acquisition of either quantita-
tive or qualitative data on slight behavioral changes at the lower exposure
levels. Acute overexposure to MC may be limited by the low odor threshold in
3
humans of 100 ppm (540 mg/m ). Others report that an obvious odor is present
3
at 500 ppm (2,700 mg/m ). It also should be noted that there are no reports
of residual adverse effects from a single exposure to MC.
Major uncertainties about the dose-response relationship estimated for MC
from the compilation of the available human reports derive from (1) the lack
of information on the length of exposure in accidental exposure and (2) the
short exposure duration (<2 hours) in most of the studies in which controlled
exposures of human volunteers were used. Although inhaled MC is retained in
the lungs in proportion to the concentration in the air, the time to reach
pulmonary steady-state conditions is relatively long (4 hours). Since the
maximum body burden from a given exposure to MC was probably not reached in
any of these studies, it is likely that exposures for longer times would
result in a greater body burden and more severe effects at a given concentra-
tion. In the studies of the inhalation toxicity of MC, there are insufficient
data to predict the results of the increased body burden of MC obtained by
extending the exposure until steady-state conditions are obtained. However,
from pharmacokinetic data presented in Figure 4-1, it can be reasonably con-
cluded that the exposure levels noted in the above dose-response table would
not shift by more than 30 percent. It should be emphasized that these approxi-
mations are only crude estimates.
5.4.1.2 Effects of Intermittent or Prolonged Exposures
There is a substantial amount of information from animal studies on the
effects of subchronic or chronic inhalation exposure to MC. There is also a
lifetime chronic study in rats. As discussed below, the only experimental
study of humans involved repeated exposure to MC 5 days per week for 3 weeks
and is of such a short duration that no information can be extrapolated from
this study as to the long-term effects of MC exposure (Stewart et al., 1969).
NOEL: is defined as the exposure level at which there are no statistically
significant increases in frequency or severity of effects between the exposed
population and its appropriate control.
5-70
-------
Observations of occupationally exposed workers exposed up to 6 years at MC
3
levels (TWA) ranging from 50 to 249 ppm (270 to 1,345 mg/m ) showed no differ-
ence between exposed and comparable control populations.
There are, however, three subchronic experimental animal studies with
exposures continuing up to 180 days which provide some information on the
effects of repeated exposures to MC. The study of Torkelson et al. (1958),
using rats, guinea pigs, rabbits, and monkeys, and the study of Eben and
Kimmerle (1974), using rats, established a no observed effect level (NOEL) for
subchronic exposure to MC for 7 to 8 hours a day, 5 days per week for 3 months
at 500 and 440 ppm (2,700 and 2,376 mg/m ), respectively. Similarly, a NOEL
o
of 370 ppm (1,898 mg/m ) was obtained by Prendergast et al. (1967) in a variety
of species (15 guinea pigs, 3 squirrel monkeys, 3 New Zealand rabbits, and 2
beagle dogs) exposed continuously to MC for 6 weeks. In this study there were
3 deaths in the low exposure group (135 ppm; 729 mg/m ), but these were attri-
buted to lung infections. These three studies would indicate that subchronic
3
exposure to MC in the range of 370 to 500 ppm (1,898 to 2,700 mg/m ) produces
no gross signs of toxicity. However, the report of McNutt et al. (1975)
indicates that exposure below this range may alter some biochemical parameters
in the liver and brain.
McNutt et al. (1975) observed mild to minimal alterations in the rough
endoplasmic reticulum, detachment of polyribosomes, microbodies, and trigly-
ceride droplets in the livers of mice exposed to 250 ppm of MC continuously
for 24 weeks. At 1000 ppm (5,400 mg/m ), the extent of these changes had in-
creased and some individual hepatocyte necrosis was observed. Although it is
unclear whether the early biochemical and histologic changes observed at expo-
o
sures of 250 ppm (1,350 mg/m ) adversely affected liver function, they may
represent the first stages in the sequence of events which leads to the hepa-
3
tocellular necrosis observed at 1000 ppm (5,400 mg/m ). The NOEL of 350 to
3
500 ppm (1,890 to 2,700 mg/m ) discussed previously may reflect the insensi-
tivity of the parameters investigated in the other studies.
The only study involving multiple experimental exposures of humans was
that of Stewart et al. (1975), in which 20 subjects were exposed to 500 ppm
3
(2,700 mg/m ) of MC for 7.5 hours a day, 5 days per week for 3 weeks. There
were no effects on the clinical chemistry of the blood or urine, nor on pul-
monary function tests. However, the female subjects did have an increased
number of complaints of odor. The authors stated that there was a lack of
5-71
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sleepiness and fatigue which had been reported in a previous study. Again,
these tests are not known to be sensitive enough to detect the slight changes
in the liver observed in mice at exposures to 250 or 1000 ppm (1,350 or 5,400
3
rog/m ) by McNutt et al. (1975). The combined evidence from the human and
animal studies would indicate that only minimal effects are produced by contin-
s
uous exposure to 250 ppm (1,350 mg/m ) of MC and that the pathologic importance
of these effects are unknown.
The studies of Maroni et al. (1977), Seki et al. (1975) and Kramer et al.
(1978) reported a NOEL for workers occupationally exposed to MC levels as high
as 350 ppm (1,890 mg/m3). The short duration of exposure, averaging less than
1 year, of the work population in the report of Kramer et al. (1978) precludes
the use of this study in human risk assessment. The study of Seki et al.
(1975) provides information on 196 male workers exposed to MC for at least 5
years at concentrations of 4, 25, 28, and 53 ppm (22, 135, 151, and 286 mg/m3).
The number of workers exposed at each concentration was relatively small, with
only 42 people exposed to the highest concentration. While no dose related
effects were observed, the exposed groups were only compared among themselves
and not with a control population. The reported details of the analytical
procedures were insufficient to determine whether the analytical data repre-
sented measurements over the entire time period (5 years) of exposure of the
study population, or whether the concentrations reported were the concentrations
in the breathing zone. Most of the deficiences in this study would probably
result in an underestimation of the exposure levels. Thus, it is difficult to
ascertain any NOEL from this study. Maroni et al. (1977) looked for neuro-
physiological abnormalities in a population of 21 women who had been exposed
for 6.5 years to an average concentration of MC between 110 and 345 ppm (594
and 1,863 mg/m3). No neurotoxicity was observed in exposed workers, as compared
to a control population of 7 unexposed women. The small size of the exposed
and control populations, and the limited number of physiological measurements
made makes the apparent NOEL from this study extremely uncertain.
5.4.2 Oral Exposure
There is little information on the toxicity of MC by the oral route.
As
summarized in Table 5-5, the acute LD,-n for MC has been determined in rats,
mice, guinea pigs, and rabbits by Torkelson et al. (1958) and the values range
from 8,600 to 14,300 mg/kg body weight. These LD5_ values could theoretically
5-72
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be used to calculate an approximate human lethal dose using the cubed root of
the body weight ratios for interspecies conversion (U.S. EPA, 1980b; Freireich
et al., 1966; Rail, 1969). However, the data obtained from the species studied
in the report of Torkelson et al. (1958) do not show a relationship between MC
toxicity and body weight. Therefore, without further information, it would
not be justified to place much reliance on an approximate human lethal dose
calculated using the above approach. However, as a crude indication of a
lethal oral dose of MC, the values were calculated from each species with the
range for a human lethal dose falling between 770 and 3940 mg of MC/kg. Also,
there is no information from human exposure to MC to indicate whether values
obtained from a calculated lethal dose are applicable to human toxicology.
For the above reasons it is considered inappropriate to attempt to predict an
approximate human lethal dose from the present data available.
There are subchronic studies on oral exposure to MC, and one chronic
study. In the NCI (1977) bioassay, rats and mice were treated by gavage with
MC in corn oil 5 days/week for 78 weeks at a level of 750 and 1500 mg/kg body
weight for the low and high dose group rats, and 2807 and 5615 mg/kg body
weight for the low and high dose group mice. Early deaths were observed in
both rats and mice, with a statistically significant dose-related trend observed
in male and female rats, and in female mice. The control animals also had
poor survival and the early mortality in this group would suggest that MC
contributed only partly to the early deaths of the treated animals. Another
difficulty with this study is that treatment of animals by gavage is not
identical to exposure through food or water since the daily dose of the compound
is administered at a single time, resulting in high body levels immediately
after treatment. As a result of the poor survival of the control animals and
the inappropriateness of the route of administration, the relevance of this
study to human risk assessment is questionable.
Both oral and inhalation studies have been performed to evaluate the
teratogenic potential. While it is not possible to define the full potential
of MC to produce human adverse teratogenic or reproductive effects, it does
not appear that short- or long-term exposure, in studies performed to date,
results in teratogenic effects in rats and mice.
5.4.3 Dermal Exposure
There is insufficient information available for quantitative risk assess-
ment of dermal exposure to MC. The pharmacokinetic data of Fukabori et al.
5-73
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(1967, 1977) would indicate that absorption of MC through intact skin can
contribute significantly to the levels of MC in the body. The extent of
dermal absorption was estimated to be 5 percent of respiratory absorption in
humans in direct contact with the liquid. The high vapor pressure of MC would
preclude dermal contact with this solvent for sufficient periods of time to
allow toxic quantities to be absorbed, except in some industrial situations.
Even so, the greater efficiency of absorption by inhalation would make this
the route of greatest concern.
5-74
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«U.S. GOVERNMENT PRINTING OFFICE: 1986-646-116-40658
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