United States Office of Health and EPA-600/8-82-004
Environmental Protection Environmental Assessment March 1 982
Agency Washington DC 20460
and Dev
Health Assessment DRAFT
Document for
Dichloromethane
{Methylene Chloride)
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Review Draft
DRAFT
Do not cite or quote
HEALTH ASSESSMENT DOCUMENT
FOR
DICHLOROMETHANE
(METHYLENE CHLORIDE)
NOTICE
This document is a preliminary draft. It has not been
formally released by EPA and should not at this stage
be construed to represent Agency policy. It is being
circulated for comment on its technical accuracy and
policy implications.
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, North Carolina 27711
Jean C. Parker, Ph.D.
Project Coordinator
'U.S. Environment?! Protection Agency.
Rc-^.on V, Library
230 Sovi'<'; Ds;:;born Street .,''
Chicago, Illinois 60604 ''"""
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DISCLAIMER
This report is an internal draft for review purposes only and does not
constitute Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
U G " ' rvotSCtion Aj 'ncy
,-Kr f*rt- "—
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PREFACE
The Office of Health and Environmental Assessment, in consultation with
an Agency work group, has prepared this health assessment to serve as a "source
document" for Agency-wide use. Originally the health assessment was developed
for use by the Office of Air Quality Planning and Standards, however, at the
request of the Agency Work Group on Solvents, the assessment scope was expanded
to address multimedia aspects. This assessment will help insure consistency in
the Agency's consideration of the relevant scientific health data associated with
methylene chloride.
In the development of the assessment document, the scientific literature
has been inventoried, key studies have been evaluated and summary/conclusions
have been prepared so that the chemical's toxicity and related characteristics
are qualitatively identified. Observed effect levels and dose-reponse
relationships are discussed, where appropriate, so that the nature of the
adverse health responses are placed in perspective with observed environmental
levels.
111
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TABLE OF CONTENTS
LIST OF TABLES vi
LIST OF FIGURES vi i
1. SUMMARY AND CONCLUSIONS 1-1
2. INTRODUCTION 2-1
3. BACKGROUND INFORMATION 3-1
3.1 PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.2 ENVIRONMENTAL FATE AND TRANSPORT 3-4
3.2.1 Production 3-4
3.2.2 Use 3-6
3.2.3 Emissions 3-7
3.2.4 Persistence of DCM 3-7
3.2.5 Products of DCM 3-9
3.2.5.1 Atmospheric Simulation Studies 3-9
3.2.5.2 Hydrolysis 3-11
3.2.5.3 Sorption 3-11
3.3 LEVELS OF EXPOSURE 3-11
3.3.1 Analytical Methodology 3-12
3.3.2 Sampling of Ambient Air and Water 3-16
3.3.2.1 Sampling and Detection in Ambient Air 3-17
3.3.2.2 Sampling and Detection in Water 3-20
3.3.2.2.1 Sample Preservation 3-20
3.3.2.2.2 Soil and Sediment 3-22
3.4 ECOLOGICAL EFFECTS 3-24
3.4.1 Effects on Aquatic Organisms 3-24
3.4.1.1 Effects on Freshwater Species 3-24
3.4.1.2 Effects on Saltwater Species 3-26
3.4.2 Effects on Plants 3-26
3.5 CRITERIA, REGULATIONS, AND STANDARDS 3-26
3.6 REFERENCES 3-29
4. METABOLIC FATE AND DISPOSITION OF DICHLOROMETHANE 4-1
4.1 ABSORPTION, DISTRIBUTION, AND ELIMINATION 4-1
4.1.1 Oral, Dermal, and Lung Absorption 4-1
4.1.2 Pulmonary Uptake and Tissue Distribution 4-2
4.1.3 Elimination 4-9
4. 2 DCM BIOTRANSFORMATION 4-12
4.2.1 Magnitude of DCM Metabolism 4-15
4.2.2 Enzyme Pathways of DCM Metabolism 4-24
4.2.3 DCM-Induced Carboxyhemoglobin Formation 4-31
4. 3 MEASURES OF EXPOSURE AND BODY BURDEN 4-38
4.4 SUMMARY AND CONCLUSIONS 4-40
4. 5 REFERENCES 4-43
5. HEALTH EFFECTS 5-1
5.1 OVERVIEW 5-1
5.2 HUMAN HEALTH EFFECTS 5-2
5.2.1 Overview 5-2
5.2.2 Acute Effects 5-3
5.2.2.1 Experimental Exposure 5-3
5.2.2.2 Accidental Exposure 5-7
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TABLE OF CONTENTS (cont.)
Page
5.2.3 Chronic Effects 5-10
5.2.3.1 Experimental Exposure 5-10
5.2.3.2 Accidental Exposure 5-11
5.2.3.3 Epidemiology 5-13
5.2.4 Relationship of CO and COHb to OCM Toxicity 5-13
5. 3 EFFECTS ON ANIMALS 5-14
5. 3.1 Overview 5-14
5.3.2 Acute Effects 5-15
5.3.2.1 Central Nervous System Effects 5-15
5.3.2.2 Carbon Monoxide Formation and
Cardiovascular Effects 5-17
5.3.2.3 Effects on the Eye 5-22
5.3.2.4 Effects on Internal Organs and Metabolism... 5-23
5.3.3 Chronic Effects 5-26
5.3.3.1 Central Nervous System Effects 5-26
5.3.3.2 Effects on COHb Levels 5-27
5.3.3.3 Effects on Internal Organs and Metabolism... 5-27
5.4 TERATOGENIC, EMBRYONIC AND REPRODUCTIVE EFFECTS 5-31
5.4.1 Animal Studies 5-32
5.4.1.1 Chicken Embryos 5-33
5.4.1.2 Mice 5-33
5.4.1.3 Rats 5-35
5. 5 MUTAGENICITY AND CARCINOGENICITY 5-37
5.6 SUMMARY OF ADVERSE HEALTH EFFECTS AND LOWEST
OBSERVED EFFECTS LEVELS 5-40
5.6.1 Animal Toxicity Studies Useful for Hazard Assessment. 5-40
5.6.2 Inhalation Exposure 5-40
5.6.2.1 Effects of Single Exposures 5-41
5.6.2.2 Effects of Intermittent or Prolonged
Inhalation Exposure 5-45
5.6.3 Oral Exposure 5-52
5. 6. 4 Dermal Exposure 5-53
5.6.5 Responses of Special Concern 5-54
5. 7 REFERENCES 5-55
6. COLLATED BIBLIOGRAPHY 6-1
7. APPENDIX: The Carcinogen Assessment Group's Carcinogen
Assessment of Methylene Chloride A-l
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LIST OF TABLES
Table Page
2-1 Consumption of dichloromethane 2-2
2-2 Selected properties of dichl oromethane 2-4
3-1 Synonyms and identifiers for dichl oromethane 3-2
3-2 Physical properties of dichl oromethane 3-2
3-3 Producers of dichl oromethane 3-5
3-4 Reaction rate data for OH + CH.CK 3-10
3-5 Ambient air levels of dichlorometnane 3-13
3-6 Effects of dlchloromethane on freshwater
species in acute tests 3-25
4-1 Absorption of DCM by human subjects (sedentary conditions) 4-7
4-2 Tissue concentrations of DCM in rats exposed to 200 ppm
for 4 days for 6 hr dai ly 4-8
4-3 Blood carboxyhemoglobin concentrations of rats exposed
to CO and DCM by inhalation 4-16
4-4 Fate and disposition of C-OCM in rats injected
intraperitoneally 4-20
5-1 COHb concentrations in nonsmokers exposed to DCM at
250 ppm for 5 days 5-11
5-2 Acute lethal toxicity of DCM 5-16
5-3 Summary of cardiotoxic action of 5% dichl oromethane 5-19
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LIST OF FIGURES
Figure Page
3-1 The effect of oxygen doping of the carrier gas on the
ECO response to several halogenated methanes at a
detector temperature of 300°C 3-21
4-1 Inspired and expired air concentrations during a 2 hr, 100
ppm inhalation exposure to DCM for a 70 kg man, and the
kinetics of the subsequent pulmonary excretion 4-5
4-2 DCM venous blood levels in rats immediately after a single
6-hr inhalation exposure to various concentrations of DCM 4-10
4-3a Carboxyhemoglobin concentrations in male nonsmokers exposed
to increasing concentrations of DCM for 1,3, or 5 hr per
day for 5 days. Pre-exposure values averaged 0.8%, but
with 3 and 7.5 hr exposures were above this baseline
value on the morning following exposure. Data
derived from Stewart and his associates (1972, 1973,
1974) 4-17
4-3b Carboxyhemoglobin concentrations in rats after exposure
to increasing concentrations of DCM for single exposures
of 3 hr. The values are corrected for pre-exposure COHb
concentration and calculated from the data of Fodor
et al. , 1973 4-17
4-4 Blood CO content of rats after 3-hr inhalation expo-
sure with 1000 ppm dichloromethane, dibromomethane,
and diiodomethane, respectively 4-18
4-5 Rates of production of CO from DCM given to rats.
Each curve represents changes above endogenous CO
rate after the dose (in umoles/kg b.w.) was given
by inhalation 4-22
4-6 Enzyme pathways of the hepatic biotransformation
of dihalomethanes 4-25
4-7 Blood COHb level in men during 8 hr exposure for
5 consecutive days to 500 ppm and 100 ppm DCM.
COHb percent saturation is equal to ug CO per
ml blood divided by 2.5 4-33
VII
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The authors of this document are:
Or. I. W. F. Davidson
Department of Physiology/Pharmacology
The Bowman Gray School of Medicine
Wake Forest University
Winston-Sal em, North Carolina
Dr. John L. Egle, Jr.
Department of Pharmacology
The Medical College of Virginia
Health Sciences Division
Virginia Commonwealth University
Richmond, Virginia
Mr. Mark M. Greenberg
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U. S. Environmental Protection Agency
Research Triangle Park, North Carolina
Dr. Jean C. Parker
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U. S. Environmental Protection Agency
Research Triangle Park, North Carolina
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The following individuals reviewed an early draft of this document and
submitted valuable comments:
Dr. Joseph Borzelleca
Department of Pharmacology
The Medical College of Virginia
Health Sciences Division
Virginia Commonwealth University
Richmond, Virginia 23298
Dr. Herbert Cornish
Dept. of Environmental and Industrial Health
University of Michigan
Ypsilanti, Michigan 48197
Dr. I. W. F. Davidson
Dept. of Physiology/Pharmacology
The Bowman Gray School of Medicine
300 S. Hawthorne Road
Winston-Sal em, North Carolina 27103
Dr. Lawrence Fishbein
National Center for Toxicological Research
Jefferson, Arkansas 72079
Dr. John G. Keller
P. 0. Box 12763
Research Triangle Park, North Carolina 27709
Dr. Marvin Legator
Professor and Director
Division of Environmental Toxicology and
Epidemiology
Department of Preventive Medicine and
Community Health
University of Texas Medical Branch
Galveston, Texas 77550
Dr. Norman Trieff
Division of Environmental Toxicology and
Epidemiology
Department of Preventive Medicine and
Community Health
University of Texas Medicine Branch
Galveston, Texas 77550
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center
New York, New York 10016
IX
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The following persons attended a review workshop to discuss EPA draft
documents on orgam'cs which included an early draft of this document.
Dr. Mildred Christian
Argus Laboratories, Inc.
Perkasie, Pennsylvania 18944
Dr. Herbert Cornish
Dept. of Environmental and Industrial Health
University of Michigan
Ypsilanti, Michigan 48197
Dr. I. W. F. Davidson
Dept. of Physiology/Pharmacology
The Bowman Gray School of Medicine
300 S. Hawthorne Road
Winston-Sal em, North Carolina 27103
Dr. John Egle
Dept. of Pharmacology
Virginia Commonwealth University
Richmond, Virginia 23298
Dr. Thomas Haley
National Center for Toxicological Research
Jefferson, Arkansas 72079
Dr. Rudolph J. Jaeger
Institute of Environmental Medicine
New York University Medical Center
New York, New York 10016
Dr. John G. Keller
P. 0. Box 12763
Research Triangle Park, North Carolina 27709
Dr. Norman Trieff
Dept. of Preventive Medicine
University of Texas Medical Branch
Galveston, Texas 77550
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center
New York, New York 10016
Dr. James Withey
Food Directorate
Bureau of Food Chem.
Tunney's Pasture
Ottawa, Canada
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Parti Gloating Members of the Carcinogen Assessment Group
Roy C. Albert, M.D., Chairman
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steve Bayard, Ph.D.
David L. Bayliss, M.S.
Chao W. Chen, Ph.D.
Herman D. Gibb, B.S., M.P.M.
Bernard H. Haberman, D.V.M., M.S.
Charahingaya B. Hiremath, Ph.D.
Robert McGaughey, Ph.D.
Beverly Paigen, Ph.D.
Dharm V. Singh, D.V.M., Ph.D.
Nancy A. Tanchel, B.A.
Todd W. Thorsland, Sc.D.
Participating Members of the Reproduction Effects Assessment Group
John R. Fowle III, Ph.D
Peter E. Voytek, Ph.D.
Carol Sakai, Ph.D.
Members of the Agency Work Group on Solvents
Elizabeth L. Anderson
Charles H. Ris
Jean C. Parker
Mark M. Greenberg
Cynthia Sonich
Steve Lutkenhoff
James A. Stewart
Wi11i am Lappenbush
Hugh Spitzer
David R. Patrick
Lois Jacob
Arnold Edelman
Josephine Brecher
Mike Ruggiero
Jan Jablonski
Charles Delos
Richard Johnson
Priscilla Holtzclaw
Assessment
Assessment
Assessment
Assessment
Assessment
Assessment
Office of Health and Environmental
Office of Health and Environmental
Office of Health and Environmental
Office of Health and Environmental
Office of Health and Environmental
Office of Health and Environmental
Office of Toxic Substances
Office1of 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
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1. SUMMARY AND CONCLUSIONS
Dichloromethane (methylene chloride, DCM) is a solvent widely used for a
variety of purposes. United States annual production of dichloromethane has
averaged around 227,000 metric tons (about 500 million pounds) in recent
years; an increase in production is expected. It is estimated that approxi-
mately 85 percent of the dichloromethane consumed in the United States is lost
directly to the environment through dispersive use, largely by evaporation to
the atmosphere. Natural sources have been proposed for dichloromethane, but
none is presently believed to contribute significantly to ambient concentra-
tions. Ambient air and water measurements, although rather scarce, indicate
that dichloromethane is found in a variety of urban and non-urban areas of the
United States and in other regions of the world. The bacKground atmospheric
concentration is about 35 parts per trillion (ppt). Concentrations of dichloro-
methane in urban areas do not seem to be significantly higher than in non-urban
areas; the maximum concentration measured in urban areas is 12 parts per
billion (ppb) detected in Los Angeles, California, 1979. Some extremely high
concentrations of DCM from nearby sources have been measured in indoor air.
Dichloromethane is not expected to accumulate in the atmosphere. Estimates of
half-life vary from 20 days to one year. Hydroxyl-free radical attack is
probably sufficiently rapid to prevent most, if not all, dichloromethane from
reaching the stratosphere. Rainout is not considered to cause a significant
reduction in atmospheric dichloromethane.
Dichloromethane has been detected in both natural and municipal waters in
various geographical areas of the United States. It has not been measured in
seawater. Concentrations of dichloromethane have been measured in surface
water and finished drinking water in the low ppb range. Dichloromethane does
005DC1/A 1-1 11-15-81
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not appear to be formed to any large extent during the chlorination process.
Its short evaporation half-life from moving water probably allows most of the
compound dissolved in water to be eventually transported into the atmosphere.
It is also readily degraded by bacteria in concentrations up to 400 parts per
million (ppm). The extent to which dichloromethane enters groundwater from
surface waters is unknown. Some dichloromethane is deposited in landfills;
where leaching is possible the compound may enter groundwater systems because
it adsorbs very little to clay, limestone, and/or peat moss, so retention in
the soil is unlikely. There is no evidence of its significant bioaccumulation
in the food chain. Little information on the ecological consequences of
dichloromethane in the environment is available.
The pharmacokinetics and metabolism of dichloromethane have not been
extensively studied in humans, although a broad outline of its absorption,
distribution, metabolism, and elimination has been established. As with other
solvents of this class, inhalation of dichloromethane in air followed by lung
absorption is the most rapid route of entrance into the body. Dichloromethane
is also well absorbed into the body after oral ingestion. Absorption through
the intact skin occurs to some extent, but is relatively a much slower process.
Dichloromethane is appreciably more water-soluble and less 1ipid-soluble than
its congeners, carbon tetrachloride and chloroform. Because of its solubility
in water and lipids, dichloromethane probably distributes throughout all body
fluids and tissues. It readily crosses the blood-brain barrier, as evidenced
by its narcotic effect. It also crosses the placenta and distributes into the
developing fetus.
Its long half-time of elimination from adipose tissue (6 to 6h hr),
together with reports that it remains in such tissue 24 hours after both
single and chronic exposures, indicate that dichloromethane may very slowly
005DC1/A 1-2 11-16-81
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accumulate in body fat with long daily exposures to high air concentrations.
The risk of accumulation might be expected to be greater for obese persons
than for lean persons.
Following oral ingestion, the absorption of dichloromethane is virtually
complete; with ambient air exposure, the amount absorbed increases in direct
proportion to its concentration in inspired air, the duration of exposure, and
physical activity. Absorbed dichloromethane is eliminated primarily by pulmo-
nary excretion of the unaltered parent compound (about 85 percent). About two
percent is excreted unchanged in the urine, while a small amount is eliminated
by other routes. Ten percent or less is metabolized before elimination from
the body. At very low concentrations essentially all of the compound absorbed
into the body may be metabolized. Dichloromethane is known to be metabolized
to carbon monoxide (CO) in man as well as in animals, primarily by the liver.
This metabolism of halogenated hydrocarbons to CO is apparently unique to the
dihalomethanes. Carboxyhemoglobin (COHb) is formed from the interaction of CO
and hemoglobin; CO dissociates at the lung and is eliminated. However, the
oxygen content of the blood is decreased, depriving the brain and heart of the
oxygen they require. Serious permanent damage may result.
The endogenous production of CO, and then COHb, from dichloromethane
metabolism is additive to COHb formed from exogenous CO. For this reason,
persons exposed to levels of dichloromethane that do not exceed the industrial
standard of 500 ppm (1737 mg/m ) may have blood COHb levels that exceed those
allowable from direct CO exposure. Dichloromethane exposure may thus result
in toxicities associated with the solvent as well as with CO. The results of
animal experimentation by several investigators indicate that carbon dioxide,
formaldehyde, and formic acid are additional metabolites of dichloromethane.
005DC1/A 1-3 11-16-81
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At least two pathways exist in rat liver for the metabolism of dichloro-
methane. Together, the microsome oxidative dehalogenation and cytosol
glutathione transferase dehalogenation systems account for the CO and CO-
generated from the metabolism of dichloromethane. The microsomal system, but
not the cytosol system, is inducible by phenobarbital and other microsomal
inducers, and by dichloromethane itself.
The adverse health effects associated with dichloromethane exposure are
primarily neurological and cardiovascular. The increased blood levels of
carboxyhemoglobin that are a consequence of metabolic transformation of di-
chloromethane to carbon monoxide may result in permanent damage to the brain
and heart. In addition, the teratogenic, mutagenic, and carcinogenic poten-
tials of this chemical are possible causes for concern. There is evidence
that exposure to dichloromethane can result in hemolytic anemia, especially in
certain populations, e.g., persons having erythrocytes deficient in glucose-6-
phosphate dehydrogenase. Liver and kidney morphological damage are probably
somewhat less likely to result from dichloromethane exposure than from exposure
to similar solvents such as chloroform and carbon tetrachloride. Contact of
the eyes, skin, and respiratory mucosa with dichloromethane will cause local
irritation.
The observed cardiotoxic properties of dichloromethane include cardiode-
pression and cardiosensitization. Several human case studies have reported
fatalities resulting from, or closely associated with, exposure to dichloro-
methane, in which myocardial infarction was diagnosed. Nonfatal exposures
have caused electrocardiographic (EKG) changes similar to CO-induced EKG
changes. The relative contributions of dichloromethane and its metabolite,
CO, to these effects are unclear. The histories of many exposed individuals
005DC1/A 1-4 11-16-81
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having symptoms of cardiotoxicity suggest the existence of underlying cardio-
vascular disease. Cardiotoxic effects may therefore be significant to this
subpopulation.
Animal studies have indicated that the primary cardiovascular effect of
dichloromethane is decreased myocardial contractility. Dichloromethane was
also capable of sensitizing the heart to epinephrine-induced arrhythmias.
While the levels of epinephrine used in these studies were somewhat higher
than those normally occurring during a stress reaction, they may suggest an
increased susceptibility of certain individuals to the effects of dichloro-
methane.
Hepatotoxicity has not been reported in any human case report, even
following fatal exposures. Animal studies have reported only minimal hepatic
changes, even at doses ranging from the L050 to "near lethal" doses. Some
species differences occurred (dogs were more susceptible than mice) but the
liver changes were nonetheless generally minor.
The only evidence of human nephrotoxicity resulting from dichloromethane
exposure was the finding of congested kidneys following a fatal exposure.
Animal studies concur, although the number of studies describing renal changes
was small. In contrast to hepatotoxicity, mice were more susceptible to
nephrotoxicity than dogs, but again, the changes reported were minimal.
There are reports of fatal and nonfatal acute human effects occurring
after exposure to dichloromethane. Inhalation exposures or concurrent inhala-
tion and dermal exposures were the most common. Although the concentration of
dichloromethane has usually not been reported, the circumstances under which
the human exposures occurred suggest that the concentrations have ranged from
very high levels in industrial accidents to more moderate levels associated
with home use of consumer products, specifically removers of paint and varnish.
005DC1/A 1-5 11-16-81
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Animal studies generally support and confirm the toxic effects notea in such
cases.
Central nervous system (CNS) effects are related to the anesthetic proper-
ties of dichloromethane. The onset of these effects is generally rapid and
they are temporary, normally subsiding within hours after the cessation of
exposure. In cases of acute human exposure, CNS effects have included death,
unconsciousness, labored breathing, headache, lassitude, and nausea. Be-
havioral and neurological alterations resulting from damage to central and
peripheral nervous systems have been reported following acute exposure. The
onset of symptoms ranged from immediate to several months after exposure.
Some of these effects were prolonged, persisting for at least 20 months after
the exposure. Progressive bilateral temporal lobe degeneration, as a conse-
quence of dichloromethane exposure, has been associated with memory loss,
general mental deterioration, headache, and dysarthria. Dichloromethane-in-
duced toxic encephalosis, resulting in visual and auditory illusions and
hallucinations, has been reported. Dichloromethane has been reported to
decrease peripheral nerve conduction and to induce abnormal rapid eye movement
(REM) sleep in rats.
There is less agreement on the human toxicology of dichloromethane from
low-level, long-term exposures. Experimental animal studies and evidence in
humans provide limited information on the correlation between chronic exposure
to dichloromethane and subsequent toxic effects. Difficulties in delineating
the toxic effects of dichloromethane are further compounded by chemical impuri-
ties. Exposure to levels of dichloromethane close to its threshold limit
value (TLV) have evoked subjective responses that suggest CNS'involvement or
are of psychosomatic origin. Studies of behavioral effects, particularly as
they affect manual and cognitive performance, have reported impairment at TLV
005DC1/A 1-6 11-16-81
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levels. Decrements in eye-hand coordination and task-related response time nave
been associated with carboxyhemoglobin levels of 3 to 5 percent.
Many studies have shown that COHb levels of 2.5 percent or greater can
adversely affect individuals with angina pectoris or cardiovascular disease.
Thus exposures to dichloromethane that increased the body burden of COHb can
further stress individuals who are already compromised by decreased oxygen
transport capabilities or cardiovascular disease. Chronic or subchronic
exposure to dichloromethane at concentrations from 50 to 200 ppm were found to
increase COHb levels to 2.9 to 4.5 percent in both workers and non-smoking
experimental volunteers.
An important and most controversial aspect of dichloromethane is its
teratogenic, mutagenic and carcinogenic potential. On the basis of the
limited data available, the teratogenic risk after exposure to dichloromethane
is minimal; however, there is a preliminary report suggesting the possibility
of delayed behavioral effects in offspring following exposure. Oichloromethane
has been demonstrated to cross the placenta, and fetal accumulation of dichloro-
methane has also been demonstrated. Higher concentrations of dichloromethane
have been found in human cord blood and fetal tissue than in maternal blood.
There are positive responses for bacterial and yeast mutagenicity, but
information on the purity of the test compounds is not yet available. Dichloro-
methane also showed an ability to transform cells using the rat embryo cell
line F1706. This was not confirmed when a reportedly purer grade of dichloro-
methane was tested.
The existing data base is inadequate for assessing the carcinogenicity of
dichloromethane. There is a marginally positive pulmonary adenoma response in
strain A mice. Two negative animal inhalation studies were inadequate because
they were not carried out for a full lifetime. Two long-term animal bioassay
005DC1/A 1-7 11-16-81
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studies are currently in progress at the National Toxicology Program (NTP)--a
gavage test nearing completion and an inhalation test recently started. The
animals in the NTP gavage study were sacrificed in December, 1980. A chronic
DCM inhalation study sponsored by industry has been conducted in rats and
hamsters. The rat study showed a small but statistically significant increase
in incidence of benign mammary tumors in female rats at all doses, and in male
rats at the highest dose. There was also a significant increase in sarcomas,
probably of salivary gland origin, in male rats. The response pattern of the
salivary gland tumors is unusual: they appeared only in males and consisted
only of sarcomas. In hamsters, there was an increased incidence of lymphosar-
comas, in females only, which was not statistically significant after correc-
tion for survival.
Only one occupational epidemiological study of dichloromethane has been
reported, and it showed no increased incidence of neoplasms that could be
related to dichloromethane at any sites. However, the study was insensitive
to latent tumor development due to the short duration of the follow-up.
Mortality studies of a worker population in which individuals had been
exposed to dichloromethane failed to reveal any signfiicant excess deaths by
major diagnostic or malignancy groupings when 751 employees exposed to the
chemical were compared with industrial workers not exposed to dichloromethane
and with New York State male populations.
A final assessment of the carcinogenicity of dichloromethane by the EPA
Carcinogen Assessment Group (CAG) will be deferred until information on the
purity of the material used in the positive mutagenicity tests is obtained and
until the results of the NTP gavage bioassay are evaluated.
005DC1/A 1-8 11-16-81
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2. INTRODUCTION
Dichloromethane is a high production industrial chemical. In view of an
apparently general belief that this compound is in many respects less toxic
than several of the other commonly used solvents, annual production may be
expected to increase. The 16,500 metric tons (36 million pounds) of dichloro-
methane produced in the United States in 1951 doubled to 33,500 metric tons
(74 million pounds) in 1955 and exceeded 227,000 metric tons (500 million
pounds) in 1976. Yearly production was only 217,000 metric tons (4-78 million
pounds) in 1977 but jumped to 297,000 metric tons (654 million pounds) in
1979, and was estimated to be 244,000 metric tons (537 million pounds) in
1980. Any leveling trend in the manufacture of this chemical is not likely to
continue, however, and overall production should increase.
Dichloromethane is used in large quantities for a variety of purposes.
Approximately 30 percent is used for paint removal. Dichloromethane is pre-
ferred for this purpose over competitive solvents because of its nonreactivity
with aluminum. Twenty percent is used as a degreasing agent and another 20
percent is used as a propellant for aerosol sprays. Since dichloromethane is
replacing many chlorofluorocarbons, the aerosol sector is expected to become
its largest market. Principal categories of dichloromethane usage during 1977
and 1978 are shown in Table 2-1.
Approximately 85 percent of the dichloromethane produced is emitted into
the environment from product manufacture and use. Most of the losses of this
chemical are through dispersion into the atmosphere, although the potential
exists for concentrations up to 1,500 ppm to occur in water effluents from its
use as an extraction solvent. The use of dichloromethane is likely to result
in measurable human exposure. In the past, the primary health concern was for
005DC1/B 2-1 11-16-81
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TABLE 2-1. CONSUMPTION OF DICHLOROMETHANE
Use
Paint remover
Metal degress ing agent
Aerosol propel 1 ant
Blowing agent for foams
(ure thane)
Exports
Other
Metric tons (103)
(1980)
73
49
46
20
44
12
Percent
(1977)
30
20
19
8
18
5
Total
(1978)
29
18
21
9
15
8
SRI International. Chemical Economics Handbook, 1980.
005DC1/B
2-2
11-16-81
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exposed industrial workers. Since dichloromethane is an ingredient in many
consumer products, however, millions of people are likely to be exposed to the
chemical at home. The concern for health effects has broadened with the
realization that most of each year's production is released into the environ-
ment and contaminates the atmosphere, raw water sources, drinking water, soil,
and possibly the food chain. The large population exposed to dichloromethane
includes various susceptible subgroups that may be particularly sensitive to
its adverse health effects, i.e., pregnant women, infants and children, the
elderly, and those with compromised cardiovascular systems. In addition, the
problem of possible additive or synergistic effects is also troublesome when
there is simultaneous exposure to other halomethanes or to certain medications
with which dichloromethane may interact adversely.
This document is intended to provide an evaluation of the current health
hazards of dichloromethane that are associated with exposure levels encountered
in the workplace as well as the lower levels expected in the environment and,
therefore, encountered by the population at large. Both the older and the more
current scientific literature has been reviewed and evaluated. The abbrevi-
ation DCM has been used for dichloromethane.
To give perspective to the evaluation of the health hazards from exposure
to DCM and a foundation for better comprehension of the data presented in
subsequent chapters, Chapter 3 provides basic background information about the
physical and chemical properties of DCM; its production, sources and emissions;
its atmospheric transport, transformation and fate; ambient air, water, and
soil concentrations; analytical methods for detection and quantification; and
ecological effects.
The physical and chemical properties of DCM are given in Table 2-2. As
can be noted from this table, DCM is highly volatile, with a vapor pressure of
005DC1/8 2-3 11-15-81
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TABLE 2-2. SELECTED PROPERTIES OF DICHLOROMETHANE
Molecular formula
Formula weight
Boiling point (760 mg Hg)
Melting point
Vapor density
Density of saturated vapor
Density
Solubility
Explosive limits in oxygen
Flash point
Autoignition temperature
Relative evaporation rate
Vapor pressure
84.94
40°C (760 mm Hg)
-95 to -97°C
2.93 (air = 1)
2.06 (air = 1)
1.326 g/ml (20°C)
2.0 g/100 ml water at 20°C;
soluble in ethanol,
ethyl ether, acetone,
and carbon disulfide
15.5-67% by volume
None
624 - 662°C
14 (water = 1)
71 (ether = 100)
Temp F Temp C mm HG
50
68
77
36
95
10
20
25
30
35
230
349
436
511
600
Conversion factors
(25°C; 760 mm Hg)
Concentration in saturated air
1 mg/liter = 1 g/cu m = 288 ppm
1 ppm = 3.474 mg/cu m = 3.474 pg/liter
550,000 ppm (25°C)
Hardie, 1969.
Weast, 1969.
American National Standards Institute Inc., 1970.
Christensen and Luginbyhl, 1974.
005DC1/B
2-4
11-16-81
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350 torr at room temperature. Since the ambient air concentration for com-
pounds of this nature is often expressed in parts per million (ppm), the
appropriate conversion factors for the metric system (mg/1 and g/m ) are also
included in this table.
Despite large emissions, DCM has been measured only a few times in the
atmosphere because of its relatively short atmospheric life of less than one
year and because of the lack of sensitivity of electron capture detectors for
a compound having only two chlorine atoms per molecule. The average back-
ground atmospheric concentration of DCM is approximately 35 ppt with little
variation between continental and marine air. Urban concentrations vary from
low background levels of 20 ppt to a maximum concentration of 12 ppb. Rela-
tively very little DCM reaches the stratosphere. The chemical has been found
in concentrations in the low ppb range in raw surface water and subsurface
water, as well as in finished drinking water, where it is formed during the
chlorination process of water treatment. The short evaporation half-life (21
min) of DCM from moving water probably allows most of the compound dissolved
in water to be eventually transported into the atmosphere. Concentrations of
up to 400 ppm DCM are also readily degraded by bacteria. Formation of DCM
from natural sources occurs but is not believed to contribute significantly to
global concentrations. DCM is deposited in landfills where some release to
the atmosphere through evaporation is likely. Where, leaching is possible, DCM
may enter groundwater systems as retention in the soil is unlikely. Elevated
levels of DCM have been measured in the indoor environment. The common sources
of exposure to DCM and their potential to affect human health are reviewed in
Chapter 3.
DCM is readily absorbed into the body, through the lungs by inhalation of
its vapors and through the gastrointestinal tract after oral ingestion in
005DC1/B 2-5 11-15-81
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water and food. Absorption through the skin from direct contact with liquid
DCM appears to be a slower process. At concentration levels allowable in the
workplace, more than 85 percent of DCM is excreted unchanged via the lungs. A
small amount of DCM is eliminated unchanged in urine and by other routes; 10
percent or less is metabolized prior to elimination. However, essentially
most of the compound absorbed may be metabolized at very low concentrations.
Biotransformation occurs primarily in the liver, and to a lesser extent in the
lungs and kidneys. DCM distributes to all body tissues. It is known to be
metabolized in animals and in man to carbon monoxide (CO), which elevates the
carboxyhemoglobin content of blood. It has a relatively long half-time of
elimination from adipose tissue. The distribution, storage, and metabolism of
DCM to CO, formaldehyde, and formic acid help to explain its human toxicity.
Toxicity may be enhanced by prior or concurrent exposure to certain
clinical drugs, exogenous CO, and some xenobiotics. These aspects of the
disposition and fate of DCM, as concluded from animal studies as well as from
methods of determining and quantifying exposure intensity and their limi-
tations, are discussed in Chapter 4.
At high concentration levels (>1000-2000 ppm), DCM is a central nervous
system depressant and an anesthetic and can cause injury to the CMS, liver, and
kidneys. It is irritating to the eyes, skin, and respiratory mucosa. Because
the affinity of its metabolite, CO, for hemoglobin is more than two hundred times
greater than that of oxygen (0?), the CO interacts preferentially with hemoglobin
to form carboxyhemoglobin, which deprives the brain and heart of the oxygen they
require, possibly resulting in serious permanent damage.
At lower level exposure, about 20 to 500 ppm DCM has behavioral and
psychological effects that alter manual dexterity and mental performance. The
005DC1/B 2-6 11-16-81
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adverse effects of low-level, chronic exposure to DCM have not been as ex-
tensively studied in either man or animals. Studies of the adverse health
affects of OCM at different levels of exposure are reviewed and evaluated in
Chapter 5.
The teratogenic, mutagenic, and carcinogenic potential of OCM is a con-
troversial major issue. Although DCM crosses the placenta, the evidence that
it is teratogenic is minimal, and the studies are few at the present time.
The possibility of delayed behavioral effects exists, but the studies are too
preliminary to be definitive. Studies relating to teratogenicity, fetal
toxicity and reproductive effects are reviewed and evaluated in Chapter 5.
The EPA Carcinogen Assessment Group (CAG) has deferred a final assessment
of the cancer-causing activity of DCM until information on the purity of the
material used in the positive mutagenicity tests on this chemical is obtained,
and until the results of the 2-year National Toxicology Program (NTP) chronic
gavage bioassay are obtained. This additional information may also help to
clarify the rather unusual results of the Dow Chemical Company inhalation studies.
Chapter 5 examines the evidence from the currently available mutagenicity studies,
animal carcinogenicity studies, and epidemiclogical data. A more in-depth review
of this information is presented in the CAG-REAG (Reproductive Effects Assessment
Group) report which is included as an appendix to this health assessment document.
DCM may be present in the water we drink, the air we breathe, and the
005DC1/B 2-7 11-16-81
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3. DICHLOROMETHANE: BACKGROUND INFORMATION
3.1 PHYSICAL AND CHEMICAL PROPERTIES
Dichloromethane (methylene chloride, DCM, CKLCIp) is one member of a family
of saturated aliphatic halogenated compounds. Other common names or synonyms
for DCM include methane dichloride, methlene bichloride, methlene dichloride,
Solaesthin, Aerothene MM, chlorure de methylene, and metlenu chlorek (see Table
3-1). The Chemical Abstracts Service Registry Number for DCM is 000075092.
Dichloromethane is a colorless, nonflammable, volatile liquid that is completely
miscible with a variety of other solvents (Anthony, 1979). Its important physical
properties are shown in Table 3-2. As can be noted from this table, DCM is
highly volatile with a vapor pressure of 350 torr at room temperature. Hence,
the most common mode of entry into the body is by inhalation. The ambient air
concentration for compounds of this nature is often expressed in parts per
million (ppm). At standard temperature and pressure, 1 part per million is
equivalent to 3.474 mg m .
In the absence of moisture at ordinary temperatures, DCM is relatively
stable when compared with its congeners, chloroform and carbon tetrachloride.
In dry air, DCM decomposes at temperatures exceeding 120°C (Anthony, 1979). At
elevated temperatures (300° to 450°C), it tends to carbonize when its vapor
contacts steel and metal chlorides. Moisture initates hydrolysis of DCM, pre-
dominantly to hydrogen chloride (HC1) with trace amounts of phosgene (Anthony,
1979). It has been reported that even trace amounts of phosgene detract from
the paint stripping qualities of DCM (De Forest, 1979). To retard production of
phosgene via hydrolysis, inhibitors are generally added to commercial prepara-
tions of OCM (De Forest, 1979). Protection against hydrolysis also is attained
by addition of phenolic compounds (Anthony, 1979).
J05DC1/C 3-1 11-16-81
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TABLE 3-1. SYNONYMS AND IDENTIFIERS FOR DICHLOROMETHANE
Chemical Abstracts Service Registry Number 000075092
Chemical Formula CH-CK
Structural Formula Cl
H - C - H
i
Cl
Dichloromethane
Methylene Dichloride
Methylene Bichloride
Methylene Chloride
Solaesthin
Aerothene MM
Chlorure de methylene
Metlenu Chlorek
TABLE 3-2. PHYSICAL PROPERTIES OF DICHLOROMETHANE
(ANTHONY, 1979)
Molecular weight
Vapor specific gravity
Boiling point
Vapor pressure
So1ubi1ity in water3
Log octane/water partition
coefficient
-1
84.94
2.93 grams liter ^ (air = 1)
40°C
511 mm Hg @ 30°C, 350 torr
13,200 (@ 20°C) to 22,700 ppm
1.25
Large discrepancies in the solubility of DCM have been reported. Dilling
(1977) reported values obtained from the literature ranging from 6,270 ppm
(@ 20°C) to 22,700 ppm (Glew and Moelwyn-Hughes, 1953).
3De Forest, 1979.
005DC1/C
3-2
11-1S-81
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Although anhydrous OCM is noncorrosive to common metals, the HC1 produced
from hydrolysis of DCM initiates corrosive action with aluminum and iron
(Anthony, 1979). The addition of epoxides to consume the HC1 affords protection
(Anthony, 1979). To minimize the decomposition of DCM, storage containers
should be galvanized or lined with a phenolic coating (Anthony, 1979). Com-
mercial grades of DCM contain a variety of stabilizers to minimize decomposition
(McKetta and Cunningham, 1979). Cyclohexane, thymol, hydroquinone, p-cresol,
and low-boiling amines have been used (De Forest, 1979). Aerosol
preparations containing DCM often use propylene oxide as a stabilizer. For
preparations designed to be used in degreasing applications, special inhibitor
mixtures are used. One such mixture includes propylene oxide, butylene oxide,
cyclohexane, and N-methyl morpholine (De Forest, 1979).
The experimentally-determined average evaporative half-life of DCM from
water is in the range of 18 to 25 minutes (Oilling, 1977). In three separate
experiments, Dilling used solutions of an average depth of 6.5 cm containing
approximately 1 ppm DCM. The solutions were stirred at 200 rpm in a 250 ml
beaker. These experimentally-determined half-lives agreed with the value (20.7
minutes) obtained by the following formula
. _ 0.6391d
\ = ~iq—'
where d is the solution depth and K, is the liquid exchange constant (cm/min).
The formula is an adaptation of the common equation for the half-life of a
substance undergoing a first order reaction.
3.2 ENVIRONMENTAL FATE AND TRANSPORT
Dichloromethane is principally used as an aerosol propellant, degreasing
solvent, and thinner in paints and lacquers. Because of its volatility and
dispersive use pattern, much of the DCM produced worldwide is emitted into the
Q05DC1/C 3-3 11-16-81
-------
atmosphere. The emissions are almost entirely due to anthropogenic sources.
Formation of DCM from natural soruces occurs but is not believed to contribute
significantly to global concentration (National Academy of Sciences, 1978).
3.2.1 Production
Oichloromethane is produced comrnercially in the United States predominantly
via the following reaction (De Forest, 1979; Anthony, 1979):
Fed- or ZnCl
HC1 + CH3OH 130a + 180*C ' CH3C1 * H2°
CH3C1 + C12 - CH2C12 + HC1
In this vapor phase reaction sequence, yields of 95 percent are generally
experienced. Dimethyl ether is the secondary byproduct of the hydrochlorina-
tion.
A less commonly used method is direct reaction of methane with chlorine at
485° to 510°C (Anthony, 1979). Methyl chloride, chloroform, carbon tetra-
chloride, and HC1 are coproducts. The coproducts represent a disadvantage of
this method. However, both reactions are used in the chemical manufacturing
industry so that the HC1 can be recycled (Anthony, 1979).
In the liquid phase, DCM can be produced by refluxing and distilling a
mixture containing methanol, HC1, and zinc chloride at 100° to 150°C. It is
not widely used (Anthony, 1979).
According to one source, production in the United States is carried out by
five major companies at seven sites (Table 3-3). However, the U.S. EPA TSCA
Public Inventory showed that in 1977 there were six manufacturers and 13 im-
porters (U.S. EPA, 1980b).
According to statistics gathered by the U.S. International Trade Commission,
the United States annual production of 36 million pounds (16,500 metric tons) in
1951 doubled to 74 million pounds (33,500 metric tons) in 1955, was 471 million
005DC1/C 3-4 11-19-81
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TABLE 3-3. PRODUCERS OF DICHLOROMETHANE
Annual Capacity (thousands of metric
Company tons annually) as of January 1, 1979
Allied Chemical, Moundsville, WV 23
Diamond Shamrock, Belle, WV 50
Dow Chemical, Freeport, TX q-
Pittsburgh, PA
Plaquemine, LA 88
Stauffer Chemical, Louisville, KY _a
Le Moyne, AL
Vulcan Materials, Geismar, LA 37
Wichita, KA 60
Source: Anon. 1979 Methylene Chloride in: Chemical Marketing Reporter.
August 6, 1979, p. 9.
005DC1/C 3-5 11-19-81
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pounds (214,000 metric tons) in 1972, exceeded 500 million pounds in 1976
(227,000 metric tons), was 478 million pounds (217,000 metric tons) in 1977, was
634 million pounds (297,000 metric tons) in 1979, and was estimated to be 537
million pounds (244,000 metric tons) in 1980. The leveling trend in annual
production is not expected to continue because DCM is believed to be less toxic
than most of the other commonly used solvents. In 1976, 42 million pounds
(19,000 metric tons) were imported, while exports totalled 97 million pounds
(44,000 metric tons) in 1975 (USITC, 1980).
3.2.2 Use
Qichloromethane is used for a variety of purposes: as a paint remover, a
urethane foam-blowing agent, a vapor degrees ing and dip solvent for metal clean-
ing, a solvent for aerosol products, a solvent in the pharmaceutical industry, a
solvent in the manufacture of polycarbonates by polymerization, and as an
extractant for caffeine, spices, and hops. It is used in the manufacture of
plastics, textiles, photographic film, and photoresistant coatings, as a solvent
carrier in the manufacture of herbicides and insecticides, and in rapid
drying paints and adhesives, carbon removers and brush cleaners. Other minor
applications include use as a low pressure refrigerant, as a low-temperature
heat transfer medium and as an air-conditioning coolant (Ahlstrom and Steele,
1979). Distribution of DCM in its major uses is shown in Table 2-1. The fastest
growing segment of the DCM market is the aerosol sector due to the substitution of
DCM for chlorofluorocarbons as a solvent, vapor pressure depressant, and
flame retardant. Consumption by the aerosol industry is expected to grow by as
much as 15 percent annually over the next several years and to become the largest
market for dichloromethane (Ahlstrom and Steele, 1979; Lowenheim and Moran,
1975).
005DC1/C 3-6 11-16-81
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Dichloromethane is expected to retain popularity as a paint remover.
Although it competes with trichloroethylene and perchloroethylene as a solvent,
it is perferred as a paint remover because of its nonreactivity with aluminum
(Lowenheim and Moran, 1975).
3.2.3 Emissions
Emissions from dispersive uses are the major source of DCM in the environ-
ment. Of the total amount of DCM produced in the United States, approximately
85 percent is estimated to be lost into the environment through sewage treatment
plants and surface waters, deposited on land or lost to the atmosphere. About
3/4 by weight of all annual halomethane emissions are thought to originate from
dispersive uses of DCM. The actual losses during production, transport, and
storage are not well documented, but it appears that such losses represent only
a very small percent of the DCM entering the environment from product manu-
facture and use. Most of the losses in production transportation and storage
are the fugitive type, that is, transient releases due to leaky pump seals,
valves, and joints. The dispersive uses of DCM are varied and widespread and
are distributed geographically approximately with the industrialized population
in the United States. Although most of the losses are to the atmosphere, DCM is
relatively soluble and, when it is used as an extraction solvent, the potential
exists for concentrations up to 1,500 ppm to occur in water effluents. The most
effective means of removal of DCM from water is air stripping, which transfers
the chemical from water to the atmosphere (NAS, 1978).
3.2.4 Persistence of DCM
Reaction with hydroxyl radicals (OH) is the principal process by which many
organic chemicals, including DCM, are scavenged from the troposphere (Crutzen
and Fishman, 1977; Singh, 1977; Altshuller, 1980). These radicals are produced
upon irradiation of ozone (0,). The resultant singlet oxygen atoms [O'(0)] then
005DC1/C 3-7 11-19-81
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react with water vapor. The tropospheric lifetime of a compound is related to
the OH concentration by the expression:
where k is the rate constant of reaction.
Recent findings (Crutzen and Fishman, 1977; Singh, 1977) imply lower tropo-
spheric concentrations of OH than previously believed. Therefore, DCM may have
a longer lifetime than commonly thought. The modelling approaches of Crutzen
and Fishman (1977) and Singh (1977) indicate that range of the average OH con-
centration as between 2 x 10 and 6 x 10 molecules cm . Using an average
5 -3
concentration of 3 x 10 molecules cm , Altshuller (1980) has calculated a
tropospheric lifetime for DCM of 1.4 years. The rate constant expression of
Davis et al. (1976) and a tropospheric temperature of 265°K were used.
Singh et al. (1979) computed a 1-year lifetime for DCM using the rate data
reported by NASA (1977) and NBS (1978) and a temperature of 265°K. An average
OH concentration of 4 x 10 molecules cm was employed in the computation.
Cox et al. (1976) on the other hand, calculated a 0.3 year lifetime in
photokinetic studies in which DCM competed with nitrous acid as the target of OH
-14 3
attack. The lifetime was derived from a rate constant of 10.4 x 10 cm
molecule sec at 298°K. An average OH concentration of 1 x 10 molecules cm
was assumed. Use of 4 x 10 molecules cm for the OH concentration would have
resulted in a calculated lifetime of 0.76 year, a value more in agreement with
those of Singh (1977) and Altshuller (1980). Davis et al . (1976) calculated a
lifetime of 0.39 years from a rate constant of 8. 7 x 10 at 265°K and an average
OH concentration of 9 x 10 molecules cm .
Determination of the rate constant for the reaction of OH with DCM has been
the focus of various investigations (Davis et al . , 1976; Cox et al., 1976;
005DC1/C 3-8 11-19-81
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Howard and Evenson, 1976; Perry et al., 1976). The values are in general agree-
ment (Table 3-4) with the exception of Butler et al. (1978).
3.2.5 Products of PCM
3.2.5.1 Atmospheric Simulation Studies— Pi 1 1 ing et al. (1976) observed that DCM
was not very reactive in a chamber atmosphere containing nitric oxide (NO) or
nitrogen dioxide (N0_). Ozone-air mixtures containing 10 ppm PCM and 5 ppm NO
or 16.8 ppm N0_ were exposed to ultraviolet radiation (UV) at an intensity about
2.6 times that of natural sunlight at noon on a summer day in Freeport, Texas.
After 21 hours of exposure, in the presence of NO, less than 5 percent of the
PCM initially present had disappeared. Similarly, less than 5 percent dis-
appeared in an N0? atmosphere after a 7.5-hour exposure. The effect of varying
UV intensity or concentration on the rate of photodecomposition was not in-
vestigated. Relative humidity in the photolysis reactor was 35 to 40 percent.
Pichloromethane was judged to contribute to oxidant formation to a lower degree
than other halogenated compounds investigated, e.g., tetrachloroethylene, tri-
chloroethy lene, and vinyl chloride.
Butler et al. (1978) have proposed that OH attack on PCM in the presence of
0- may result in formation of phosgene (COd,) via the reaction sequence:
CH2C12 + OH *
•CHC12 + 02 -»
+ OH
This pathway was suggested to account for a low rate constant of the reaction of
OH with PCM in the presence and absence of CO in the test atmosphere. Production
of CO,, was followed.
Chlorine-sensitized photooxidation of PCM in the presence of Cl? in dry air
resulted in CO and C0~ as the major carbon-containing products (Spence et al . ,
005DC1/C 3-9 11-16-81
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TABLE 3-4. REACTION RATE DATA FOR OH +
CO
I
o
k x I0"14cm3 molecule"1 sec"1
14.5 ± 2.0
15.5 ± 3.4
11.6 ± 0.5
8.7
10.4
2.7 ± 1
—
°K Arrhenius Expression
298.5
296
245 to 375°K 4.27 ± 0.63 x Ifl"12
exp [-(1094 ± 81/T)]
265
298
302
5.2 x 10~12exp(-1094/T)
Reference
Perry et al. , 1976
Howard and Evenson, 1976
Davis et al. , 1976
Cox et al. , 1976
Butler et al. , 1978
NASA,* 1977; National
Bureau of Standards,
1978
*
NASA preferred value; reliability of log k judged to be ± 0.2 at 230°K.
-------
1976). After 5 minutes of irradiation of 20 ppm DCM and 5 ppm Cl? in air, 19
ppm CH-Cl- was consumed. The product distribution was: CO (5 ppm); HC1 (38
ppm); phosgene (2 ppm); formylchloride (0 ;pm); and CO- (12 ppm). The product
distribution is illustrative of a chain reaction:
•CHC1202 - CIO + HCOC1
HCOC1 + Cl - HC1 + COC1
coci + o2 - co2 ->- cio
COC1 -> CO + Cl
Chlorine-sensitized photooxidation of DCM is not expected to be significant
under real atmospheric conditions as Cl will react with species other than
halocarbons (Spence et al., 1976).
3.2.5.2 Hydrolysis—The hydrolysis of DCM in natural waters is influenced by
acidic and basic conditions. When hydrolyzed in water at temperatures ranging
from 80 to 150°C, the hydrolysis in an acidic solution was reported to proceed
at a rate corresponding to a measured half-life of 13.75 days (Fells and Moelwyn-
Hughes, 1958).
In a review, Radding et al. (1977) reported a maximum hydrolytic half-life
of 704 years (100 to 150°C at pH 7); this is in sharp contrast to the aqueous
reactivity results found by Oil ling et al. (1975) in which the half-life was
about 18 months (25°C).
3.2.5.3 Sorpti on—Dill ing et al. (1975) found that DCM could be adsorbed to dry
bentonite clay and peat moss when these absorbents were added to a sealed solu-
tion containing DCM. However, the DCM which leaches from landfills adsorbs very
little to clay, limestone, and/or peat moss, so retention in the soil is unlikely.
3.3 LEVELS OF EXPOSURE
Dichloromethane has been detected in ambient air and in surface and drink-
ing waters at numerous locations throughout the United States.
005DC1/C 3-11 11-16-81
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Average background mixing ratios are approximately 30 to 50 ppt. Ambient
air levels of DCM at various locations are shown in Table 3-5. Singh et al.
(1979) reported that the average northern hemisphere background mixing
ratio is approximately 45 ppt. The Washington State University group reported
free troposphere values between 30 and 40 ppt (Cronn et al., 1977; Robinson,
1978; Cronn and Robinson 1979; Grimsrud and Rasmussen, 1975; Rasmussen et al.,
1979).
Pellizzari and Bunch (1979) have compiled a list of the sites in the
United States at which Pellizzari and coworkers have identified DCM. DCM was
sampled by adsorption onto Tenax GC, coupled with analysis by high resolution
GC-MS. The highest level reported in New Jersey was at a waste disposal site
in Edison. A level of 360 ppb was measured during an 11 minute sampling
period in March 1976. During a 75 minute sampling period (Nov. 19.77), 55 ± 0.1
ppb was detected at a site in Staten Island, New York. Longer sampling times
(up to 8 hours) were required at sites in Virginia, West Virginia, and
Pennsylvania to detect DCM. During a 7 hour and 15 minute period, about 70
ppb were detected at a site in Front Royal, Virginia (Oct. 1977). In the
southwest, a level of about 1 ppb was reported at sites in Houston, Texas
during a 3-hour sampling period (Oct. 1977). Lower levels over considerably
longer sampling periods (up to 24 hours) were reported for sites in Louisiana,
e.g., Baton Rouge and Geismar. Sampling during a 48-hour period in Upland,
California (Aug. 1977) indicated levels of about 12 ± 9 ppb.
3.3.1 Analytical Methodology
There are four practical methods to measure air concentrations of the
halogenated hydrocarbons:
1. gas chromatography with an electron-capture detector (GC-EC);
2. gas chromatography-mass spectrometry (GC-MS);
005DC1/C 3-12 11-19-81
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Location
TABtE 3-5. AMBIENT AIR LEVELS OF 01CHLOROMETHANE
Type of site
Date of measurement/
analytical method
Mixing Ratio
ppb
Reference
Arizona
Phoenix Urban
CO
I
Cali fornia
Mill Valley
San Jose
Apr 23 to May 6, 1979
GC-EC
Background subject Jan 11 to 27, 1977
to urban trans- GC-EC
port
Riverside Urban
Badger Pass High altitude
Point Arena Marine coastal
Urban
Point Arena Marine coastal
Los Angeles Urban
Apr 25 to May 4, 1977
GC-EC
May 5 to 13, 1977
GC-EC
May 23 to 30, 1977
GC-EC
Aug 21 to 27, 1978
GC-EC
Aug 30 to Sept 5, 1978
GC-EC
Apr 9 to 21, 1979
GC-EC
Max 5.1552
Min 0.0859
Avg 0.8936 ± 0.9886
Max 0.087
Min 0.038
Avg 0.055 ± 0.014
Max 0.473
RTn 0.033
Avg 0.111 ± 0.094
Max 0.126
HTn 0.009
Avg 0.044 ± 0.025
Max 0.102
Min 0.013
Avg 0.045 ± 0.022
Max 1.920
Min 0.060
Avg 0.401 ± 0.352
Max 0.080
Min 0.016
Avg 0.039 ± 0.017
Max 12.0288
Min 0.6014
Avg 3.7511 ± 2.6203
Singh et al. , 1(J81
Singh et al., 19/9
Ibid
Ibid
Ibid
Ibid
Ibid
Singh et al., 1981
-------
TABLE 3-5. (continued)
Location
Panama
Type of site
Canal Zone
Southern Hemisphere
0° to 42°S
77° to 90°S
32° to 55°S
77° to 90°S
Marine
Remote
Marine
Remote
Date of measurement/
analytical method
July 1977, GC-MS
Oct 1976, GC-MS
Jan 1977, GC-MS
Oct 1977, GC-MS
Nov 1977, GC-MS
Mixing Ratio
Reference
Max - Cronn and Robinson,
fiTn - 1979
Avg 0.034
Avg 0.035 ±0.003 Robinson, 1978
Avg 0.034 t 0.004
Ayj 0.040 ± 0.002
Avg 0.033 ± 0.001
-------
[ABLE 3-5. (continued)
CO
I
» - . . - -
Location
Oakland
Kansas
Jetmar
Nevada
Reese River
North Pacific
3/°N
0°N - 33°N
Washington
I'ul Iman
Pul Iman
Type of si te
Urban
Remote
High altitude
Ocean
Marine
Marine
Rural
Rural
Date of measurement/
analytical method
June 28 to July 10, 1979
GC-EC
June 1 to 7, 1978
GC-EC
May 14 to 20, 1977
GC-EC
Apr 1977, GC-MS
Oct 1976, GC-MS
Dec 1974 to Feb 1975
GC-MS
Nov 1975, GC-MS
Mixing Ratio
ppb
Max 2.4058
Min 0.0859
Avg 0.4155 ± 0.3146
Max 0.105
Min 0.033
Avg 0.054 ± 0.015
Max 0.099
Min 0.015
Avg 0.052 ± 0.022
Avg 0.030 ± 0.008
Avg 0.033 ± 0.046
Max -
Min -
Avg <0.005
Avg 0.035
Reference
Singh et al . ,
Singh et al . ,
Singh et al . ,
Cronn et al . ,
1981
1979
1979
1977
Robinson, 1978
Grimsrud and
1975
Rasmussen et
Rasmusser
al. , 1975
-------
3. long-path infrared absorption spectroscopy, usually with preconcen-
tration of whole air and then separation of the compounds by gas
chromatography (MC-IR); and
4. infrared solar spectroscopy, using the solar spectrum at large
zenith angles to obtain great path lengths through the atmosphere.
Each method has advantages and disadvantages and applications for which
it is best suited. A major drawback with these techniques is that they do not
allow real-time continuous measurements of the halocarbons at ambient levels
in the environment.
The two most widely used systems for identifying and measuring trace
amounts of OCM that occur in ambient air are (1) gas chromatography-mass spec-
trometry (GC-MS) and (2) gas chromatography-electron capture detection (GC-EC).
Both systems have a limit of detection below 30 parts per trillion (ppt). The
GC-EC method has been reviewed by Pellizzari (1974) and by Lovelock (1974).
The electron capture detector is specific in that halogenated hydrocarbons are
quantitated while non-halogenated hydrocarbons do not respond. Thus, high
background levels of non-halogenated hyrocarbons in ambient air or water samples
do not interfere with measurements of halogenated hydrocarbons. In a complex
mixture in which several compounds may have similar retention times, alteration
of the operating parameters of the GC-EC system will usually provide separation
of the components. Linearity over a wide concentration range is achieved when
the electron capture detector is used in the constant current mode. In this
mode, the change in pulse frequency is linearly related to sample concentra-
tion. Nitrogen, or a 95 percent argon/5 percent methane mixture, is commonly
used as the carrier gas.
3.3.2 Sampling of Ambient Air and Water
The methods used to date to analyze air and water for DCM content have
the problems of contamination, absorption, and adsorption. There are four
general approaches used to collect samples of air for analysis of trace gas
005DC1/C 3-16 11-19-81
-------
concentration: cryogenic sampling in which liquid helium or liquid nitrogen
is used to cool a container to extremely low temperatures; pump-pressured
samples, in which a mechanical pump is used without cryogenic assistance to
fill a sampler to a positive pressure relative to the surrounding atmosphere;
ambient or subambient pressure sampling in which an evacuated container is
simply opened and allowed to fill until it has reached ambient pressure at the
sampling location; adsorption of selected gases on such adsorbants as molecular
sieves or activated charcoal. Contamination and other problems are more
serious in low-pressure sampling than in high pressure sampling systems
(National Academy of Sciences, 1978).
Water samples are subject to the same possibility of contamination and
other problems that exist in air sampling. Particular care must be devoted to
the sampling, transportation, and storage of aqueous samples of OCM because of
its volatility and the complexity of the samples, especially those containing
chlorine or other oxidants. A technique that is often used involves filling and
sealing a serum bottle without air space and storing it just above freezing
(Kopfler et al., 1976). Water samples generally require additional preparation
before analysis. Normally, they may be concentrated by various water analysis
techniques but direct aqueous injection is used occasionally in GC analysis.
3.3.2.1 Sampling and Detection in Ambient Air—Several common approaches are
used to sample ambient air for trace gas analysis, including (National Academy of
Sciences, 1978):
1. 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.
2. Ambient pressure samples: An evacuated chamber is opened and allowed
to fill until it has reached ambient pressure at the sampling location. If
005DC1/C 3-17 11-19-81
-------
filling is conducted at high altitude, the sample may become contaminated upon
return to ground level.
3. Adsorption on molecular sieves, activated charcoal, or other sorbents.
4. Cryogenic samples: Air is pumped into a container, liquefied, and a
partial vacuum is created which allows more air to enter. This method allows
for the collection of several thousand liters of air.
Singh et al. (1979) have satisfactorily measured ambient levels of DCM by
analysis with GC-EC. Samples were pressurized in stainless steel vessels, then
fib
preconcentrated by freezeout on 100/120 mesh glass beads (Tenax monomer was
discontinued since oxygen was found to oxidize the monomer and interfere with
electron capture detection). Separation was performed on a column containing
0.2 percent Chromosorb W 1500 80/100 mesh on Carbopack C. A post-column Ascarite
water trap was used to remove water prior to electron capture detection. Dual
detectors were used to provide a coulometric response.
Harsch et al. (1979) used GC-EC to identify and measure the level of DCM in
samples of ambient air. A 500 ml sample was preconcentrated using the freezeout
concentration method (Rasmussen et al., 1979). Halocarbons were desorbed onto a
stainless steel column packed with 10 percent SF-96 on 100/120 mesh Chromosorb
W. The reported detection limit was 26 ppt. Good separation from chlorofluoro-
carbon 113 (trichlorotrifluoroethane) was reported.
Cronn and Harsch (1979) reported a GC-MS detection limit of 6 ppt for a 500
ml aliquot of DCM. Cronn et al. (1977) also reported a detection limit of 20
ppt for a 100 ml aliquot. Pressurized air samples were separated on a column of
Durapack n-octane on 100/120 mesh Porasil C. Cronn and coworkers (1976) have
compared GC-MS with GC-EC in terms of precision and sensitivity. In general,
GC-MS offered great specificity but could not equal GC-EC in reproducibility for
the 11 halocarbons studied. With mass spectrometry, the detection limit for DCM
005DC1/C 3-18 11-19-81
-------
was 9 ppt with a percent standard deviation of 13. The detection limit with'
temperature programmed GC-EC was 4 ppt and a percent standard deviation of
7.3.
Pellizzari and Bunch (1979) reported an estimated detection limit of 200
ppt using a high resolution GC-MS system in which OCM was first adsorbed onto
Tenax GC. The accuracy of analysis was reported as ±30 percent. The inherent
analytical errors are a function of several factors, including: (1) the
ability to accurately determine the breakthrough volume; (2) the accurate
measurement of the ambient air volume sampled; (3) the percent recovery of OCM
from the sampling cartridge after a period of storage; and (4) the reproduci-
bility of thermal desorption from the Tenax cartridge and its introduction
/6\
into the analytical system. Oxidation of the Tenax monomer was not reported.
Difficulties in using a coulometric approach to the GC-EC quantification
of DCM in air samples have been reported by Lillian and Singh (1974). These
investigators were unable to measure DCM accurately with detectors in series
due to a greater-than-coulometric response. Dichloromethane was reported to
have a very low ionization efficiency. It was suggested that the observed
response is attributed to the products of ionization having greater electron
affinities than the reactants.
Cox et al. (1976) reported that polyglycol stationary phase chemically
bonded to porous glass (Durasil Low Kl) was the only material found to separate
OCM from other halocarbons during a GC-EC analysis.
Satisfactory separation and analysis of DCM was reported by Grimsrud and
Rasmussen (1975) with a 50-foot SCOT OV-101 column by GC-MS.
When the freezeout concentration method of Rasmussen et al. (1979) was
applied to a 500 ml aliquot of air, the detection limit for DCM with GC-EC
analysis was 4 ppt and a percent standard deviation of 26.2. The GC column
contained 10 percent SF-96 on 100/120 mesh Chromosorb W.
005DC1/C 3-19 11-19-81
-------
The National Institute for Occupational Safety and Health (NIOSH) method
P & CAM 127 (NIOSH, 1974) is recommended for measurement of DCM in samples
when the concentration is greater than 0.05 mg in each sample. This method
utilizes adsorption on charcoal followed by desorption with carbon disulfide.
Analysis is made by gas chromatography with flame ionization detection. The
mean relative standard deviation of the method is 8 percent.
Grimsrud and Miller (1979) have reported an improved GC-EC method by
which the detector response of DCM is enhanced by the addition of 0- in the
carrier gas. At the highest 02 doping (5 parts per thousand, ppth) the response
of the detector to OCM (960 ppb) was enhanced 57-fold. The enhancement is
depicted in Figure 3-1. A constant current electron capture detector was
used.
3.3.2.2 Sampling and Detection in Watei—A gas purging and trapping method
suitable for DCM has been described by Bellar et al. (1979). Water samples
are purged by bubbling with helium or nitrogen at 23°C. The halocarbons are
adsorbed onto a porous polymer trap as the gas is vented. Quantification is
(&t
made by GC-MS. Tenax GC (60/80 mesh) was considered an effective adsorbent
for compounds that boil above approximately 30°C. A recommended general
purpose column is an 8-foot by 0.1 inch (i.d.) stainless steel or glass tube
packed with 0.2 percent Carbowax® 1500 on Carbopack® - C (80/100 mesh). For a
sample volume of 5 ml, the range of the limit of detection is 0.1 to 1.0 ug
per liter.
3.3.2.2.1 Sample Preservation (Water). Bellar et al. (1979) recommended that
water samples be stored in narrow mouth glass vials. Vials are filled to zero
head space and covered with a Teflon -faced silicone rubber septum. Screw
caps are suitable seals. The presence of chlorine in water samples has been
shown to result in an increase in the concentration of certain halomethanes
(not including DCM) upon storage.
005DC1/C 3-20 11-19-81
-------
12 3 4 5
O2 CONCENTRATION, ppth
Figure 3-1. The effect of oxygen doping of the carrier
gas on the ECO response to several halooenated
methanes at a detector temperature of 300° C.
From Grimsrud and Miller (1979).
3-21
-------
3.3.2.2.2 Soil and Sediment (Water). Dichloromethane has been found in
drinking and surface water at a variety of locations in the United States. In
a recent National Academy of Sciences report (NAS, 1977) DCM was reported to
be formed as a result of ch Tori nation treatment of water.
In a survey for volatile organics in five drinking water supplies, Coleman
et al. (1976) found that DCM was common to all cities evaluated (Cincinnati,
Miami, Philadelphia, and Ottumwa, Iowa). Concentrations were not reported.
Analysis was performed using GC-MS.
In a 1975 survey by the U.S. Environmental Protection Agency (1975a), DCM
was detected in 9 of 10 water supplies. Lawrence, Massachusetts, had the
highest concentration (1.6 ug/1). A mean concentration of <1 ug/1 in finished
water was reported in a survey of Region V water supplies (U.S. EPA, 1975b).
This survey indicated eight percent of the finished-water supplies contained
detectable DCM.
Dichloromethane was not among the major halogenated hydrocarbons detected
by Dowty et al. (1975a) in New Orleans drinking water. In another report, DCM
was detected in finished waters in the New Orleans area by GC-MS (Dowty et
al . , 1975b). It was also found in Mississippi River clarifier effluent. The
chlorocarbon was sorbed onto poly p-2,6-diphenyl phenylene oxide (35/60 mesh).
Vapors were desorbed onto a capillary chromatographic column and quantitated
by mass spectrometry. Raw water influent was purified after clarifier treat-
ment (sedimentation and some chemical treatment) to an extent that OCM concen-
tration dropped 32 percent. However, the concentration in finished water
increased after chlorination. Dichloromethane also was detected in commerci-
ally bottled artesian well water.
A slight increase in DCM concentration in chlorinated finished water was
observed by Bellar et al. (1974). Finished water having 2.0 ^g/£ DCM resulted
005DC1/C 3-22 11-19-81
-------
following complete treatment process of raw river water containing no detectable
DCM. Water samples also were collected from various locations in a sewage treat-
ment plant. Before treatment the water contained 2.36 ppb (8.2 ug/1). Before
chlorination and after preliminary treatment, the water contained 0.8 ppb
(2.9 ug/1). After chlorination the effluent contained 1 ppb (3.4 ug/1). It
was concluded that DCM, along with other chlorocarbons, may have formed as a
result of the chlorination treatment. The most notable chlorocarbon was chlo-
roform, for which an increase of 7.1 ug/1 to 12.1 ug/1 was observed subsequent
to chlorination. GC-MS analysis was performed using a headspace preconcen-
tration technique.
Oichloromethane was detected at 32 of 204 surface water sites from which
samples were collected (Ewing et al., 1977). Sites were located near heavily
industrialized river basins across the United States. Concentrations were
reported as being greater than 1 ppb (1 ug/1). Samples were collected from
July 1975 to December 1976. Of the 204 sites, 91 were among major rivers and
57 were in tidal areas and estuaries. Samples (125 ml) were held at 60°C and
/&
stripped. Volatiles were sorbed onto Tenax GC and desorbed onto Carbowax 1500
columns and analyzed by mass spectrometry.
Dichloromethane was not among the contaminants detected by Sheldon and
Hftes (1978) in raw Delaware River water collected from August 1976 to March
1977.
Pellizzari and Bunch (1979) reported detecting DCM in untreated Mississippi
River water (Jefferson Parish, Louisiana)' at a mean concentration of 2.581 ug/1.
The highest value reported was 15.8 ug/1. Determinations were made from February
7 to August 5, 1977. A mean concentration of 0.13 ug/1 was reported by Pellizzari
and Bunch (1979) in tap water from Jefferson Parish. The highest level was
1.1 ug/i.
005DC1/C 3-23 11-19-81
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3.4 ECOLOGICAL EFFECTS
3.4.1 Effects on Aquatic Organisms
Dichloromethane has been tested for acute toxicity in a limited number of
aquatic species. The information presented in this chapter focuses upon observed
levels that were reported to result in adverse effects under laboratory condi-
tions. Such parameters of-toxicity are not easily extrapolated to environmental
situations. Test populations themselves may not be representative of the entire
species in which susceptibility to the test substance at different lifestages
may vary considerably.
Guidelines for the utilization of these data in the development of criteria
levels for DCM in water are discussed in the Ambient Water Quality Criteria for
Halomethanes (U.S. Enviornmental Protection Agency, 1980b).
The toxicity of OCM to fish and other aquatic organisms has been gauged
principally by flow-through and static testing methods. The flow-through method
exposes the organism(s) continuously to a constant concentration of DCM while
oxygen is continuously replenished and waste products are removed. A static
test, on the other hand, exposes the organism(s) to the added initial concen-
tration only. Results from both types of tests are commonly used as initial
indications of the potential of substances to cause adverse effects.
3.4.1.1 Effects on Freshwater Species—Results of flow-through and acute static
tests with DCM and freshwater species (fish and invertebrates) are shown in
Table 3-6.
Alexander et al. (1978) used both flow-through and static methods to in-
vestigate the acute toxicity of several chlorinated solvents, including DCM,
to adult fathead minnows (Pimephales promelas). Studies were conducted in
accordance with test methods described by the U.S. Environmental Protection
005DC1/C 3-24 11-16-81
-------
TABLE 3-6. EFFECTS OF DICHLOROMETHANE ON FRESHWATER SPECIES IN ACUTE TESTS
Species
Type of Test
LC50
EC50
Reference
bluegill (Lepomis macrochirus)
static
224,000
U.S. EPA, 1980a
fathead minnor (Pimephales
promo las)
static
3iO,000 ug/1
Alexander et al., 19/8
10
I
ro
in
fathead minnow (Pimephales
promelas)
Daphnia roagna
flow-through 193,000 ug/1
statis
Ibid
224,000 pg/1 U.S. EPA, 1980a
96-hour
48-hour
-------
Agency. No irreversible effects were observed at concentrations below the
LD50.
Chronic test data concerning life cycle or embryo-larval tests are not
available.
3.4.1.2 Effects on Saltwater Species—Static tests with mysid shrimp resulted
in an LC50 value of 256,000 ug/1. There are no chronic data for DCM.
In a 96-hour static test with Sheepshead minnow (Cyprinodon variegatus) the
LC50 value was 331,000 ug/1 (U.S. EPA, 1980a).
3.4.2 Effects on Plants
The 96-hour EC50 values for DCM, based upon chlorophyll a and cell numbers
of the freshwater alga, Selenastrum capricornutum. were above the highest test
concentration (662,000 ug/1).
The 96-hour EC50 value, based on chlorophyll a and cell numbers of the
saltwater alga, Skeletonema costatum. was above the highest test concentration
(662,000 ug/1) (U.S. EPA, 1980a).
3.5 CRITERIA, REGULATIONS AND STANDARDS
Permissible levels of DCM 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 DCM at no time
exceed 500 ppm (1,737 mg/m ) time-weighted average in any 8-hr work day of a 40-hr
week, with an acceptable ceiling concentration of 1000 ppm (3,474 mg/m ), that
should not exceed 2,000 ppm (6,948 mg/m ) for more than 5 minutes in any 2 hours.
The American Conference of Government Industrial Hygiene (ACGIH) threshold limit
value (TLV) for inhalation exposure of 200 ppm (695 mg/m ), proposed for pre-
vention of narcotic effects or liver injury and for protection against excessive
carboxyhemoglobin formation, has recently been lowered to 100 ppm (347 mg/m ).
The 8-hour time-weighted average value in the Federal Republic of Germany is
005DC1/C 3-26 11-19-81
-------
500 ppm; in the German Democratic Republic and Czechoslovakia, 144 ppm; and in
Sweden, 100 ppm. The acceptable ceiling concentration in the USSR is 14 ppm
(49 mg/m3). The National Institute for Occupational Safety and Health (NIOSH)
has recommended that occupational exposure to DCM not exceed 75 ppm (261 mg/m ),
determined as a time-weighted average for up to a 10-hour work day of a 40-hour
week, in the absence of exposure to carbon monoxide above a time-weighted
average of 9 ppm for up to a 10-hr work day.
The U.S. Environmental Protection Agency water quality criteria are scien-
tific assessments of ecological effects and human health effects for incorpora-
tion into water quality standards (U.S. EPA, 1980a). The available data for
halomethanes including dichloromethane indicate the acute toxicity to freshwater
aquatic life occurs at concentrations as low as 11,000 ug/1; and acute( and chronic
toxicity to saltwater aquatic life occurs at concentrations as low as 12,000 and
6,400 mg/1, respectively. Toxicity would be expected to occur at lower concen-
trations among species that are more sensitive than those tested. A decrease in
algae cell numbers occurs at concentrations as low as 11,500 Mg/1•
Because positive results for mutagenic endpoints correlate with positive
results in j_n vivo bioassay for oncogenicity, mutagenic data for the halomethanes
suggest that several of the compounds might be carcinogenic. The U.S Environmental
Protection Agency Carcinogen Assessment Group is currently deferring a final cancer
assessment of DCM until further information becomes available. However, in the
Ambient Water Quality Criteria for Halomethanes (U.S. EPA, 1980a) it was noted
that for the maximum protection of human health from the potential carcinogenic
effects due to exposure to DCM, or combinations of this chemical with chloro-
methane, bromomethane, bromodichloromethane, tribromomethane, dichlorodif'luo-
romethane, and/or trichlorofluoromethane, through ingestion of contaminated
water and contaminated aquatic organisms, the ambient water concentration should
005DC1/C 3-27 11-19-81
-------
be zero based on the assumption of no threshold for these chemicals. However,
since zero level may not be attainable the levels that may result in incremental
increase of cancer risk over the lifetime are estimated at 10 , 10* , and 10 .
The corresponding recommended criteria, based upon data for chloroform, are 1.9
ug/1, 0.19 ug/1, and 0.019 ug/1, respectively. If the above estimates are made
for consumption of aquatic organisms only, excluding consumption of water, the
levels are 157 ug/1, 15.7 ug/1, and 1.57 ug/1, respectively. Estimates for water
consumption only were not made.
In cases such as halomethanes where one criterion is derived for an entire
class of compounds, the Agency does not state that each chemical in the class is
a carcinogen. The intended interpretation of the criterion is that the risk is
less than 10 whenever the total concentration of all halomethanes in water is
less than the criterion. In a hypothetical case where all of the halomethanes
in a sample are non-carcinogenic, the criterion would be too strict; however,
this situation seldom occurs. In most cases where halomethanes are detected, a
mixture of compounds occurs and in calculation of the criterion the assumption
is made that all components have the same carcinogenic potency as chloroform.
The derived water quality criterion based on noncarcinogenic risks, assum-
ing a daily water intake of 2 liters and the consumption of 6.5 g of fish and
shellfish per day (bioconcentration factor 0.91), would be 12.4 mg/1.
The available data for halomethanes indicate that acute and chronic toxicity
to saltwater aquatic life occur at concentrations as low as 12,000 and 6,400
fjg/1, respectively, and would occur at lower concentrations among species that
are more sensitive than those tested. A decrease in algal cell numbers occurs
at concentrations as low as 11,500 ug/1.
005DC1/C 3-28 11-19-81
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atmosphere. I_n: National Bureau of Standards Special Publication 519,
Trace Organic Analysis: A New Frontier in Analytical Chemistry, Proceed-
ings of the 9th Materials Research Sy-,osium, April 10-13, 1978. Issued
April, 1979, pp. 143-151.
Grimsrud, E. P. , and R. A. Rasmussen. Survey and analysis of halocarbons in
the atmosphere by gas chromatography-mass spectrometry. Atmos. Environ.
9:1014-1017, 1975.
Harsch, D. E. , D. R. Cronn, and W. R. Slater. Expanded list of halogenated
hydrocarbons measurable in ambient air. J. Air Pollut. Control Assoc.
29(9):975-976, 1979.
Howard, C. J., and K. M. Evenson. Rate constants for the reactions of OH with
ethane and some halogen substituted ethanes at 296K. J. Chem. Phys.
64:4303, 1976.
Keith, L., ed. Identification and Analysis of Organic Pollutants in Water.
Ann Arbor Science Publishers, Inc., Ann Arbor, MI, 1976. pp. 87104.
Kopfler, F. C., R. G. Melton, R. D. Lingg, and W. E. Colman. Identification an
analysis of organic pollutants in water. L. Keith, ed., Ann Arbor Science
Publishers, Inc., Ann Arbor, MI, 1976. pp. 87104.
Lillian, D., and H. B. Singh. Absolute determination of atmospheric halocarbons
by gas phase coulometry. Anal. Chem. 46(8).-1060-1063, 1974.
Lovelock, J. E. The electron capture detector: Theory and practice. J.
Chromat. 99:3-12, 1974.
Lowenheim, F. A. , and M. K. Moran. Methyl Chloride methylene chloride, ^n:
Faith, Keyes, and Clark's Industrial Chemicals, Fourth Edition, John
Wiley, New York, 1975. pp. 530-538.
McKetta, J. J. and W. A. Cunningham, eds., Encyclopedia of Chemical Processing
and Design, pp. 267, 1979.
National Aeronautics and Space Administration. Chlorofluoromethanes and the Stra-
tosphere. Robert 0. Hudson, ed. National Aeronautics and Space Administration
Reference Publication 1010, 1977.
National Academy of Sciences. Drinking Water and Health. National Research
Council, 1977. pp. 743-745.
National Academy of Sciences. Non-fluorinated halomethanes in the environment.
Panel on low molecular weight-halogenated hydrocarbons. Coordinating
Committee for Scientific and Technical Assessments of Environmental
Pollutants, 1978.
National Bureau of Standards, Special Publication 513 "Reaction rate and photo-
chemical data for atmospheric chemistry," 1977, R. F. Hampson, Jr. and
D. Garvin (eds.). National Bureau of Standards, Washington, DC, 1978.
005DC1/C 3-31 11-19-81
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National Institute for Occupational Safety and Health (NIOSH). NIOSH Manual
of Analytical Methods, HEW Publication No. (NIOSH) 75-121, 1974.
Pellizzari, E. 0. and J. E. Bunch. Ambient Air Carcinogenic Vapors: Improved
Sampling and Analytical Techniques and Field Studies. EPA-600/2-79-081,
May, 1979.
PeUizzari, E. 0. Electron capture detection in gas chromatography. J.
Chromat. 98:323-361, 1974.
Perry, R. A., R. Atkinson, and J. N. Pitts. Rate constants for the reaction
of OH radicals with CHFC12 and CH3C1 over the temperature range 298-493K
and with CH2C12 at 298K. J. Chem. Phys. 64:1618, 1976.
Radding, S. 8., 0. H. Liv, H. L. Johnson, and.T. Mill. Review of the environ-
mental fate ofcffelected chemicals. EPA 560/5-77-003, U.S. Environmental
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Rasmussen, R. A. , 0. E. Harsch, P. H. Sweany, J. P. Krasnec, and D. R. Cronn.
Determination of atmospheric ha1 carbons by a temperature programmed gas
chroraatographic freezeout concentration method. J. Air Poll. Control
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Robinson, E. Analysis of Halocarbons in Antarctica. ' Report 78/13-42 prepared
for the National Science Foundation, December 1978.
Sheldon, L. S., and R. A. Hites. Organic compounds in the Delaware River.
Environ. Sci. Techno 1. 12(10):1188-1194, 1978.
Singh, H. B. Atmospheric halocarbons. Evidence in favor of reduced average
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Singh, H. B., L. J. Salas, A. J. Smith, and H. Shigeishi. Measurements of
some potentially hazardous organic chemicals in urban environments.
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Cavanagh. Atmospheric distributions, sources and sinks of selected
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submitted to the U.S. Environmental Protection Agency by SRI Inter-
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Spence, J. W. , P. L. Hanst, and B. W. Gay, Jr. Atmospheric oxidation of
methyl chloride, methylene chloride, and chloroform. J. Air Pollut.
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U.S. Environmental Protection Agency. Region V Joint Federal/State Survey of
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U.S. Environmental Protection Agency. Ambient Water Quality Criteria for
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005DC1/C 3-33 11-19-81
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4. METABOLIC FATE AND DISPOSITION OF DICHLOROMETHANE
4.1 ABSORPTION, DISTRIBUTION, AND ELIMINATION
4.1.1 Oral, Dermal, and Lung Absorption
Dichloromethane (DCM) is a colorless liquid with a pleasant smell and phy-
sical properties that make it an excellent organic solvent for many uses in
industry and in certain consumer products. Because of its relatively high
vapor pressure at room temperatures (350-400 torr), DCM is readily absorbed into
the body following inhalation. Although it can be detected in air by its odor
at about 200 ppm (May, 1966; Leonardos, 1969), the great majority of severe
poisonings from this solvent occur from inhalation exposure; reports of poison-
ing by oral ingestion are rare (Llewellyn, 1966; Stewart and Hake, 1976;
Friedlander et al., 1978). While absorption through the intestinal mucosa
after oral ingestion appears to be rapid and complete, total recovery has
occasionally followed the swallowing of quite large doses (Roberts et al.,
1976).
The limited information available indicates that absorption of DCM through
the skin from direct liquid contact or by immersion of hands or arms is a slow
process. Early animal studies by Schutz (1958) showed that DCM does penetrate the
skin and can be absorbed into the body by this route. Schutz exposed the shaved
skin of rats to direct liquid contact for up to 20 minutes and found that only 2
minutes of exposure produced kidney damage probably through solvent action leading
to decreased urine formation and the appearance of blood in the urine. Stewart
and Dodd (1964) attempted to quantify the rate of absorption through human skin
by immersing the thumbs of volunteers into liquid OCM and then determining the
appearance and concentration of DCM in the breath. An estimate of the amount
entering the body was made by comparison with breath concentrations obtained
following controlled inhalation exposures. They concluded from breath analysis
005DC5/A 4-1
-------
after 10 minutes of immersion (1.4 to 2.4 ppm, 4.86 to 8.34 mg/m ) and after 30
minutes of immersion (2.4 to 3.6 ppm, 8.34 to 12.51 mg/m ) that DCM is very
slowly absorbed. They also determined that although the amount absorbed also
depends on the skin area involved, the slow rate of absorption would be unlikely
to result in toxic quantities of DCM being taken into the body from direct contact
with the skin of the hands and forearms. Indeed, immersion in DCM was found to
be accompanied by excruciating pain within a few minutes that would no doubt serve
as an effective deterrent.
In spite of the long use of DCM as a general industrial solvent spanning
at least six decades (Lehmann and Schmidt-Kehl, 1936), and although its narcotic
and anesthetic properties have been known to clinical medicine for over 50
years (Bourne and Stekle, 1923), few controlled and definitive studies have
been made of iis pharmacokinetics and metabolism in humans exposed to low
concentrations. Most available information is derived from studies appearing
within the last 8 years, stimulated by the resurgence of interest in DCM
following the demonstration of its metabolism to carbon monoxide (CO) (Stewart
et al., 1972). Of interest also is the question of the contribution of this
metabolic transformation to the well-known neurotoxicity and cardiotoxicity of
DCM. These studies have been greatly facilitated by the development of gas
chromatographic methods for the determination of DCM and its metabolites in
alveolar air and body fluids and tissues (Latham and Potvin, 1976; DiVincenzo
et al., 1971).
4.1.2 Pulmonary Uptake and Tissue Distribution
Dichloromethane is appreciably more water soluble (2 g per 100 ml) and less
lipid soluble than its congeners, chloroform and carbon tetrachloride.
005DC5/A 4-2 12-9-81
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At 37°C, DCM has a partition coefficient (.olive oil/water) of 130 (Lehmann ana
Schmidt-Ken 1, 1936; Morgan et al., 1972). Morgan et al. (1972) suggest, from
correlation of lipid solubility and toxic effects of halohydrocarbons, that
compounds with lower lipid partition coefficients may not achieve sufficient
levels in tissues to produce acute liver and kidney damage like chloroform and
carbon tetrachloride.
Because of its water and lipid solubility and the very large lung alveolar
surface area, inhaled DCM rapidly equilibrates across the alveolar endothelium.
The blood/air and water/air Ostwald coefficients of 7.9 and 7.2 at 37°C,
respectively, indicate that OCM is largely dissolved in plasma and cellular
water rather than lipid components of blood (Morgan et al., 1972; Lindqvist,
1978). Therefore, DCM probably distributes throughout the body water and has
been detected in urine (MacEwen et al., 1972; Divincenzo et al. , 1972) and
breast milk (Vozovaya et al. , 1974). Because of its lipid solubility, it
distributes to all body tissues and cellular lipids. It readily crosses the
blood-brain barrier even at relatively low vapor exposure concentrations as
evidenced by its impairment of manual and mental performance at 500 ppm (1737
mg/m ) (Winneke and Fodor, 1976). It also crosses the placenta and may affect
fetal development (Schwetz et al., 1975). Tissue concentrations of DCM in-
crease with exposure concentration and duration and, for any given tissue, are
dependent also on the largely unknown tissue partition coefficients. Engstrom
and Bjurstrom (1977) determined that the Ostwald coefficient for subcutaneous
adipose tissue from human buttocks is 51 at 60°C, a value which indicates that
the partition coefficient for this tissue/blood may be about 7 at body tem-
perature.
The magnitude of DCM uptake into the body (dose, burden) primarily depends
on several parameters: inspired air concentration, pulmonary ventilation,
C05DC5/A 4-3 12-9-81
-------
duration of exposure, and the rates of diffusion into ana soluo^ity in the
various tissues. The concentration of DCM in alveolar air, in equilibrium
with pulmonary venous blood content, approaches a minimum difference with the
concentration in the inspiratory air until a steady-state condition is reached.
After tissue and total body equilibrium is reached during exposure, uptake is
balanced by elimination through the lungs and by other routes, including
metabolism. The difference between alveolar and inspiratory air concentrations,
together with the ventilation rate (about 6 1/min at rest), provides a means
of calculating uptake during exposure:
Q = (C- - C , ) V • T
y v insp alv
where Q is the quantity absorbed; C, the concentration in mg/1; V, alveolar
ventilation rate in 1/min; T, duration of exposure in minutes. The percent
retention is defined as (C. - Caiv)/Cinsp x 100. and % retention ,x quantity
inspired (V • T • C. ) is equal to uptake.
Figure 4-1 illustrates the overall time-course of absorption and elimi-
nation during and after a 2-hour inhalation exposure of 100 ppm (347 mg/m )
DCM for a 70-kg man. During exposure, the alveolar air concentration of DCM
can be described by an exponentially rising curve with three components. At
the beginning of exposure an initial rapid rate of uptake occurs [0 to 50 ppm
(0 to 173 mg/m ) alveolar air], followed by a second slower uptake [50 to 65
ppm (173 to 208 mg/m ) alveolar air] and finally a very slow rate as equilib-
rium is approached at 70 ppm (243 mg/m ) alveolar air concentration. The
total quantity of DCM absorbed and retained in the body during exposure is
represented by the area between the alveolar and inspired environmental air
concentration curves. Complete equilibrium or steady-state conditions are not
attained by the end of the 2-hour exposure to DCM as shown by the slowly
rising alveolar concentration curve. The three uptake compartments of the
005DC5/A 4-4 12-9-81
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DURING
EXPOSURE
AFTER EXPOSURE
100
30
a
a
of
a
5
o
UJ
Z
IU
RESPIRATORY
ABSORPTION
i I 7
ROOM CONCENTRATION
ABSORPTION AND EXCRETION
OF METHYLENE CHLORIDE
EXHALED AIR CONCENTRATIONS
20
RESPIRATORY EXCRETIONS
_• • f. y-^v--,^ff-fffyi,y:lj,.if^,..mi^ 1.1 LIVf^H^^MK "
L—.— T T
TIME, hours
Figure 4-1. Inspired and expired air concentrations during a 2 hr, 100 ppm
inhaiatton exposure to OCM for a 70 kg man, and the kinetics of the subsequent
pulmonary excretion. From Riley et al., 1966.
4-5
-------
exponential alveolar curve correspond to equilibrium attained bv r;rst order
passive diffusion of DCM from blood first through a vessel-rich group (VRG) of
tissues with high blood flow (VRG: brain, heart, kidneys, liver, endocrine
and digestive systems), then more slowly through the lean body mass (muscle
group, MG: muscle and skin) and lastly through adipose tissues (fat group,
FG). With termination of exposure, blood and alveolar air DCM concentrations
decline in parallel in an exponential manner with three components of pulmonary
elimination and desaturation from VRG, MG, and FG body compartments. The area
under the alveolar elimination curve in Figure 4-1 is proportional to the
quantity of DCM absorbed during the 2-hour exposure.
The retention of DCM as a percentage of inspired air concentration is
independent of that concentration at equilibrium. Retention values for DCM,
reported by different investigators, are shown in Table 4-1. These values
have a large range and vary with duration of exposure. As expected, the
values are higher for short exposures of 20 to 30 minutes (75 percent reten-
tion). Variation in the values is also due, in part, to differences in body
weights of the subjects and differences in body composition (proportion of
adipose to lean mass). For exposures greater than one hour, the mean reten-
tion approximates 42 percent of uptake of DCM or approximately 125 mg/hr for
an exposure of 100 ppm, assuming a resting ventilation rate of 6 1/minute.
The quantity (dose) of DCM absorbed into the body is, for short exposures,
theoretically directly proportional to the concentration of DCM in the expired
air. This relationship has been confirmed experimentally (Lehmann and Schmidt-
Kehl, 1936; Astrand et al., 1975). The body burden of DCM also increases with
exposure duration and with physical activity (increased ventilation and cardiac
output) at a given inhaled air concentration (Engstrom and Bjurstrom, 1977;
Astrand et al., 1975). Astrand et al. (1975) found that physical activity
005DC5/A 4-6 12-9-81
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TABLE 4-1. ABSORPTION OF DCM BY HUMAN SUBJECTS (SEDENTARY CONDITIONS)
Investigator
Lehmann and
Schmidt- Ken 1 . 1936
Riley et al. , 1966
DiVincenzo et al. , 1972
Astrand et al. , 1975
Engstrom and
Bjurstrom, 1977
Inhalation
concentration,
ppm
662
806
1,152
1,181
44-680
100
100
200
250
500
750
Exposure,
hr
0.30
0.50
0.50
0.50
2.00
2.00
4.00
2.00
0.50
0.50
1.00
Retention,
%
74
75
72
70
31
53
41
51
55
55
34
during exposure to 250 and 500 ppm (869 and 1,737 mg/m3) DCM for 0.5 hour
decreased retention from 55 percent in a resting state to 40 percent during
activity, but doubled the amount of OCM absorbed because of a three-fold
increase of ventilation rate (6.9 to 22 1/minute).
The quantity of DCM absorbed is dependent also on body weight and fat
content of the body. Engstrom and Bjurstrom (1977) showed that for an exposure
to 750 ppm (2,606 mg/m ) for 1 hour the amount of DCM absorbed into the body
was directly proportional to body weight and to body fat content expressed as
a percentage of body weight. Obese subjects (avg. body fat 25 percent of body
weight) absorbed 30 percent more DCM than lean subjects (avg. body fat 8
percent of body weight). Biopsy analysis of subcutaneous adipose tissue of
obese subjects revealed concentrations of 10.2 and 8.4 mg DCM/kg tissue weight
at 1 and 4 hours postexposure, respectively. These concentrations, although
lower than found in adipose tissue of lean subjects, represented a greater
005DC5/A 4-7 12-9-81
-------
total storage amount in obese subjects since the;r total rat stores were
greater. Significant amounts of DCM were found in adipose tissue (1.6 mg/kg)
of obese subjects 22 hours after exposure, indicating that elimination of DCM
from the FG compartment proceeds at a slow rate, and that accumulation, par-
ticularly in obese people, may occur with repeated daily exposure. This
conclusion is supported by the findings of Savolainen et al. (1977), who
exposed rats chronically to DCM [200 ppm (695 mg/m ), 6 hours daily, for 5
days] and determined OCM concentrations in perirenal fat and in other tissues.
Their data, shown in Table 4-2, indicate that significant amounts of DCM
remained in perirenal fat 18 hours after the previous exposure of day 4, and
markedly increased further with a 6-hr exposure on day 5.
TABLE 4-2. TISSUE CONCENTRATIONS OF DCM IN RATS EXPOSED TO 200 PPM
FOR 4 DAYS FOR 5 HR DAILY*
Exposure
on the 5th
day, hr Cerebrum
0
2 73 ± 20
3 119 ± 33
4 57 ± 8
6 83
nmoles/q
Cerebel Turn
-
57 ± 20
36
95 ± 8
90
tissue
wet weight ±
Blood
-
90 ±
79 ±
120 ±
100 ±
10
3
10
1
S.D.
Liver
-
85 ±
82 ±
101 ±
83 ±
2
1
13
10
Perirenal
fat
113 ±
526 ±
537 ±
608 ±
659 ±
29
94
33
58
77
^Derived from Savolainen et al. (1977).
Previous work by DiVincenzo et al. (1972) in man, and Carlsson and Hultengren
(1973) in rats, had indicated that little uptake by adipose tissue occurs.
However, in their studies single, short exposure periods of 2 hours were used,
005DC5/A
4-3
12-9-81
-------
and of the smaller amounts absorbed, 95 percent was probably accommodated in
VRG and MG compartments since the FG compartment receives only 5 percent of
the cardiac output.
The blood concentration of DCM during inhalation and in the elimination
phase after exposure parallels alveolar DCM concentration. This predictable
relationship is defined by the solubility of DCM. Astrand et al. (1975)
showed that for men exposed to 250 and to 750 ppm (869 and 2,606 mg/m ) DCM
for 1.5 hour, the arterial blood (mg/1) : alveolar air (mg/1) concentration
ratios were constant and averaged 10.3 and 11.1, respectively, over three-fold
changes in alveolar concentrations. These j_n vivo Ostwald coefficients are in
good agreement with the value (8) found by Lindqvist (1978) for blood/air at
37°C j_n vitro. MacEwen et al. (1972) determined the DCM blood concentration
in dogs continuously exposed for 16 days to 1,000 and 5,000 ppm DCM. Blood
levels found were 36 and 183 mg/1, respectively, in direct proportion to
exposure concentration. Total equilibrium can be assumed to have occurred in
these animals. Ostwald coefficients of 10.4 and 10.5 for the two exposure
concentrations are in close agreement with the above values noted in man.
Similar values can be calculated from the data of Latham and Potvin (1976);
Figure 4-2 shows the proportional relationship they found in rats between
blood and inspired air concentrations over a range of 1,000 to 8,000 ppm
(3.474 to 27.792 g/m3).
4.1.3 Elimination
The evidence in the literature indicates that any significant elimination
of DCM is only by the pulmonary route. MacEwen et al. (1972) found DCM in
urine collected from dogs 6 and 24 hours after a 5,000 ppm inhalation exposure.
Analysis revealed 51 and 33 mg/1 urine, respectively, demonstrating that there
is some excretion of DCM by this route. However, these amounts represent less
005DC5/A 4-9 12-9-81
-------
* 0.60
o
e*
O
O 0.40
Q
O
O
0.20
INHALATION
1000 2000 4000
EXPOSURE CONCENTRATION, ppm
8000
Figure 4-2. DCM venous blood levels in rats immediately after a single
6-hour inhalation exposure to various concentrations of DCM. From
Latham and Potvin, 1976.
4-10
-------
than 2 percent of the orobable body aose. DiVincenzo et al. (1972) found an
average of 22.6 ug of DCM excreted in the urine of four subjects during a
24-hour period after exposure to 100 ppm (347 mg/m ) for 2 hours, and an
average of 66.4 ug for seven subjects exposed at 200 ppm (695 mg/m ) for 2
hours. These very small amounts are less than one percent of the expected
body retention of DCM.
Figure 4-1 also shows schematically the time-course of pulmonary elimina-
tion of DCM after exposure. The parameters of elimination equilibration of
the body are the same as those of assimilation equilibration. After termina-
tion of exposure, DCM immediately begins to be eliminated from the body via
the lungs. Alveolar air equilibrates with pulmonary venous blood whose concen-
tration becomes a function of the first order diffusion of DCM from tissues,
the arterial blood flow/tissue mass, and the relative solubilities of DCM in
tissues. Figure 4-1 shows that alveolar DCM concentration follows an exponen-
tial decay curve with three major components reflecting de-saturation of the
VRG, MG, and FG compartments, respectively. The half-times of elimination of
DCM from these compartments have not been firmly established. DiVincenzo et
al. (1972), who exposed subjects to 100 ppm and 200 ppm (347 and 695 mg/m3)
for 2- and 4-hour periods, felt that "very little vapor" reached the fat
stores and muscle tissues under these conditions. They found DCM to have a
half-time value in blood of 40 minutes following 2 hours of exposure, and pro-
longing exposure to 4 hours had no significant effect on the half-time.
However, the low exposure concentrations, combined with the poor recovery of
their gas chromatographic method for blood DCM, suggests that these investi-
gators were not able to follow the complete blood decay curve. Riley et al.
(1966) measured expired air concentrations after termination of exposure and
found half-times of 5 to 10 minutes for the VRG compartment, 50 to 60 minutes
005DC5/A 4-11 12-9-81
-------
for the MG Compartment, and 400 minutes for the FG comoartment. Morgan et al.
(1972), using isotopically labeled DCM, estimated the half-time of DCM in the
VRG compartment as 23 minutes. Engstrom and Bjurstrom (1977) reported the
half-time of the MG compartment for lean subjects as about 60 minutes with a
longer value in obese subjects. For both lean and obese subjects, they found,
by biopsy, residual concentrations of DCM in adipose tissue nearly 24 hours
after exposure, suggesting that the half-time of elimination from the FG
compartment is fairly long. From the postexposure alveolar concentration
curves prepared by Stewart et al. (1976a) for subjects exposed to inhalation
concentrations of 50 to 500 ppm (173 to 1,736 mg/m ) DCM for 1 to 7.5 hours
per day for 5 successive days, the half-time of elimination for MG and FG can
be estimated as 60 to 80 minutes, and 240 minutes, respectively. Thus, the
best guess from these studies for the half-times of elimination from the
vessel rich tissues (VRG), the muscle mass (MG), and the adipose tissue (FG)
are 8 to 23 minutes, 40 to 80 minutes, and 6 to 6.5 hours, respectively. The
long half-time of elimination of the adipose tissue compartment, together with
reports that DCM remains in this compartment 24 hours after single and chronic
exposures indicates that DCM may very slowly accumulate in body fat with long
daily exposures to high air concentrations. The risk of accumulation might be
expected to be greater for obese persons.
4.2 DCM BIOTRANSFORMATION
DCM is known to be metabolized in man to carbon monoxide (CO) primarily
by the liver. The CO production results in an elevation of blood carboxy-
hemoglobin (COHb) content, from which CO dissociates at the lung and is then
eliminated. Experiments in animals suggest that the metabolism is limited by
hepatic enzyme saturation at low tissue concentrations of DCM. The extent of
005DC5/A 4-12 12-9-81
-------
metabolism in man, as a percentage of retained DCM dose, has not been deter-
mined by experiment, although estimates from extrapolation of blood COHb
levels during and after inhalation exposure indicate a metabolism of less than
10 percent of the body dose. The details of hepatic biotransformation of OCM
and the kinetics of blood COHb formation and elimination are reviewed below.
Before 1972, most of the absorbed dose of DCM was thought to be excreted
unaltered in exhaled air, while a small amount was found in the urine (MacEwen
et al., 1972; OiVincenzo et al. , 1972; Heppel et al., 1944). Metabolism of
DCM to CO was not known to occur. In 1972, however, Stewart et al. discovered
that the COHb concentration in blood increased in persons exposed to 200 to
1,000 ppm (695 to 3,474 mg/m3) DCM for 1 to 2 hours. The COHb levels continued
to rise beyond cessation of exposure and decreased more slowly than when
similar levels of COHb were induced by breathing CO., Stewart and his asso-
ciates (1972,1972a) proposed that CO was the end product of DCM metabolism.
In the following year, Fodor et al. (1973) showed that COHb blood levels from
DCM exposure were further elevated by concomitant exposure to diiodomethane
and dibromomethane, indicating that these dihalomethanes are also metabolized
to CO. At first this unique halocarbon metabolism was not generally accepted,
since the increased COHb levels might well reflect a change in the rate of
endogenous CO production or excretion that is associated with heme degradation
by the microsomal hepatic heme oxygenase system in man (Coburn, 1973; Tenhunen
et al., 1969). Stewart et al. (1972,1972a), however, observed no evidence of
an enhanced metabolism of hemoglobin in their subjects exposed to OCM, and the
subjects did not excrete increased amounts of urobilinogen in their urine
either during or after exposure.
A second hypothesis put forth to explain the origin of the excess COHb
postulated a DCM-induced conformational change in hemoglobin that increased CO
Q05DC5/A 4-13 12-9-21
-------
affinity. Hence more COHb would be formed, with a longer biological half-life,
from endogenous and exogenous CO sources at the same ambient concentrations.
Settle (1971) had shown that xenon and, to a much larger degree, cyclopropane,
bind to myoglobin and increase CO affinity. Following this observation, Nunes
and Schoenborn (1973), using X-ray diffraction, demonstrated that DCM also
binds to sperm whale myoglobin and suggested that this binding might increase
CO affinity. Settle (1975) then investigated the CO binding to human hemoglobin
(Hb). He observed that CO binding to human Hb HI vitro at 37°C is increased
in the presence of DCM (1,000 ppm, 3,474 mg/m ). The amount of COHb formed at
a given CO concentration is doubled. Also, measurements of the P™ values of
Hb (partial pressure of 0- or CO at which 50 percent saturation occurs at
37°C) in the presence and absence of DCM (1,000 ppm) indicated a six-fold
increase in CO affinity. From these observations, Settle (1975) suggested
that the increased COHb seen _ui vivo may be due to an increase in CO affinity
and not to metabolism. More recently, Collison et al. (1977) have determined
the Haldane affinity constant for CO for both human and rat blood equilibrated
at 37°C with air containing only CO and CO plus DCM (10,000 ppm). No differ-
ence in the Haldane constants for human blood (mean value, 227) or rat blood
(mean value, 179) was found. Negative results were also obtained when the
absolute affinity of CO was measured in a nitrogen-OCM atmosphere. Dill et
al. (1978) re-determined the PSQ value for human and rat blood in the presence
and absence of CO (2,500 ppm) and DCM (800 ppm). DCM was found to have no
effect on the P5Q values.
The biotransformation of OCM to CO and CO™ has now been confirmed by
several metabolism studies. The hepatic metabolism of DCM has been unequivoc-
ally shown to be the origin of the CO responsible for increased COHb blood
concentrations. Independently, several groups have shown by administering
005DC5/A 4-14 12-9-31
-------
C-DCM or C-OCM to ^ats that labeled CO subsequently appears ;n COHb *ith
essentially the same specific activity (Carlsson and Hultengren, 1975; Miller
et al., 1973; Kubic et al., 1974; Zorn, 1975). Furthermore, Fodor and co-
workers (1973,1976) demonstrated that rats exposed to CO and DCM, singly and
then in combination, effected an additive increase in blood COHb levels (Table
4-3). In addition, many investigators have shown a dose-response relationship
between injected or inhaled DCM and increased blood COHb levels in both experi-
mental animals and men (Figure 4-3) (DiVincenzo et al., 1972; Astrand et al.,
1975; Fodor et al. , 1973; Ratney et al. , 1974; Stewart et al. , 1973, 1976;
Forster et al. , 1974; Roth et al., 1975; Ciuchta et al., 1979; Hake et al.,
1974).
The metabolism of halogenated hydrocarbons to CO is apparently unique to
the dihalomethanes. It is not observed with chloroform, carbon tetrachloride,
methyl chloride, methyl iodide, trichlorofluoromethane, dichlorodifluoromethane,
carbon disulfide, formaldehyde, formic acid or methanol (Miller et al., 1973;
Kubic et al., 1974; Fodor and Roscovanu, 1976; Rodkey and Collison, 1977).
Fodor and Roscovanu (1976) state, without giving data, that chloroform, bromo-
form and iodoform are metabolized to CO in the rat, thus increasing blood COHb
levels, although this has not been the observation of other investigators.
According to Fodor and Roscovanu (1976), of the dihalomethanes, the bromo-,
iodo-, and bromo-iodo-halides are more extensively metabolized to CO than is
DCM (Figure 4-4), thus increasing COHb to a significantly greater extent than
DCM (Miller et al., 1973; Kubic et al. , 1974; Fodor and Roscovanu, 1976;
Rodkey and Collison, 1977).
4.2.1 Magnitude of DCM Metabolism
Balance studies with isotopically labeled DCM to determine the extent of
biotransformation have not been attempted in man; however, several groups have
005DC5/A 4-15 12-9-31
-------
TABLE 4-3. BLOOD CARBOXYHEMOGLOBIN CONCENTRATIONS OF RATS
EXPOSED TO CO AND DCM BY INHALATION
DCM
Exposure
100
1000
0
100
1000
CO
concentration ppm
(0.5 - 2)*
(0.5 - 2)*
100
100
100
COHb
^Saturation
6.2
12.5
10.9
16.4
19.0
*Ambient air CO concentration. Data abstracted from Fodor et al., 1973.
005DC5/A 4-16 12-9-81
-------
I
ZOO
300
400
500
INHALED AIR. ppm
Figure 4-3a. Carboxynemoglobin concentrations in male nonsmokers exposed
to increasing concentrations of DCM for 1, 3, or 5 hr per day for 5 days. Pre-
exposure values averaged 0.8%, but with 3 and 7.5 hr exposures were above
this baseline value on the mornings following exposure. Data derived from
Stewart and his associates (1972, 1973,1974).
4 -
200
400
500
BOO
1000
INHALED AIR. ppm
Figure 4-3b. Carboxynemoglobin concentrations in rats after exposure to
increasing inhaled concentrations of DCM for single exposures of 3 hr. The
values are corrected for pre-exposure COHb concentration and calculated
from the data of Fodor et at., 1973.
4-17
-------
65
60
55
50
. 40
tu
% 35
0 30
0
§ 25
O
m 20
15
10
5
m
-
•
:
-
-
•
t
• i
* *
• i
-
•
-
-
M
-
-
-
A 1 A 1 A 1
CH2CI2 CH28r2
Figure 4-4. Blood CO content of rats
after 3-hour inhalation exposure with
1000 ppm dichloromethane, dibromo-
methane, and diiodomethane, respec-
tively. From Fodor and Roscovanu,
1976.
4-
18
-------
carried out such investigations in experimental animals. OiVincenzo and
Hamilton (.1975) provided the first information on the extent to which OCM is
metabolized in rats. These investigators injected rats intraperitoneally with
14C-DCM in corn oil and determined fate and disposition of radioactivity in
exhaled air, urine, feces and carcass 2, 8, and 24 hours after single doses
ranging from 412 to 930 mg/kg. Volatile compounds in exhaled air were col-
lected, identified and quantified by GC and radiotracer assay. Recovery of
radioactivity was essentially 100 percent 24 hours after administration.
About 98 percent of total radioactivity was eliminated in exhaled air, and
less than 2 percent in urine or feces (Table 4-4). Some 90 percent of in-
jected DCM was eliminated unmetabolized in exhaled air. Most of this elimina-
tion (95 percent) occurred within 2 hours. Only 2 percent of the dose was
metabolized to CO, 3 percent to C0?, and 1.5 percent to an unidentified vola-
tile compound (Table 4-4). These results indicate that less than 7 percent of
the dose is metabolized in the rat.
Rodkey and Collison (1977) considered that the small proportion of OCM
found by DiVincenzo and Hamilton (1975) to be metabolized could be due to the
high dose of DCM used; i.e., metabolic transformation of DCM may be limited by
DCM excretion being more rapid than metabolism. These investigators carried
out balance studies with small doses of C-OCM administered to rats (17
mg/kg) by either inhalation or intraperitoneal injection. The animals were
placed in a closed rebreathing system with traps for CO^ and CO after conver-
sion to CO,, by passing through a catalyst bed of Hopcalite'3. Some 76 percent
of 14C-radioactivity was recovered as 14CO (46.9 percent) and " C02 (28.9
percent). The remaining 24 percent unaccounted for could have been exhaled as
14
unchanged C-OCM, since no radioactivity was recovered in carcass tissues.
Their results also showed that DCM is directly metabolized to CO without
005DC5/A 4-19 12-9-81
-------
TABLE 4-4. FATE AND DISPOSITION OF 14C-OCM IN RATS
(412-430) INJECTED INTRAPERITONEALLY*
As % of dose (averages)
2 hr 8 hr 24 hr
Breath
unchanged 14C-OCM 84.5 94.0 91.5
14CO 0.14 1.43 2.15
14C02 ,. 0.55 1.53 3.04
unidentified l C Q.40 0.80 1.49
85.59 97.76 98.18
Urine
unidentified 14C <0.01 <0.01 1.06
Feces
unidentified 14C <0.01 <0.01 0.07
Carcass
unidentified 14C 3.09 2.06 1.53
(mainly in liver)
*Derived from DiVincenzo and Hamilton, 1975.
005DC5/A 4-20 12-9-31
-------
isotopic dilution. The extent of the conversion was surprisingly great at
this low dose, and independent of the mode of administration. For each mole
of OCM metabolized, about 0.5 mole of CO and 0.3 mole of C02 were produced.
Rodkey and Collison (1977), in a second experiment, investigated the
relationship between dose and extent of metabolism. DCM was administered to
rats by intraperitoneal injection or by vaporization of the dose in their
closed rebreathing system without a CO trap. Doses from 6.8 to 69 mg/kg were
given. CO production and DCM disappearance were calculated from the change in
gas phase composition as determined by GC. A control period was used to
measure the endogenous rate of CO production. When DCM was added to the
system there was an immediate increase in the initial rate of CO production
(about 35 times endogenous for all doses), and a rapid disappearance of DCM
from the gas phase (90 percent in 30 minutes). CO continued to be produced
for more than 2 hours after nearly complete disappearance of gaseous DCM.
Figure 4-5 shows the rates of CO production for various doses of DCM. The
initial rates [about 25 umole/hr/kg body weight (b.w.)] are similar for all
doses, but for lower doses they progressively decrease after 1 to 2 hours to
the endogenous rate as the DCM dose is metabolized. For a very high dose of
DCM (68 mg/kg), CO was produced at a nearly constant rate over a 6-hr period
(COHb, 44 percent). These observations suggest a saturation of the meta-
bolizing enzymes even at the lowest dose (6.8 mg/kg), giving initially zero-
order kinetics followed by first-order kinetics as the DCM concentration in
the inhaled air is decreased below enzyme saturation. The total amount of CO
produced was related to the dose of DCM given. For lower doses, the moles of
CO produced per mole of DCM were similar and averaged 0.48; at high DCM dose
(68 mg/kg), the ratio was 0.62 after 10.5 hours of exposure, suggesting that
substrate-induced enzyme formation may occur with long exposure to high doses
005DC5/A 4-21 12-9-81
-------
200
o
74
150
tu
O
o
o
z
ui
LU
O 100
CO
<
o
o
50
793
I
246
TIME AFTER CHjClj ADDITION, hours
Figure 4-5. Rates of production of CO from DCM given to rats.
Each curve represents changes above endogenous CO rate after
the dose (in /^moles/kg b. w.) was given by inhalation. From
Rodkey and Collison, 1977.
4-22
-------
of DCM. Similar results were obtained with germ-free rats obviating intestinal
bacteria as a source of CO. In normal rats, DCM in fact inhibited methane
production by intestinal bacteria. The same results were also obtained with
dibromomethane, dichloromethane, bromochloromethane, and diiodomethane in
respective order of magnitude and rate of CO production.
The important finding by Rodkey and Collison (1977) that the metabolism
of DCM to CO in rats is rate limited by enzyme saturation to about 25 pmole/hr/kg
b.w. explains the seemingly low conversion observed by DiVincenzo and Hamilton
(1975) in this same species. Recalculation of their data (Table 4-4) gives a
DCM metabolism to CO of about 19 umole/hr/kg b.w. Comparable results for mice
were also obtained by Yesair et al. (1977). These investigators administered
14
by intraperitoneal injection 1.0 and 100 mg/kg C-DCM in corn oil. Exhaled
14 14 14 14
CO, C02, and unmetabolized C-OCM were trapped (CO after oxidation with
Hopcalite9 to CO-, CO,, in aqueous potassium hydroxide, and C-DCM on
coconut charcoal) and quantified by gas chromatography and radiotracer assay.
The 1 mg/kg dose (11.76 umole/kg) was quantitatively metabolized to CO (0.45
mole/mole DCM) and C02 (>0.50 mole/mole DCM). The larger dose (11.76 umole/kg)
yielded, in the exhaled air collected for 12 hours, 470 umole/kg of unmetabo-
lized 14C-DCM (40 percent dose), and 0.20 mole 14CO and 0.25 mole 14C02.
Hence, at least in mice under these experimental conditions, a 12 umole/kg
dose of DCM (1 mg/kg) does not saturate the metabolizing enzymes, whereas 1200
umole (100 mg) DCM/kg saturates the enzymes and is metabolized at a constant
rate of about 20 umole/hr/kg b.w. The remainder of the dose is excreted
unchanged in the exhaled air. Recently McKenna et al. (1979) exposed rats to
50, 500, and 1,500 ppm (174, 1,737, and 5,211 mg/m3).14C-OCM for 6 hours and
also found the net uptake and metabolism of DCM to CO and C00 did not increase
C-
in proportion to the incremental increase of DCM exposure concentration.
005DC5/A 4-23 12-9-31
-------
Increasing amounts of unchanged DCM in exna'ec air «ere "'curia with '."creasing
exposure concentration. McKenna et al. (1979) were able to describe the
relation of total metabolism of DCM to exposure concentration by nonlinear
Michaelis-Menten kinetics. While these estimated rates of DCM metabolism
observed in the rodent cannot with absolute certainty be extrapolated to man,
there is little reason to doubt that the factors governing the metabolism of
DCM in rodents are generally similar in man.
4.2.2 Enzyme Pathways of DCM Metabolism
Figure 4-6 summarizes current knowledge of the enzyme pathways involved
in the biotransformation of DCM. The scheme is based on studies j_n vivo and
j_n vitro with hepatocytes and microsomal preparations. The preponderance of
evidence as noted above from j_n vivo experiments indicates that these enzyme
pathways are unique to the dihalomethanes and give rise to both CO and CCu in
nearly equimolar amounts.
The primary reaction the dihalomethanes undergo appears to be an oxi-
dative dehalogenation first described by Kubic and Anders (1975). These
workers found that DCM and other dihalomethanes were metabolized by rat liver
microsomal fractions to CO with inorganic halide release. The system required
both NAOPH and 0? for maximal activity. These experiments were carried out in
a closed vessel, substrates were added without carrier solvent, and CO was
determined by the gas chromatographic headspace method. With dibromomethane
as the substrate, 3.6 mole bromide was produced per mole CO. In the absence
of NADPH, microsomal fractions dehalogenated the methanes without CO for-
mation. Anaerobic conditions substantially reduced the rate of conversion,
although some CO formation (20 percent maximal) occurred. Equimolar substrate
concentrations of dichloromethane, bromochloromethane, dibromomethane and
diiodomethane added to microsomes produced the least amount of CO for dichloro-
methane, while diiodomethane yielded the greatest amount (7 times DCM). Liver
005DC5/A 4-24 12-9-81
-------
CH2C12
MICROSOMAL
MIXED
FUNCTION
OXIDASE
NAOPH
BINDING TO CELLULAR
MACROMOLECULES
CYTOSOL
GLUTATHIONE
TRANSFERASE
HCHO
COHb
CO
PULMONARY
ELIMINATION
Figure 4-6. Enzyme pathways of the hepatic biotransformation of
dihalomethanes.
4-25
-------
mi cro somes were f i ^e tiroes Tiers active than '^ng ana chi"T. . fines mo'~e active
than kidney microsomes. Hogan et al. (1976) also found OCM to be converted to
CO by rat liver microsomes requiring aerobic conditions and a NADPH generating
system. These workers noted a high correlation between j_n vitro CO production
and microsomal cytochrome P.,-0 content.
Further evidence of the participation of the P-TQ mixed function oxidase
system in the metabolism of dihalomethanes is the observation that dibromo-
methane and dichlorotnethane added to microsomal cytochrome P.™ preparations
produce type I binding spectra (Kubic and Anders, 1975; Cox et al . , 1976).
However, Cox et al . (1976) found that the affinity for P^Q is less for DCM
(K,., 10 mM) than for chloroform (K<-, 3 mM) or carbon tetrachloride (1C, 1.5
mM) although carbon tetrachloride and chloroform do not give rise to CO J_n
vivo (Miller et al., 1973; Kubic and Anders, 1975; Rodkey and Collison, 1977).
Both Kubic and Anders (1975) and Hogan et al. (1976) found that phenobarbital
J_n vivo pretreatment induced additional j_n vitro CO production, while cobaltous
chloride, which depletes microsomal cytochrome P^CQ, reduced CO microsomal
production. Furthermore, SKF 525A, ethylmorphine and hexobarbital (type I
substrates) inhibited j_n vitro microsomal conversion of dibromomethane to CO
(Kubic and Anders, 1975).
Neither the details of the mechanism of the enzymatic oxi dative dechlo-
rination of dihalomethanes to CO, nor the identity of transient intermediates
are known. Carbon dioxide, an end product of metabolism j_n vivo, has not been
reported to be a product with CO of microsome oxidative dechl ori nation. Kubic
and Anders (1975) found that rat microsomal fractions debrominate dibromo-
methane in the absence of NADPH, which suggests that this compound may also be
converted to one-carbon metabolites other than CO. Van Dyke and coworkers
(1970,1971) have described a microsomal Pn oxidative dechlorination of
005DC5/A 4-26 12-9-81
-------
chloroethanes and chloropropanes. The system requires NADPH and 0-,, and is
c_
inducible by phenobarbital administration. Dechlorination occurs optimally in
the presence of CL, although some dechlorination may also occur under anaerobic
conditions. Optimal conditions also require inclusion in the system of some
unknown (not glutathione) 105,000 g cytosol factor. Unfortunately, the pro-
ducts of the dechlorination reaction were not usually characterized, since the
reaction was followed by release of C1. However, in the case of 1,1,2-tri-
chloroethane, the products were identified as mono- and dichloroacetic acid
and mono- and dichloroethanol, of which the last compound was the major meta-
bolite.
At least two pathways exist in rat liver for the biotransformation of the
dihalomethanes (Figure 4-6). Kuzelova and Vlasak (1966) detected formic acid
in the urine of DCM-exposed workers and suggested that DCM was metabolized via
formaldehyde to formic acid. Originally Heppel and Porterfield (1948) reported
the conversion of dibromomethane and bromochloromethane to stoichiometric
amounts of formaldehyde and inorganic halide by a 9,000 g supernatant fraction
of rat liver, and also by liver slices and homogenates. The system did not
require 02 but was glutathione dependent. Kubic and Anders (1975) have recently
confirmed these findings and localized this metabolic pathway to the cytosol.
Ahmed and Anders (1976) extended these findings and showed that the cytosol
system is a glutathione transferase found only in the liver which requires no
other cofactor than glutathione (cysteine is not a substitute), and is not
inducible by phenobarbital or by repeated exposure to DCM or dibromomethane.
The substrate order of activity is diiodo > dibromo = bromochloro >dichloro-
methane, the same .order as found for oxidative dehalogenation by Kubic and
Anders (1975). It is unlikely that this pathway contributes to CO production
via a metabolism of formaldehyde to CO since formaldehyde administration does
005DC5/A 4-27 12-9-31
-------
not oroduce an increase of COHb in animals or -nan (Kub^'c el 5' , l?7^, °cd'
-------
1970). However, the amount of C09 generated by oxidation of CO is very small,
since the rate of conversion is less than 5 percent endogenous CO from heme
catabolism (Tzagoloff and Wharton, 1965). On the other hand, the metabolic
rate of conversion of CO to CO- appears to increase as a function of body
stores of CO and thus blood COHb levels. In dogs, at 10 to 15 percent COHb,
the metabolic conversion rate equals the formation of endogenous CO (Luomanmaki
and Coburn, 1969). Increased CO production and elevated COHb resulting from
catabolism of OCM may stimulate the metabolic production of CO- from CO, thus
contributing to the total CO,, produced by DCM metabolism.
Together, microsome oxidative dehalogenation and cytosol glutathione
transferase dehalogenation systems (Figure 4-6) account for the CO and CO-
generated from the metabolism of the dihalomethanes. Since the microsomal
system is apparently saturated and rate-limiting at low doses (supra vide),
the relative molar amounts of CO and CO- produced should provide an index of
the activity of the two pathways. However, Yesair et al. (1977) found nearly
equal molar amounts of CO and CO- with both low and high doses of DCM in mice.
Rodkey and Collison (1977) found, for low doses in rats, 1.6 times as much CO
produced as C0?, suggesting greater metabolism by the microsomal oxidative
pathway. While it has been observed that equimolar doses of dibromomethane
and diiodomethane produce greater COHb levels than DCM produces in rats (Fodor
and Roscovanu, 1976; Roth et al., 1975; Rodkey and Collison, 1977), in micro-
somal preparations (Kubic and Anders, 1975), and in cytosol preparations
(Ahmed and Anders, 1976), no information is available on the ratio of CO:CO-
produced by these compounds. The use of isolated hepatocytes may prove useful
and avoid many of the difficulties inherent with 'whole animal' experiments.
Cunningham et al. (1979) have used isolated rat hepatocytes to investigate
the binding to cellular macromolecules of "reactive" intermediates (possibly
005DC5/A 4-29 12-9-31
-------
, aenyae; •>om m
14
binding of nonextractaole C to cellular proteins ana lipids, although less
than that observed *ith trichloroethylene or carbon tetrachloride. Binding to
RNA and DNA was insignificant compared to trichloroethylene. Phenobarbital-
stimulated rat hepatocytes showed increased binding of trichloroethylene and
carbon tetrachloride metabolites to cell lipids Out decreased the binding of
14
DCM metaoolites. Reynolds and Yee (1S67) studied labeling patterns of C-DCM
14
and C-formaldehyde in rat liver and found that they have similar patterns.
Binding occurred most at the amino acid locus corresponding to serine, and on
the acid soluble cell constituents, with smaller amounts in lipid and nucleic
acids.
The metabolism of the dihalomethanes by the microsomal oxidative dehalo-
*
genation patnway (but apparently not the cytosol pathway) can be modulated by
inducers of microsomal mixed-function oxidase system. Pretreatment of animals
with phenobarbital was found by some workers to increase blood COHb levels and
microsomal production of CD (Kubic et al., 1974; Kubic and Anders, 1975; Hogan
et al., 1976), but other investigators found no effect or observed a decrease
(Miller et al., 1973; Roth et al., 1975). Roth et al. (1975) suggested that
enhanced metabolism may initially be induced by phenobarbital, but the result-
ing increase in "local microsoma1 levels of CO may be sufficient to inhibit
cytochrome P.,-0 oxidative dechlorination. Of particular interest to human
exposure in the industrial setting is the finding that chronic daily exposures
of rats to DCM substantially increased metabolism and COHb blood concentrations.
suggesting that this dihalomethane can induce its own metabolism (Kubic et
al., 1974; Rodkey and Collison, 1977). Heppel and Porterfield (1948) also
reported that repeated administration of bromochloromethane to rats led to an
increased rate of dehalogenaticn. However. Haun et al. (1972) found that
005DC5/A 4-30 12-9-21
-------
continuous exposure of mice to 100 ppm (34-7 mg/mJ) DCM for 4 to 12 weeks
decreased hepatic content of cytochrome P^cn- Initially, repeated exposures
may result in an increase of "reactive" binding of metabolites to cellular
components, and in cytochrome inhibition by CO, thus producing cellular
functional changes contributing to the hepatotoxicity of OCM.
4.2.3 DCM-Induced Carboxyhemoglobin Formation
Blood COHb accumulates when the amount of endogenous or exogenous derived
CO in the body exceeds that of pulmonary elimination. Since Stewart and his
associates (1972,1972a) reported the remarkable increase of blood COHb (up to
15 percent from 0.6 percent pre-exposure) in persons acutely exposed by inhala-
tion to DCM vapor, numerous investigations of the phenomenon have been made in
experimental animals and man. Studies have been undertaken to determine the
dose-response relationship of blood COHb level with OCM air concentration,
with duration of exposure, with time-course of COHb blood concentration rise
and decline, and with the magnitude of its occurrence in the industrial setting.
Because of its metabolism, DCM as a new endogenous source of CO, is additive
to exogenous environmental CO as a health hazard. Of particular concern are
smokers who maintain significant constant levels of COHb, i.e., 4.6 to 5.2
percent (Stewart et al., 1974; Kahn et al., 1974), and others who may have
increased sensitivity to CO toxicity, such as pregnant women and persons with
cardiovascular disease. Indeed, Stewart et al. (1972,1972a) have noted that
exposure to concentrations of OCM that do not exceed the industrial TLV (200
ppm, 695 mg/m ) may yield COHb levels exceeding those allowable from exposure
to CO itself (35 ppm, 38.5 mg/m3). Ratney et al. (1974) studied the blood
COHb levels (calculated from alveolar concentrations) of a group of young male
adults in their workplace where large quantities of DCM were used (a plastic
film plant). Workroom air concentrations of DCM averaged 200 ppm (695 mg/m )
005DC5/A 4-31 12-9-81
-------
•vith no measurable CD. At the beginning of the work :ay cicod COHb levels
averaged 4.5 percent. After an 8-hour exposure COHb levels rose to about 9
percent, then declined exponentially to 4.5 percent by the next working day
(16 hours later) with an apparent half-time of CO pulmonary elimination of 13
hours. In contrast, 35 ppm (38.5 mg/m ) CO in the ambient air of nonsmokers
produces a blood COHb level at the end of 8 hours of 5 percent or less with a
half-time of pulmonary elimination in man of 4 to 5 hours (Peterson and Stewart,
1970; NIOSH, 1972; Lambertsen, 1974).
Stewart and his associates, in a series of studies (Stewart et al.,
1972,1972a,1973; Forster et al. , 1974; Hake et al., 1974) have shown that
blood COHb levels achieved in response to DCM exposure are proportional to the
inhaled concentration and to the duration of exposure. Male nonsmokers were
exposed to DCM for for 1, 3, and 7.5 hours daily, 5 days weekly. Blood COHb
levels were determined for daily pre-exposure and post-exposure times. Their
data, which are replotted in part in Figure 4-3, indicate that maximum COHb
levels occur with 400 to 500 ppm (1390 to 1737 mg/m ) exposure and increase
with duration of exposure. Similar results were reported in women nonsmokers
exposed 1, 3, and 7.5 hours to 250 ppm (869 mg/m ) DCM for 5 consecutive days
(Hake et al., 1974), and (Fodor and Roscovanu, 1976) in male volunteers exposed
to 100 and 500 ppm (347 and 1737 mg/m3) DCM daily for 5 days. In each of
these studies, the time course of decay of COHb levels to pre-exposure levels
occurred within 24 hours so that a consistent increment in COHb with daily
exposure was not observed (Figure 4-7); however, the half-time of COHb dis-
appearance was significantly longer than expected. The longer apparent half-
time of pulmonary elimination of CO produced by hepatic metabolism of DCM
results from storage of DCM in adipose tissue (supra vide), with conversion to
CO continuing subsequent to termination of exposure. In these circumstances,
005DC5/A 4-32 12-9-81
-------
.a
X
10 -
EXPOSURE, days
Figure 4-7. Blood COHb level in men during 8 hr exposure for 5 consecutive
days to 500 ppm and 100 ppm DCM. COHb percent saturation is equal to
nq CO per ml blood divided by 2.5. From Fodor and Roscovanu, 1976.
4-33
-------
the biological half-Mfe of CCHb derived from QCM common1;, observed as 10 to
15 hours is proportional to the body burden of OCM, in contrast to the constant
half-life of 4 to 5 hours from CO inhalation (Stewart et al., 1972, 1972a,
1976a, 1976b; Fodor et al., 1973; Ratney et al., 1974; Peterson, 1970).
Physical activity or exercise during exposure to DCM markedly increases
pulmonary absorption and body retention (supra vide), but tends to diminish
the maximum blood COHb level achieved by the end of the exposure period,
although high blood COHb levels are attained 3 to 4 hours after exposure when
compared with control sedentary subjects (Stewart and Hake, 1976; Astrand et
al. , 1975). These findings may be explained by the decrease in half-life of
pulmonary CO elimination effected by the increased pulmonary ventilation rate
and increased cardiac output with physical activity (Lambertsen, 1974).
The proportionality between DCM inhalation concentration and resultant
blood levels of COHb observed in man has been confirmed by animal studies,
although with evident species differences. Figure 4-3b shows the relationship
between exposure concentration and COHb levels in rats reported by Fodor et
al. (1973). For a 3-hour exposure, maximal levels of COHb (12.5 percent) are
achieved with about 1,000 ppm (3,474 mg/m ). Fodor and Roscovanu (1976) noted
that a 3-hour exposure with 200 ppm (695 mg/m ) DCM in man produced CQHb
levels of about 4.3 percent, but found nearly twice this level in the rat
(Figure 4-3b), suggesting that metabolic capacity for CO formation is greater
in rats than man. Hogan et al. (1976) found that rats exposed to 440 ppm
(1,529 mg/m ) for 3 hours had maximal COHb levels of about 7 percent, and
3
exposure to 2,300 ppm (7,990 mg/m ) produced no further increase. Pretreat-
ment of the animals with phenobarbital increased the rate of rise of COHb
levels, increased the time the maximum level was maintained, but did not
005DC5/A 4-34 12-9-81
-------
increase the highest level itself. These investigators suggest that endo-
genous CO inhibition of the P45Q metabolizing system by CO binding to the
cytochrome may occur at very high levels of DCM exposure.
Concentrations of COHb in the blood of rabbits after very short exposures
(20 minutes) to DCM inhalation concentrations ranging from 2,000 to 12,000 ppm
(6,948 to 41,688 mg/m ) were found by Roth et al. (1975) to be a linear function
of DCM exposure concentration. COHb levels were approximately 5.5 percent at
2,000 ppm (6,948 mg/m3) and 13 percent at 12,000 ppm (41,688 mg/m3). With 4
hours of exposure at about 7,000 ppm (24,318 mg/m ), steady-state blood COHb
concentrations of 14 percent were attained. Phenobarbital pretreatment of the
rabbits decreased the blood level of COHb achieved with a given DCM exposure,
although in parallel experiments phenobarbital stimulated rabbit microsomal
benzene hydroxylase and benzphetamine N-demethylase activity. Roth et al.
(1975), therefore, also suggested that increased CO production from DCM at the
microsome in response to phenobarbital-induced increase of cytochrome P45Q
system inhibits further DCM metabolism.
Haun et al. (1971,1972), in a comparison of dogs and monkeys continuously
exposed to 25 and 100 ppm (87 and 347 mg/m ) DCM for 6 to 13 weeks, found that
steady-state levels of blood COHb were maintained throughout the exposure
period and were proportional to the exposure concentration. While dogs had
higher steady-state blood concentrations of DCM than monkeys at these inhala-
tion concentrations, monkeys had the higher COHb blood levels, suggesting that
monkeys have a greater hepatic capacity for CO formation.
Ordinarily, the sole endogenous source of CO and hence COHb is from the
physiologic catabolism of heme by the hepatic microsomal heme oxygenase pathway
(Coburn, 1973; Tenhunen et al., 1969). The endogenous rate of CO production from
this source in normal man is about 20 umole/hr, producing a blood COHb level of
005DC5/A 4-35 12-9-81
-------
aporox imately 0.4- percent. There ;s 3 c^ose 1;near rcr^e1 ati on between the iiolar
rate of CO production and percent COHb saturation; only 10 to 15 percent of the
total body CO is not associated with hemoglobin. Most of this 10 to 15 percent is
bound to hemoproteins such as myoglobin and heme cytochromes; about 1 percent is
dissolved in body water. The rate of endogenous CO production from heme cata-
bolism is markedly inhibited by exogenous CO sources producing COHb levels of
approximately 12 percent saturation, suggesting that blood levels of 12 percent
saturation are sufficient to inhibit, by CO binding, hepatic microsomal oxidase
systems involved in hemoglobin degradation to CO (Coburn, 1970). The half-life
of COHb (4 to 5 hours) arising from heme catabolism or exogenous environmental CO
is decreased by increased alveolar ventilation or increased inspired partial
pressure of oxygen (Lambertsen, 1974).
To the present time, no studies in man have been reported on either the
rate(s) of endogenous formation of CO during DCM exposure or the extent of meta-
bolism of DCM to CO as a percentage of retained dose. It is also not known if
DCM is metabolized in man by both the oxidative and nonoxidative enzyme pathways,
shown in Figure 4-6, as it is in the rat. Compared with the endogenous rate of CO
formation from heme degradation, COHb levels produced by DCM suggest that the
rates of CO formation from DCM are in the order of thirty-fold higher than endo-
genous rates from heme.
Observations in the rat and mouse indicate that their hepatic capacity to
metabolize DCM is rate-limited by saturation of the metabolizing enzyme systems at
relatively low concentrations of OCM exposure and blood levels (supra vide). A
consideration of the data of Stewart et al. (Figure 4-3), relating inhalation
exposure concentration of DCM and percent COHb saturation, and the fact that the
retained dose of DCM is directly proportional to the level and duration of
exposure, shows that the percentage of the retained dose metabolized to CO, and
OQ5DC5/A 4-36 . 12-9-81
-------
hence COHb, is not constant but decreases at higher doses. Thus, at an expo-
sure of 500 ppm (1,737 mg/m ) for 7.5 hours in male nonsmokers (Figure 4-3a),
the percent COHb saturation and hence metabolism is apparently rate-limited in
a manner similar to that in the rodent (Figure 4-3b). Since it can be expected
that complete equilibrium exists with inspired air concentration of OCM at 7.5
hours, and for an Ostwald coefficient of 10.5 for blood/air concentrations,
then the blood concentration will approximate 0.21 umole OCM/ml. Assuming
that this blood concentration of DCM does indeed saturate hepatic metabolism
of DCM, and therefore a zero-order rate of conversion to CO occurs, then as a
first approximation a one-compartment open kinetic model with zero-order input
can be used to describe the time course of blood COHb as follows:
k •+ -» k
o „ e
where V is the volume of the Hb compartment (90 percent CO is distributed in
this compartment (Luomanmaki and Coburn, 1969), k , zero order rate of CO
formation, k , first order rate constant for pulmonary elimination of CO from
COHb, and C, the concentration at any time t of COHb formed; then
k -k t
c = ° r e i
Lt Vk Ll-e J
This equation (Wagner, 1975) describes the time course of rising COHb concen-
tration with zero-order formation of CO. With long periods of exposure to
DCM, the relation becomes
t o e
that is, a steady-state plateau concentration of blood COHb is reached. Using
the data of Stewart et al., Figure 4-3, and assuming that a value of 11 percent
005DC5/A 4-37 12-9-81
-------
;atjrgfon cor-esoonas to trie steady state concentration ror 500 ppm
(1,737 mg/m ) DCM exposure, then for a 70 kg man with a 5 liter blood volume
and a normal Hb content a value for k of 13 umole CO/hr/kg can be calculated.
This value agrees well with estimates for the rodent (20 to 25 umole CO/hr/kg)
and corresponds to a hepatic clearance of 72 ml/min or about 6 percent of the
amount of DCM in the minute blood flow through the liver.
While the accumulation of blood COHb with DCM exposure may be more appro-
priately described by other first order linear models, and possibly by nonlinear
Michaelis-Menton kinetics (McKenna et al., 1979), nonetheless this approxima-
tion indicates that the extent of metabolism of an inhaled OCM dose is probably
less than 10 percent for a 500 ppm (1,737 mg/m ) exposure. An adequate kinetic
model describing in man the parameters of absorption, distribution, and elimina-
tion of DCM, as well as metabolism to CO and COHb, would be of considerable
value in evaluating the effects of physical activity, pulmonary and cardio-
vascular diseases, and concomitant environmental xenobiotic exposures includ-
ing drugs such as alcohol and barbiturates, of which very little is known.
Hake (1979) has developed a computer simulation model, based on the Coburn-
Forster-Kane equation, describing the kinetics of CO metabolism using the
experimental data of Stewart and his associates. He was able to show that
physical activity during exposure to DCM led to a lower blood COHb than the
sedentary level, because while increased ventilation increased DCM uptake, it
also increased the pulmonary elimination rate constant (k ) for CO.
4.3 MEASURES OF EXPOSURE AND BODY BURDEN
In the controlled laboratory setting, estimating the DCM absorbed into
the body by comparing inspired and alveolar air concentrations, or by measuring
blood levels of DCM and then extrapolating these parameters to body dose still
remains an imprecise task. The goal, however, is to develop a sufficient data
005DC5/A 4-38 12-9-31
-------
base and knowledge of the kinetics of absorption, disposition, elimination,
and metabolism of DCM to enable assessment of the body burden of DCM from
acute or chronic exposure in the industrial setting where air concentrations
and exposure periods vary widely. Air monitoring, an important control measure,
cannot be used as a reliable index of body burden. At present, monitoring
levels in blood or alveolar air, and measuring blood COHb levels are the
available approaches to estimating body burdens from recent exposures. However,
in addition to the lack of reliable pharmacokinetic knowledge necessary to
interpret these determinations into accurate and reliable measures of body
burden, they are also subject to unknown inter-individual variation from
factors such as anthropometric differences, metabolism and work load, age and
sex, as well as modifications from drugs and environmental xenobiotics.
Stewart and his associates (1976a) advocate the use of breath analysis to
monitor DCM exposure, in part because it is a noninvasive method and avoids
the problems associated with multiple blood sampling required for determina-
tions of blood DCM and COHb levels. In addition, breath samples can be readily
collected with little inconvenience in the immediate post-exposure period and
also at later periods. Analysis of DCM in those samples by infrared spectros-
copy or gas liquid chromatography provides both an identification and a measure-
ment of the magnitude of exposure.
Stewart and his associates have constructed a "family" of post-exposure
breath decay curves spanning 20 hours from controlled and known inhalation
exposures of volunteers in the laboratory. The concentration of DCM in the
alveolar air during and after exposure is directly related to the average
inhalation exposure concentration. Provided duration of exposure and work
intensity are known, the total body burden can be estimated by reference to
"standard" breath concentration curves. Because of large variations between
005DC5/A 4-39 12-9-81
-------
individuals exposed under 'denticai conditions, on;v ;ery approximate DCM sodv
doses can be estimated. The first 2 hours after exposure appear to be the
most reliable breath analysis sampling time for estimating the time-weighted
average OCM exposure concentration. Stewart et al. (1976a) provide little
information on the statistical reliability or the inter-individual variation
expected in the predictive values for exposure burdens, although they point
out the desirability of constructing "individualized" breath decay curves for
every person expected to be exposed to DCM. Recently, Petersen (1978), using
the same experimental exposure data base as Stewart et al., has developed
empirical equations relating post-exposure breath concentrations with exposure
time, duration, and blood COHb levels.
4.4 SUMMARY AND CONCLUSIONS
The kinetics of absorption, distribution and excretion of DCM in man has
not been extensively studied; few well-designed, controlled studies are avail-
able, particularly for repeated chronic daily exposures to low vapor concentra-
tions relevant to workplace or environmental conditions. Nonetheless, the
broad outlines of the pharmacokinetics have been established. Inhalation and
lung absorption of DCM vapor in air is the most important and rapid route of
absorption into the body. Absorption through the skin by vapor contact or by
immersion is slow and does not contribute significantly to body burden.
Pulmonary absorption is directly related to the blood/air partition coefficient
which, for DCM, is three times greater than for methylchloroform and similar
to that of trichloroethylene. Hence, for similar vapor exposure concentrations,
pulmonary uptake of DCM will nearly equal trichloroethylene uptake and will be
three times greater than methylchloroform uptake. However, as total body dose
of DCM is related also to solubility in tissues, at 200 ppm (695 mg/m ), which
was until recently the TLV value for an 8-hr exposure, less than 2 g may be
005DC5/A 4-40 12-9-81
-------
expected to be absorbed into the body of a normal sedentary 70 kg man. The
total body dose of OCM increases in direct proportion to inspired air concen-
tration, duration of exposure, and body weight and fat content; it is also
increased by physical activity during exposure. Because of its lipid solubi-
lity, DCM is distributed widely throughout the body, readily crossing the
blood-brain and placenta! barriers, and concentrating into breast milk. Blood
and tissue concentrations achieved during exposure are directly proportional
to inspired air concentration and total body dose. OCM has the lowest tissue/
blood partition of all structurally related solvents and, for given blood
levels, the tissue concentrations for OCM are lower than for other solvents.
After exposure (inhalation, oral, or skin absorption), more than 85
percent of DCM is excreted unchanged via the lungs; less than 2 percent is
eliminated in urine, 10 percent or less by metabolism, and the remainder by
other routes of elimination. Alveolar air concentration and blood concentra-
tion decline in a parallel exponential fashion showing three major components
of elimination with half-times of approximately 15, 60, and 360 min. The long
half-time of elimination of the adipose tissue compartment (6 hr) together
with reports that OCM remains in this compartment 24 hr after exposure indi-
cates that DCM may very slowly accumulate in body fat with chronic daily
exposure, particularly in obese persons.
Although less than 10 percent of a body dose of DCM is biotransformed,
the metabolism of DCM to CO and CO,, is unique to and characteristic of dihalo-
methanes. Biotransformation occurs primarily in the liver but also in the
lung and kidney. Metabolism is rate-limited by substrate saturation of metabo-
lizing enzymes and by product (CO) inhibition. In the liver, CO is produced
by oxidative dechlorination of DCM by microsomal P.-.. mixed-function oxidase
system. A second cytcsolic glutathione transferase system dehalogenates DCM
005DC5/A 4-41 12-9-81
-------
to oroduce forr^a1 den'/de «hicK ; ~ '•jrthe*' :x-'d-';ed to 20-, T~e -vc^csoma1
system (but not the cytosol system) is inducible oy pnenooarbital ana other
microsomal inducers and Dy DCM itself.
The endogenous production of CO and thence COHb from DCM metabolism is
additive to COHb formed from exogenous CO. The formation of COHb from DCM
exposures that do not exceed the industrial TLV may yield blood COHb levels
exceeding those allowable from exposure to CO itself; hence DCM exposure may
result in health hazards and toxicities associated with the solvent itself as
well as with CO at the cytochrome level. The kinetics of COHb formation and
CO elimination are interdependent with the metabolism of DCM. A functional
relationship exists between DCM inhalation concentration and duration of
exposure and the time course and peak blood COHb level. The blood COHb level
achieved is the result of CO formation from DCM and the kirfetics of pulmonary
elimination of CO from COHb. Physical activity decreases blood COHb levels by
increasing CO pulmonary elimination.
Further research on the disposition and fate of OCM after low chronic
vapor exposure is needed and would help in understanding: (1) bioaccumulation
in adipose tissue. (2) enzyme mechanisms of biotransformation and intermediate
reactive metabolites. (3) kinetics of CO and COHb formation, (4) interactions
of DCM and CO metabolism and toxicities with ethanol and other common drugs,
and (5) binding to tissue macromolecules.
005DC5/A
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chloroethanes and propanes j_n vitro. Biochem. Pharm. 20:463-470, 1971.
Vozovaya, M. A., L. R. Malyarova, and K. M. Yenikeyera. Levels of methylene chloride
in biological fluids of pregnant or lactating workers of an industrial
rubber products company. Gig. Truda Prof Zobol 4:42-43, 1974 (Russian).
Wagner, J. G. Fundamentals of Clinical Pharmacokinetics. Drug Intelligence
Publications, Inc., Hamilton, IL, 1975.
Winneke, G. and G. G. Fodor. Dichloromethane produces narcotic effects. Int. J.
of Occup. Hlth. and Safety. 45(2):34-37, 1976.
Yesair, D. W. , P. Jaques. P. Shepis, and R. H. Liss. Dose-related
pharmacokinetics of C-methylene chloride in mice. Fed. Am. Soc. Exptl.
Biol. Proceed. 36:998 (abstr), 1977.
Zorn, H. In: Bericht uber die 14 Jahrestagung der Deutschen Gesellschaft fur
Arbeitsmedizin e. V., G. Lehnert, D. Szadkowski and H. J. Weber, eds., p.
343, Gentner Verlage, Stuttgart, 1975.
005DCS/A 4-48 12-9-81
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5. HEALTH EFFECTS OF QICHLOROMETHANE
5.1 OVERVIEW
The primary results of an acute exposure to dichloromethane (DCM) are
central nervous system (CNS) depression, cardiotoxic effects, and increased
levels of blood carboxyhemoglobin (COHb), which are a consequence of the meta-
bolic transformation of DCM to carbon monoxide (CO). Hepatic and renal
toxicity may result in liver and kidney damage that may or may not be rever-
sible, depending on dose and length of exposure. Ocular toxicity can result
in damage to the eye, but complete healing usually would be expected. Few
dermal effects have been reported, although dermal exposure may result in more
severe systemic effects. DCM may be capable of producing hemolytic anemia in
susceptible subpopulations such as those having erythrocytes deficient in
glucose-6-phosphate dehydrogenase, although there is little existing evidence.
Fatal and nonfatal acute human effects have occurred upon exposure to DCM.
Inhalation exposure or concurrent inhalation and dermal exposures have been
the most common. The concentration of DCM in human exposures has usually not
been reported. However, the circumstances under which the exposures have
occurred suggest that the concentrations have ranged from very high levels
(>5,000 ppm, 17.37 g/m ), in industrial accidents where large volumes of DCM
were involved in operations at elevated temperatures, to more moderate levels
(100 to 1000 ppm, 347 to 3474 mg/m ), i.e., those associated with home use of
consumer products, specifically paint and varnish remover. Animal studies
generally support and confirm the findings noted in cases of acute human expo-
sure. DCM has been found to be mutagenic in bacteria and a mammalian cell line.
Extensive information is either not presently available or has not been fully
appraised for an assessment of the carcinogenic potential of DCM. The EPA
005DC4/A 5-1 12-9-81
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Carcinogen Assessment Grouo ^as der~er>-ed a *-'nal assessment of the :arc-,no-
gem'city of DCM until additional data can be thoroughly evaluated.
5.2 HUMAN HEALTH EFFECTS
5.2.1 Overview
CNS effects are related to the narcotic properties of DCM. The onset of
these effects is generally rapid, and their persistence is of short duration
(normally subsiding within hours after the cessation of exposure). In cases
of acute human exposure, these effects have included death, unconsciousness,
labored breathing, headache, lassitude, and nausea.
Behavioral and neurological alterations resulting from damage to the
nervous system have been reported following acute exposure to DCM. The onset
of symptoms ranges from immediate to a latency of several months. The persist-
ence of some of these effects is prolonged, some for at least 20 months after
the exposure. The effects include mental depression (which has been known to
result in suicide in exposed individuals), personality changes, psychoneurotic
reactions, and dysarthria.
The observed cardiotoxic properties of DCM include cardiodepression and
cardiosensitization. Human case studies have been reported which included
fatalities resulting from, or closely associated with, exposure to DCM, in
which myocardial infarction was diagnosed. Nonfatal exposures have caused
electrocardiographic (ECG) changes that were similar to those induced by CO.
(It is as yet unclear what the relative contributions of DCM and its metabo-
lite, CO, are to those effects.) The case histories of certain exposed indi-
viduals suggest the existence of underlying cardiovascular disease. This effect
may, therefore, be significant to this human subpopulation.
Hepatotoxicity has not been reported in any human case report, even follow-
ing fatal exposures. The only evidence of human nephrotoxicity resulting from
OCM exposure was a finding of congested kidneys following a fatal exposure.
005DC4/A 5-2 12-9-31
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Ocular toxicity other than eye irritation and congested conjunctivas has
not been reported in humans exposed to DCM.
Human studies have reported few hematologic changes as a result of
exposure to DCM. Iji vitro studies of human blood from sickle cell patients
indicated that DCM could produce hemolysis. Furthermore, it appeared to pre-
*
ferentially lyse sickle cells and other abnormal cell forms. These studies
suggest that OCM may produce anemia in predisposed individuals (e.g., those
with sickle cell anemia or glucose-6-phosphate dehydrogenase deficiency).
The biotransformation of DCM to CO, and the subsequent formation of COHb,
is an important clinical measure of exposure. CO production, resulting from
DCM exposure, occurs intracellularly, in close proximity to the biochemical
sites of CO toxicity. The CO derived from DCM may therefore be more toxic than
that resulting from inhalation of CO, if the usual comparative measure of ex-
posure (COHb level1;) is considered. There is no general consensus concerning
the relative contributions of the parent compound, DCM, and its metabolite,
CO, to the overall toxicity of OCM.
5.2.2 Acute Effects
5.2.2.1 Experimental Exposure—Several experiments have been carried out in-
volving the acute exposure of human subjects to DCM. Fodor and Winneke (1971)
and Winneke (1974) used two behavioral tests as indications of CNS function in
subjects exposed to DCM: (a) a visual critical flicker frequency (CFF) test
to determine the frequency at which intermittent flashes of light appear as a
steady or continuous light; and (b) an auditory vigilance test (AVT), which
involves the detection of faint and frequently occurring auditory signals.
A study involving female volunteers aged 20-30 years (Fodor and Winneke,
1971) found that when these volunteers were subjected to 4-hr exposures of DCM
at concentrations of 300 and 800 ppm (1,042 and 2,779 mg/m ) (1 weeK apart),
005DC4/A 5-3 12-9-81
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there was a significant decrease n; n trie c^'f; ;a: '"^cKe^ *requenc_, VCF^ of
approximately the same degree at both concentrations. The ability to detect
sound signals in the AVT test was also reduced by exposure to DCM, especially
during the mid-interval of the 4-hour exposure. Winneke (1974) also found that
exposure of male subjects to concentrations of 370, 470, and 751 ppm (1,285,
1,633, and 2,609 mg/m ) for 3 to 4 hours resulted in a decreased CFF, a de-
creased AVT, and a decreased performance in most psychomotor tasks, especially
at 751 ppm (2,609 mg/m ). These consequences appear to be mediated via direct
effects on the CNS, since in the same study 18 subjects exposed to either 50
or 100 ppm (174 or 347 mg/m ) of the DCM metabolite CO displayed no sign of
impaired CNS function.
In a chamber with known dynamic characteristics, Winneke and Fodor (1976)
exposed 12 women (ages 22 to 31) to a DCM vapor concentration of 500 ppm
(1,740 mg/m ). Behavioral performance was evaluated by an AVT test and a CFF
test to determine cortical alertness. Latency of response and percentage of
missed signals were noted for three 1-hour periods composed of a 45 minute
vigilance task followed by a 15 minute CFF task. A decreased response in both
tests became evident within 30 minutes and the impairments increased progres-
sively through termination of the experiment 60 minutes later. Alveolar and
blood DCM concentrations were not measured in these studies, nor were COHb and
alveolar CO.
Gamberale et al. (1975) investigated the effect of DCM on psychomotor and
cognitive performance in 14 men, ages 20 to 30 years, divided at random into
two groups. The first group was initially exposed to progressively increased
concentrations of DCM—870, 1,740, 2,600, and 3,470 mg/m3 (250, 500, 748 and
997 ppm)—through a face mask. Seven days later they were observed under con-
trol conditions. The second group was studied under identical conditions, but
005DC4/A 5-4 12-9-81
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in ^averse order. The subjects were exposed to eacn concentration leve1 for
30 minutes with no break in exposure; total exposure time for each individual
was two hours. At each concentration a peak plateau of alveolar DCM occurred
in about 10 minutes. Heart rate showed no change. During the two hours of
exposure to OCM, no impairment in any measured performance was observed that
was statistically significant, although reaction time appeared to be more
irregular during exposure than during control conditions. Numerical ability
and short-term memory were unaffected. The experiment did not consider any
latency effects.
Putz et al. (1976) exposed six men and six women (ages 18-40 years) to
700 mg/m3 (201 ppm) of 99.5% pure DCM or to 80 mg/m3 (7 ppm) CO, for four hours
in an 8 m chamber with unspecified dynamics. Alveolar concentrations were
measured hourly, and peripheral blood from a finger was taken before and after
exposure. Peak alveolar CO after four hours of exposure to DCM was about
60 mg/m (17 ppm) and COHb was 5.1%. Alveolar CO after CO exposure for four
hours was 50 mg/m (4 ppm) and COHb was 4.85%. Thus, there was COHb and
alveolar CO equivalence between the two compounds. Eye and hand coordination
at the end of the exposure period was depressed in subjects exposed to either
compound compared with controls, with DCM depression being greater than CO
depression. The authors concluded that CO may have'been primarily responsible
for the decreases in performance after exposure to both compounds.
In one study, Stewart et al. (1972b) observed CNS depression in humans
exposed to 3,400 mg/m (977 ppm) 99.5% pure DCM for two hours. However, in a
later study (1973), some of these same investigators reported that results from
behavioral tests (Romberg equilibrium test, alertness testing, coordination
tests, arithmetic tests) were inconclusive in individuals exposed to levels
005DC4/A 5-5 12-9-81
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of 170, 350 and 370 mg/m (-9, 101, and 250 cpm) DCM for f = -,e days oe^ week
for up to five weeks.
Stewart and Dodd (1964) examined 11 male subjects from 23-43 years of age,
exposed to 741 to 3,431 mg/m (213 to 986 ppm) DCM for 2 hours. Several, but
not all, of the subjects exposed to 1,789 mg/m (514 ppm) or higher reported
some light-headedness. Impaired CNS function, as represented by measurement
of the Visual Evoked Response (VER), or changes in electroencephalograms (EEC)
in response to a flashing light, were observed in these same subjects.
Stewart et al. (1972a, 1972b) reported that 1 to 2-hour exposures to con-
centrations of DCM ranging from 1,740 to 3,480 mg/m (500 to 1,000 ppm) (in
one study), and from 741 to 3,431 mg/m (213 to 986 ppm) (in another study),
resulted in elevated carboxyhemoglobin (COHb) and alveolar CO values at all
dose levels examined. In both reports, the peak COHb levels as well as the
amount of COHb remaining 17-24 hours after the DCM exposures were directly
proportional to the exposure concentrations.
Stewart and Dodd (1964) studied groups of three to five men and women
(25-62 years old) who immersed their thumbs in various chlorinated aliphatic
solvents. Within the first 2 minutes of immersion in DCM, all subjects re-
ported an intense burning sensation on the dorsal surface of their thumbs.
Within 10 minutes, a feeling of coldness or numbness, alternating with the
burning sensation, was reported; the slightest movement of the thumb triggered
waves of searing and excruciating pain. Following removal from the solvent,
their thumbs were described as "numb and cold" or "asleep." A very mild
erythema and a hint of white scaling were the only overt signs of irritation.
Within one hour, both the erythema and parathesia subsided.
005DC4/A 5-6 12-9-81
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5.2.2.2 Accidental Exposure—In a case of acute exposure resorted by Stewart
and Hake (1976), a 66-year-old recently retired executive who refinished furni-
ture as a hobby, worked with a commercial paint and varnish remover containing
DCM for 3 hours in his basement. One hour later, he experienced chest pains.
Two hours later, he was admitted to a coronary care unit with severe, crushing
retrosternal pain. Acute anterior myocardial infarction was diagnosed. The
man recovered after an unspecified period of time and returned home. Two weeks
after returning home he resumed paint stripping. Again, severe retrosternal
pain developed and he was readmitted to a hospital with cardiogenic shock,
dysrhythmia, and heart failure. Six months after his second discharge from
the hospital, he once again returned to his paint stripping activities. He
experienced chest pain and died. No mention of carboxyhemoglobin levels was
made in the report.
It is likely that DCM exposure was a factor in the acute myocardial
ischemia and death of this man. Although mixed exposure occurred, DCM is
strongly implicated as a causative agent by: (1) the high correlation between
the cardiovascular effects of pure OCM and a commercially formulated paint
stripper (see Aviado et al., 1977, below); (2) the well-established metabolism
of this halogenated hydrocarbon solvent to carbon monoxide; (3) the known
similar effects of carbon monoxide and DCM on cardiovascular function; and (4)
the striking sequence of events that occurred in each episode.
This exposure is considered to be acute because of the close temporal
relationship between the exposures and the onset of clinical manifestations
and because of the relatively long time between successive exposures. This
does not, however, preclude the possibility that cumulative effects occurred.
005DC4/A 5-7 12-9-81
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Kuzelova et al. (.1975) ^sported a -~atal case of DCM exposure in a film
plate production factory. OCM vapors (concentration unspecified) were suffi-
cient to produce narcosis in a 30-year-old workman who, as a result, fell into
a vat containing the solvent. He was unconscious and exhibited acrocyanosis,
shivering, labored breathing, and severe chilblains over 40 percent of his body.
He suffered repeated anterolateral myocardial infarctions and died 26 hours
after the incident.
Another case of accidental human exposure to OCM occurred in a plant where
OCM was used to extract oleoresin from dried plant materials (Moskowitz and
Shapiro, 1952). Four men were found unconscious after excess DCM vapor
apparently escaped from a condensing system. One of the men was dead upon
arrival at the hospital. Death was attributed "presumably" to CNS depression.
At autopsy OCM was found in tissues, and the kidneys and lungs* were congested.
The three other men recovered. The only remarkable observation that can be
made from the case reports — which were unfortunately neither detailed nor
complete (no blood work was done until the 2nd day of hospital ization) — is
that hemoglobin levels were depressed (76-79% normal values) and red blood
cell counts were low (3,550,000 - 3,950,000 mm ) 2 days after admission.
Irritation of eyes, respiratory passages, bronchi, and lungs were also noted.
•Studies of human blood _i_n vitro from individuals homozygous for sickle
cell anemia (Matthews et al., 1977; 1978) have shown that DCM produced
hemolysis, preferentially of sickle and other abnormal cell types. These
reports suggest that acute exposure to relatively high concentrations of DCM
may result in anemia, particularly in individuals who are predisposed to
hemolytic anemias, e.g., persons with sickle cell anemia or glucose-6-phosphate
dehydrogenase deficiency. However, more compelling evidence is required to
ascertain that such an effect exists.
005DC4/A 5-8 12-9-81
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A fatal case of a 13-year-old boy who was remov'ng paint from a bathtub
using a paint remover that contained DCM was reported (Bonventre et aj_, 1977).
The victim was discovered dead no more than 9 hours after exposure. Obser-
vations at autopsy included congested conjunct!vae, cyanosis in the brain, and
acute lung congestion and edema. Toluene as well as OCM was detected in the
liver, blood, and brain. The concentrations of OCM found in the liver, blood,
and brain (14.4 mg/100 g, 51 mg/100 ml, and 24.8 mg/100 g, respectively)
indicated to the authors that OCM was the primary agent responsible for death,
although they also stated that toluene may have had a contributory and/or
concurrent effect. Unfortunately, COHb levels were not reported.
Two individuals were exposed to an unknown concentration of DCM fumes
resulting from an accidental leak in a degreasing factory (Benzon et al.,
1978). One of them, a 40-year-old male, was found unconscious. When examined
1.5 hours later, he was conscious, although amnesic. His initial arterial
blood carboxyhemoglobin (COHb) was 19 percent, decreasing to 11 percent after
20 hours, and to 4 percent after 28 hours. He was discharged from the hospital
with no further complications.
The other individual, a 50-year-old man with a history suggestive of
ischemic heart disease did not lose consciousness. When examined, his COHb
level was 11 percent and an early mid-diastolic heart murmur was found at the
lower left sternal border. The ECG showed left anterior fascicular block and
sinus bradycardia. The following day the ECG showed bundle branch block and
nonspecific ST-T wave changes; at this time the COHb level was 6 percent. The
myocardial fraction of creatine phosphokinase (CK-MB) was not elevated,
suggesting no myocardial injury. Subsequent ECGs showed no further changes.
Thirty-four hours after admission to a hospital, blood levels of COHb had
005DC4/A 5-9 12-9-81
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decreased to 3 percent. He -/as subsequently discharged; no *urther data were
presented. The authors commented that the greatest threat to their patient's
health was the continued high levels of COHb, resulting from metabolic con-
version of DCM to CO.
Another report of acute toxicity in humans following exposure to DCM con-
cerned a man who worked approximately 4 hours over an open drum of DCM, dipping
his bare hands into the liquid to clean copper gaskets (Hughes, 1954). There
was no provision for air ventilation in the workroom. Upon exposure, symptoms
of excessive fatigue, weakness, sleepiness, lightheadedness, chilly sensations,
and nausea were reported. About 2 hours after the exposure, cough and substernal
pain developed, and pulmonary edema was diagnosed upon admission to a hospital.
DCM was considered to be the pronounced respiratory irritant. In striking
contrast to the observations of Stewart and Dodd (1964), no adverse dermal
effects were noted in this case.
5.2.3 Chronic Effects
5-2.3.1 Experimental Exposure—Stewart and his colleagues extended their
studies of the effects of DCM on human subjects to include longer exposure.
In one study (Hake et al., 1974), male volunteers were exposed for 5 days/wk
for 5 weeks to DCM concentrations of 50 ppm in week 1, 250 ppm in week 2,
250 ppm in week 3, 100 ppm in week 4, and 500 ppm in week 5 (1 ppm = 3.474
mg/m ). Three subjects were exposed for 1 hr/day, 3 subjects were exposed for
3 hr/day, and 4 subjects were exposed for 7.5 hr/day. This complex experimental
design was further confounded by attempts to separate smoking from nonsmoking
populations. The information that can be gathered from this report is summarized
in Table 5-1. Alongside is a summary of a companion report from Stewart's group
(Forster et al., 1974), which exposed 9 female subjects to 250 ppm DCM for 1,
3, or 6.5 hr/day for 5 days.
005DC4/A 5-10 12-9-81
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TABLE 5-1. COHb CONCENTRATIONS IN NON-SMOKERS EXPOSED
TO DCM AT 250 ppm (869 mg/m3) FOR 5 DAYS
Exposure Time
hr/day
P re- expo sure
1
3
7.5
Mean of Daily Maximum
Male (n)a
0.9 (5)
3.3 (1)
7.0 (1)
9.6 (3)
Observed Average COHb (%)
Female (n)
1-4 (8)
3.5 (3)
5.1 (1)
10.1 (4)
aHake et al., 1974 bForster et al., 1974
Carboxyhemoglobin levels up to 10 percent were reported in 10 men (20 to
39 years old) and 9 women (20 to 41 years old) exposed to OCM concentrations
between 40 and 500 ppm (139 and 1,737 mg/m3) by inhalation (Peterson, 1978).
Exposure durations were between 1 hour and 7.5 hours per day for not more than
5 successive days.
5.2.3.2 Accidental Exposure—Degenerative nervous system disorder was reported
in a 39-year-old chemist occupationally exposed for about 5 years to air con-
centrations of pure OCM estimated to range from 660 to 900 ppm (2,293 to 3,127
mg/m ) (Weiss, 1967). No liver or kidney damage or ECG alterations were re-
ported. Dermal contact had also occurred and erythema and fissures appeared
on the hands and forearms. This individual's progressive visual and auditory
illusions and hallucinations were correlated with exposure to DCM. Neurologic
and psychiatric examinations excluded an underlying psychosis. Toxic
encephalosis due to occupational exposure to DCM was diagnosed.
005DC4/A 5-11 12-9-81
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Collier (1936) reported four cases of occuDaf'cnai exposure to pa^nt re-
mover containing approximately 96 percent DCM. The men, all of whom were pro-
fessional painters, had been exposed to lead for 5-14 years. During one autumn,
while they were engaged in removing paint, the workers complained of loss of
appetite, dullness, faintness, and giddiness, while using the remover and dur-
ing the following few hours. Two of the workers were examined by the author
and the findings were reported in detail. One painter, aged 42, complained of
leg and arm pains, precordial pain, great fatigue, and blurred vision. The
author diagnosed the symptoms as slight chronic lead intoxication and acute
dichloromethane-induced toxemia, since the acute symptoms subsided upon cessa-
tion of working with the paint remover. The second painter, aged 45, experi-
enced tingling of the hands and feet, in addition to symptoms of fatigue and
drowsiness. Upon cessation of exposure to DCM, all of the symptoms subsided.
Barrowcliff (1978) and Barrowcliff and Knell (1979) reported that an in-
dividual exposed to 300 to 1,000 ppm (1,042 to 3,474 mg/m ) DCM for 3 years
developed bilateral temporal lobe degeneration. This was thought to result
from chronic CO intoxication as a result of exposure to DCM.
There is one epidemiological study of workers exposed to DCM. In this
study, Friedlander et al. (1978) compared the mortality experience of 751 em-
ployees exposed to DCM with industrial workers not exposed to DCM and with New
York State male populations. The authors asserted that DCM exposure had no
adverse effect on mortality. While the data support this conclusion, there
are certain limitations in the experimental design that may limit the validity
of the findings: (a) use of a broad definition of exposure, which may diminish
some of the differences between internal ("industrial") controls and the ex-
posed population; (b) use of subject groups that permitted the "healthy worker"
005DC4/A 5-12 12-9-81
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ohenomenon to influence comparisons; and (c) use of small sample sizes in specv
fie mortality categories, despite a rather large total population.
5.2.3.3 Epidemiology—Friedlander et al. (1978) conducted an epidemiological
study of Eastman Kodak Company (Rochester N.Y.) male workers exposed continu-
ously to 104 to 4,176 mg/m (30 to 1,200 ppm) DCM for up to 30 years. No in-
crease was found in the incidence of cancer-related deaths when compared with
three control groups consisting of other Kodak employees, and subjects taken
from New York State and United States male mortality tables.
5.2.4 Relationship of CO and COHb to DCM Toxicity
It was first observed by Stewart et al. (1972b) that inhalation of DCM is
followed by a sustained elevation of COHb concentration. Several studies demon-
strate a direct relationship between peak levels of COHb formation and both
length of exposure to and concentration of DCM (Hake et al., 1974; Forster et
al., 1974).
Investigations of the source of the CO induced by OCM exposure have
centered around two possible explanations. The first of these was proposed by
Settle (1975), who suggested that the rise of COHb levels seen with DCM expo-
sure was due to a change in the affinity of Hb for CO induced by DCM. Several
subsequent reports have indicated that this is not the case but rather that
OCM is biotransformed into CO (Dill et al., 1978; Collison et al., 1977). It
was further demonstrated that DCM had no effect on the kinetics of the oxyhemo-
globin dissociation curve (ODC), and that any shift of the ODC to the left was
due solely to CO formation from DCM metabolism. It also appears that the pul-
monary loss of CO formed from DCM exposure is slower than when subjects are
directly exposed to CO (Ratney et al., 1974).
According to case studies reported by Stewart and Hake (1976), the use of
a paint and varnish remover for 3 hours produced a level of about 9 percent
005DC4/A 5-13 12-9-81
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tions greater than 5 percent can adversely affect a patient with angina
pectoris or other cardiovascular diseases (Aronow and Isbell , 1973; Anderson
et al., 1973; Scharf et al., 1974). Stewart and Hake (1976) state further
"... because it is so sustained following exposure, the cardiovascular
stress produced by elevated COHb levels, derived from C1-LC1,, metabolism,
is greater than that resulting from equally high COHb levels derived from
CO."
and:
"The COHb resulting from the metabolism of DCM is additive to the COHb
level attained from exposure to other exogenous sources of CO. For
example, a paint remover exposure that results in a 10 percent COHb
saturation level when added to a heavy smoker's pre-existing COHb level
of 10 percent will produce headache and nausea in the healthy, and suf-
ficient cardiovascular stress in the patient with coronary heart disease
to be dangerous."
A comprehensive discussion of CO effects on the CNS and cardiovascular
system is also presented in the EPA Document, "Air Quality Criteria for Carbon
Monoxide" (Environmental Criteria and Assessment Office, 1979).
5.3 EFFECTS ON ANIMALS
5.3.1 Overview
Animal studies generally support and confirm the findings noted in cases
of human exposure to DCM. This is true of the depressant effects of DCM on
the central nervous system. The primary cardiovascular effect of DCM has been
shown by animal studies to be decreased myocardial contractility. DCM is also
capable of sensitizing the heart to epinephrine-induced arrhythmias.
Animal studies have reported few hepatic changes, even at doses ranging
from the LD,-0 to "near lethal" doses. Some species differences have occurred
(dogs were more susceptible than mice), but the liver changes, nonetheless,
have been generally minor.
005DC4/A . 5-14 12-9-81
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The number of studies describing renal changes was small. In contrast to
hepatotoxicity, mice were more susceptible to nephrotoxicity than dogs, but
again, the changes reported were minimal.
Two reports, both from the same laboratory, suggested that DCM may affect
pancreatic function.
In rabbits, moderate to severe conjunctiva! changes occurred following
instillation of DCM, and corneal thickness and intraocular tension increased.
These changes persisted for 1-2 weeks.
Animal studies reported minimal hematologic changes. However, one sub-
chronic study using dogs noted a variety of changes that suggested OCM
exposure could result in hemolytic anemia.
5.3.2 Acute Effects
Studies investigating mortality following short-term exposure to OCM are
summarized in Table 5-2. Good agreement between doses causing the reported
effects is seen within each route of administration category with the
exception of one intraperitoneal value reported by Zakhari (1977). The oral
LDcQ data are also similar to intraperitoneal ID™ data. Oral and
intraperitoneal data cannot be compared with inhalation data due to the "first
pass effect" which occurs following oral or intraperitoneal administration.
Morbidity resulting after short-term exposure includes effects on several
different organs. Ocular damage, liver damage, kidney damage, changes in car-
diac parameters and increased pancreatic bile duct flow all occur.
5.3.2.1 Central Nervous System Effects—A NIOSH report (1976) records that
Berger and Fodor (1968) exposed rats to very high DCM concentrations ranging
from 2,800 to 28,000 ppm (9.7 to 97.27 g/m3). In these exposures an initial
period of excitation was followed by a deep narcosis accompanied by a decrease
in muscle tone and" a reduction in electroencephalogram (EEG) activity. Rats
005DC4/A 5-15 12-9-81
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TABLE 5-2. ACUTE LETHAL TOXICITY OF OCM
Route of
Administration
Oral
Intraperi toneal
Inhalation
Species
mice
rats
rats
mice
mice
mice
mice
mice
dogs
mice
mice
rats
rats
guinea
pigs
Dose
1987 mg/kg
4368 mg/kg
2388 mg/kg
448 mg/kg
1330 mg/kg
1995 mg/kg
1990 mg/kg
1900 mg/kg
1260 mg/kg
14,100 ppm
16,100 ppm
ppm
25,600-28,000
ppm
16,100-18,150
ppm
11,550 ppm
Duration of
Exposure
-
-
-
—
-
6 hours
7 hours
1.5 hours
6 hours
6 hours
Effect
LD50
LD50
LD50
LD50
100%
survival
20%
survival
LD50
LD50
i n
LU50
LD50
i n
LU50
(a)
(a)
LD50
Reference
Zakhari (1977)
Ugazio et al. (1973)
Kimura et al. (1971)
Zakhari (1977)
Plaa and Larson (1965)
Plaa and Larson (1965)
Klaassen and Plaa (1966)
Gradiski et al. (1974)
Klaassen and Plaa (1967)
Gradiski et al . (1974)
Svirbely et al . (1947)
Berger and Fodor (1968)
Berger and Fodor (1968)
Balmer et al. (1976)
(a) Cessation of brain electrical activity in all animals. 1 ppm = 3.474 mg/m"
005DC4/A
5-16
12-9-81
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exposed to concentrations of 25,000 to 28.000 ppm (86.9 to 97.27 g/m ) ceased
to exhibit electrical activity after only 1.5 nr; those exposed to concentra-
tions between 16,000 and 18,000 ppm (55.58 and 62.53 g/m ) ceased electrical
activity after 6 hr. Lower concentrations 5,000 to 9,000 ppm (17.37 to 31.27
g/m ) produced long sleeping periods without the frequent desynchronization
phases characteristic of normal sleep. Sleep behavior was even altered at the
lowest dose examined 2,800 ppm (9,727 mg/m ), where a 14-hr exposure resulted
in abnormal rapid eye movement (REM) sleep. Other studies have also utilized
sleep behavior-as an index of CNS function. Fodor and Winneke (1971) have
demonstrated in the rat a linear dose-response relationship between REM sleep
patterns and 24-hr DCM exposures. They found that total sleep time and time
between two REM periods increased with increasing DCM concentrations of 500,
1,000, and 3,000 ppm (1,737, 3,474, and 10,422 mg/m ). A concentration of
5,000 ppm (17,370 mg/m ) administered for 1 hr to male rats was sufficient to
demonstrate a decrease in running activity (Heppel and Neal, 1944). Running
activity increased after exposure, but was lower than activity in the same
male rats when they had not been exposed to DCM. Similarly, Thomas et al.
(1971) reported that the spontaneous activity of mice was decreased by a 3-hr
exposure to a 1,000 ppm (3,474 mg/m ) concentration of DCM.
5.3.2.2 Carbon Monoxide Formation and Cardiovascular Effects—Much of the
definitive work concerning the formation of COHb as a result of DCM exposure
and metabolism has been performed in animal model systems.
In 1973, Fodor et al. examined COHb levels in the rat after a 3-hour
exposure to DCM at a concentration of 100 ppm (347 mg/m ). The COHb level after
treatment was estimated at 10.9 percent vs 0.4 percent as the control COHb value.
Blood COHb levels in rabbits exposed for 4 hours to DCM levels of between 2,000
and 12,000 ppm (6.9 and 41.7 g/m ) were found to be a linear function of DCM
005DC4/A 5-17 12-9-81
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concentration, approximately 5.5 percent at 2,000 ppm (6.9 g/m ) and 13 percent
at 12,000 ppm (41.7 g/m3) (Roth et al., 1973).
Several investigators have demonstrated with radiolabelled DCM that CO
formed during exposure to DCM is a metabolite of the parent compound (Roth et
al., 1973; Carlsson and Hultengren, 1975; MacEwen and Vernot, 1971). DiVincenzo
and Hamilton (1975) administered radiolabelled DCM to rats intraperitoneally
(i.p.); 24 hours later it was found that 91.5 percent of the DCM was eliminated
unchanged in the expired air and only 2 percent was eliminated as CO. Hogan
et al. (1976) have determined that the mixed function oxygenase system of
hepatic microsomes is responsible for the metabolic conversion of DCM to CO.
They have also suggested that the apparent non-inducibility of CO formation
may be due to endogenous CO inhibition of the metabolizing enzyme system.
Aviado et al. (1977) used pentobarbital-anesthetized, artificially venti-
lated, open-chested dogs to examine the cardiovascular effects of exposure to
0.5, 1.0, 2.5, and 5 percent pure DCM (5,000, 10,000, 25,000, and 50,000 ppm)
(1 ppm = 3.474 mg/m ), as well as to the same concentrations of a paint stripper
composed of 90.2 percent DCM, 4.2 percent methanol, 3.2 percent isopropanol,
and 2.4 percent toluene. Pure DCM or paint stripper was administered via an
endotracheal catheter for a period of 5 minutes. At the higher doses (2.5 per-
cent and 5 percent), all hemodynamic effects were consistent with primary de-
pression of myocardial contractility (Table 5-3): left ventricular (LV) dp/dt
(the time-dependent rate of rise of ventricular pressure which is a measure of
myocardial contractility), fell, as did LV pressure. Left ventricular end-
diastolic pressure (LV filling pressure) rose. However, cardiac output fell.
There was a fall in mean arterial pressure; yet, calculated peripheral vascular
resistance (MAP-central venous pressure/cardiac output) rose. Heart rate fell
nonsignificantly when 5 percent DCM (50,000 ppm) (.174 g/m ') was employed.
005DC4/A 5-18 12-9-81
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TABLE 5-3. SUMMARY OF CARDIOTOXIC ACTION OF 5% OICHLOROMETHANE
Function3
HR
MAP
LVP
CVP
LVEDP
LVdP/dt
CO
SV
VR
Taylor et al. (1976)
1% r (NS)
0%
0%
0%
9% r (NS)
17% 4-
14% 4-
14% *
25% f
Aviado et al ,
11% 4.
4% 4.
4% 4.
125% !•
22% 4-
36% 4.
33% *
44% t
. (1977)b
(NS)
(NS)
(NS)
(NS)
Comments:
Rabbits
Pentobarbi tal-anethesti zed
Spontaneously breathing
Closed-chested
1-min. exposure (except
1.5 min. for CO)
Static dose (5%)
Dogs
Pentobarbi tal-anestheti zed
Artificially ventilated
Open-chested
5-min. exposure
Increasing doses (0.5, 1.0,
2.5, and 5.0%)
Key: HR - Heart Rate
MAP - Mean Arterial Pressure
LVP - Left Ventricular Pressure
CVP - Central Venous Pressure
LVEDP - Left Ventricular End-Oiastolic Pressure
LVdP/dt - Left Ventricular Rate of Time-Dependent Pressure Change
CO - Cardiac Output
SV - Stroke Volume
VR - Vascular Resistance (Peripheral or Systemic)
RR - Spontaneous Respiration Rate
MV - Minute Volume
NS - Not significantly different from control values at p <0.05
DResults presented are for the highest dose (5%) of dichloromethane
005DC4/A
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12-9-81
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The authors concluded that the effects //ere compouna--e' atea ana tnat 3 CM
exerted a negative inotropic action.
The effects of DCM on cardiovascular function were studied in groups of
six male New Zealand white rabbits (Taylor et al., 1976). The animals (2.4-
3.4 kg b.w.) were anesthetized with pentobarbital and bilaterally vagotomized;
results were recorded for 1-minute exposures to 5 percent (50,000 ppm, 174 g/m )
DCM in air (except for cardiac output measurements which were over a period of
1.5 min.), enriched to 40 percent oxygen and delivered via an endotracheal tube
to spontaneously breathing animals. No changes were observed in the mean arterial
pressure, left ventricular pressure, left ventricular end-diastolic pressure,
or heart rate. However, significant depression of left ventricular dP/dt (rate
of rise in ventricular pressure, an index of myocardial contractility), cardiac
output, and stroke volume occurred. Exposure to DCM resulted in increased
peripheral vascular resistance, apparently to compensate for the decreased car-
diac output and to maintain arterial pressure (Table 5-3).
Aviado's study in dogs correlates well with Taylor's findings in rabbits.
At 5 percent, DCM decreased left ventricular dP/dt, cardiac output, and stroke
volume. An increase in left ventricular end-diastolic pressure and peripheral
vascular resistance was observed in both animal models. The mean arterial
pressure and left ventricular pressure were not affected in either study.
One difference between the studies of Aviado and Taylor was the lack of
change in the heart rate of rabbits compared with an apparent decrease in dogs.
This could be due to a difference in the length of exposure, since rabbits were
exposed for only 1 minute, and dogs were exposed for 5 minutes. Also, vagotomy
of the rabbits may have prevented a parasympathetically-mediated fall in heart
rate. The only other difference between the two studies was that left ventri-
cular end-diastolic pressure did not change in rabbits, but significantly in-
creased in dogs.
005DC4/A 5-20 12-9-31
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Trained rnale beagle dogs (size and sex unspecified) were ^epeatedly exposed
to 37,000 and 70,000 mg/m3 (10,650 and 20,150 ppm) pure DCM (Reinhardt et al.,
1973). After 5 minutes of exposure to OCM, 0.008 mg/kg epinephrine was injected
intravenously. Sensitization to epinephrine did not develop. Higher concentra-
tions of DCM could not be given because the animals became hyperactive, similar
to Stage II general anesthesia.
Aviado and Belej (1974) exposed Swiss mice (25-35 g) five times to DCM
concentrations of 20 and 40 percent v/v in oxygen. A face mask of (unspeci-
•
fied) description was used. Cardiac arrhythmia was produced by the higher
concentration. Intravenous epinephrine 0.006 mg/kg sensitized the heart to
arrhythmias induced by the lower dose.
Loyke (1973) induced chronic renal hypertension in 13 Sprague-Oawley rats
(100 g, sex not specified). Both poles of one kidney were ligated and the con-
tralateral one was removed. After maintenance of high systolic pressure for
three months, 11 experimental rats and three controls were injected subcutane-
ously with 2 mg/kg unspecified DCM, biweekly for 15 doses. Two hypertensive
rats were used for positive controls and 11 normotensive rats as naive controls.
DCM reduced the systolic blood pressure of the hypertensive rats from 200 to
160 mm Hg. The positive controls remained hypertensive. DCM was ineffective
in reducing blood pressure in normotensive rats. No changes ascribable to DCM
were seen in the liver.
Douglas et al. (1976) and Wilkinson et al. (1977) used spontaneously hyper-
tensive rats weighing 400 mg (sexes not specified). Six were injected sub-
cutaneously with 90 mg/kg unspecified DCM. Six others were used as controls.
OCM reduced the blood pressure of the hypertensive rats by about 10 percent
but was ineffective in reducing blood pressure of the normotensive rats.
005DC4/A 5-21 12-9-81
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Adams and Erickson (1976) exoosed eight trained mongre1 dogs (size and
sex not specified) to 1,700, 3,400, 6,800 and 17,400 mg/m3 (489, 979, 1,957
and 5,009 ppm) OCM for two hours via a permanent tracheotomy. The animals
were exposed repeatedly but randomly to all concentrations. Frequency of ex-
posure was not given. COHb, which continued to increase for two hours after
exposure, was both time- and dose-responsive. The slopes of the increase
showed that the higher blood levels increased faster at higher concentrations
than at lower concentrations. Cardiovascular effects resembled those induced
«
by epinephrine, except that the heart rate did not change. DCM increased blood
pressure, increased coronary flow, increased inotropic action and induced
arrhythmias.
Pryor et al. (1978) reported an experiment with an open-chest mongrel dog
(size and sex unspecified). The blood concentration of OCM after 1.5 minutes
of exposure to vaporized DCM of unspecified concentration with oxygen supple-
mentation was 2,320 ug/ml and without oxygen supplementation was 2,680 ug/ml.
A second exposure to DCM was given one hour after the first. After both ex-
posures, heart rate slowed, blood pressure fell and aortic blood flow decreased.
During recovery after the first exposure, tachycardia and hypertension developed.
After the second exposure, the animal died.
Differences in the above studies in terms of the effects of DCM on blood
pressure, inotropic action, and induction of arrhythmias may be due to the use
of different species of animals and different doses of DCM.
5.3.2.3 Effects on the Eye—Ballantyne et al. (1976) studied the effects of
liquid DCM on various ocular parameters by instilling 0.1 and 0.01 ml of DCM
in rabbits' eyes. Lachrymation persisted for a week, inflammation of the lids
and conjuctivae for two weeks, conjunctiva! edema for a week, sloughing for
three days, and increased corneal thickness for nine days, [ritis and keratitis
005DC4/A 5-22 12-9-81
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appeared within six hours and lasted for seven ana fourteen days, respectively.
Intraocular pressures increased also. Increased corneal thickness developed
in rabbits exposed to OCM vapor at concentrations of 1,750 and 17,500 mg/m
(504 and 5,040 ppm).
5.3.2.4 Effects on Internal Organs and Metabolism—Harms et al. (1976) cannu-
lated the bile duct of male Sprague-Oawley rats (350-450 g) after pretreatment
with intraperitoneal injections of 670 mg/kg OCM dissolved in corn oil. H-
Insulin was instilled into the duct 24 hours later. OCM seemed to induce an
increase in pancreatic bile duct flow which was unrelated to any observed
hepatic effect. Hamada and Peterson (1977), in a follow-up study, intraperi-
toneal ly injected 860 mg/kg DCM (dissolved in corn oil) in male Sprague-Oawley
rats (280-320 g). The bile duct was cannulated and bile duct pancreatic flow
and its contents were measured and compared with controls. OCM induced in-
creased pancreatic bile duct flow, decreased protein concentration, and in-
creased chloride, sodium and potassium. Bicarbonate was unaffected. There
was no statistically significant difference in wet weight of the pancreas or
in total bile flow; this may indicate a reduced hepatic bile flow. An experi-
ment with secretin indicated that these changes were not related to that sub-
stance or, after an ancillary experiment with atropine, to any cholinergic
effect.
Plaa and Larson (1965) injected 10 male Swiss mice (18-30 g) intraperitone-
ally with 1330 mg/kg of DCM (source and quality unidentified) dissolved in corn
oil. No glycosuria or proteinuria was detected 24 hours later. Two surviving
mice of ten that had been injected with 1,995 mg/kg OCM had proteinuria but
not glycosuria. No renal histopathology was seen after the lower dose.
Proteinuria following the higher dose suggested that there might have been
some tubular damage.
005DC4/A 5-23 12-9-81
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Klaassen and Plaa (1966) also injected male Swiss-Webster mice (25-35 g)
intraperitoneally with 13.3 g/kg analytical grade DCM. These mice showed no
glycosuria, proteinuria or changes in Bromsulphalein (BSP) excretion 24 hours
after injection. Two groups of ten mice each were gavaged for three days with
5 g/kg 60 percent ethanol and equicaloric solutions of dextrose. All animals
were then injected with DCM, and urine was collected and analyzed 24 hours
later. BSP excretion remained within control limits, indicating the absence
of a renal lesion, but the authors reported "a few kidneys exhibited hydropic
degeneration with minimal necrosis of the convoluted tubules."
Klaassen and Plaa (1966) injected Swiss-Webster mice (25 to 35 g) intra-
peritoneally with analytical grade DCM. Doses of 13,300 mg/kg had no effect
on BSP retention or on SGPT activity 24 hours after injection. No histopatho-
logy was seen upon examination of the liver.
In the dog, however, effects on the liver by DCM were reported by Klaassen
and Plaa (1967). They derived an ED5Q for SGPT elevation of 798 mg/kg (the
LD50 was 1,260 mg/kg). Histopathology showed moderate neutrophilic infiltra-
tion in the sinusoids and portal areas. Necrosis was not seen. At "near lethal"
doses there was vacuolization of centrilobular hepatocytes.
Reynolds and Yee (1967) gavaged fasted male Charles River rats (100 to
300 g) with doses of C-QCM up to 2,210 mg/kg, dissolved in mineral oil. The
rats were sacrificed 24 hours later. No liver necrosis was seen and there was
14
no change in glucose-6-phosphatase activity. Labelled DCM ( C) was found in
hepatic lipids and proteins, minimally in the lipids and in high concentrations
in proteins. Similar patterns of incorporation were found in liver microsomes.
In contrast to Reynolds and Yee's findings of minimal lipid incorporation
of 14C after gavage, Bergman (1978) found that after 10 minutes of inhalation
005DC4/A 5-24 12-9-31
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14
of C-labelled DCM by mice (strain, sex, weight, unspecified; DCM unspecified)
whole body autoradiography showed OCM to have ". . .a strong fat affinity".
Metabolites appeared immediately in the oronchi, the liver and the kidney and
persisted for as long as 48 hours. The formation of CO also produced radio-
activity in blood and protein. Differences between the two studies may have
been due to differences in animals, route of administration and dose.
Weinstein et al. (1972) exposed female ICR mice (13 to 20 per group, 23
to 27 g) continuously to approximately 17,000 mg/m (4,893 ppm) vaporized tech-
nical grade DCM in Thomas Domes, at pressures slightly below ambient. Tempera-
ture, humidity, C0? and air flow were monitored and controlled. During a 24-
hour period, there was a progressive decrease of spontaneous activity. 8ody
weight decreased, liver weight increased absolutely and as a ratio to body
weight, liver triglycerides increased,(indicating liver toxicity), glycogen
decreased, and protein synthesis was reduced as shown by reduction of incorpora-
tion of H-leucine.
Many histological changes appeared. Fatty infiltration after 24 hours
involved the entire lobule; centrilobular hepatocyte nuclei became smaller and
dense, and ballooning developed. Staining showed glycogen reduction. Progres-
sive changes began after 12 hours of exposure. Smooth (SER) and rough (RER)
endoplasmic reticulum showed changes: polysomes broke down, ribosomal particles
detached from RER in the centrilobular cells, RER membranes broke up into
vesicle, pernuclear cisternae dilated, and lipid droplets increased. Mitochondria,
however, were unaffected. The authors concluded that the pattern of liver damage
was similar to that following carbon tetrachloride exposure.
Morris et al. (1979) exposed male Hartley guinea pigs weighing 500 to 750 g
for six hours to an 18,000 mg/m (5,181 ppm) vapor concentration of reagent
grade DCM. Animals were sacrificed immediately after exposure and liver samples
005DC4/A 5-25 12-9-81
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,vere taken. Liver and serum were analyzed for tf'glyce^des *nicb ^ncreased
markedly in the former and decreased in the latter. Liver phospholipids
showed no change nor did serum-free fatty acid. Liver slices were incubated
14 14
with C-palmitic acid. The uptake of C was similar to controls. Uptake
14
after Oleucine did not differ between controls and exposed samples.
Differences in the above two studies in protein synthesis may be due to
different periods of exposure and the use of different animal species.
5.3.3 Chronic Effects
5.3.3.1 Central Nervous System Effects—Few studies have examined the effect
of chronic DCM exposure on CNS function. One assumes the reason for this to
be that chronic DCM exposure would elicit many of the same types of responses
produced in acute studies. This appears to be borne out by the few chronic
studies that have looked at CNS function. For example, Heppel et al. (1944)
exposed dogs, monkeys, rabbits, guinea pigs, and rats to DCM at a concentration
of 34,800 mg/m3 (10,000 ppm), 4 hr/day, 5 days/wk for 8 weeks. They found that
all animals became inactive during each exposure, but some went through an
initial excitement stage, very similar to the findings in the acute study of
Berger and Fodor (1968). Weinstein et al. (1972) had also reported decreased
activity in female mice at the 24-hour juncture of a 7-day, continuous study
of 17,400 mg/m (5,000 ppm) of DCM. Along with the lethargy, the mice were
also observed to assume a hunched posture and to develop yellow, greasy, rough-
appearing hair coats. After 96 hours of exposure, normal activity, for the
most part, resumed. At 168 hours of exposure, the only abnormal sign that
remained was the appearance of emaciation and dehydration. Thomas et al. (1972)
reported a paradoxical increase in the spontaneous activity of female mice that
had been continuously exposed to DCM at 87 mg/m (25 ppm) for 14 weeks. Raising
005DC4/A 5-26 12-9-81
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the exposure concentration of 348 mg/m (100 ppm) -esuited in a ^esponse no
different from control. Raising the exposure level to 3,480 mg/m (1,000 ppm)
for a continuous 4-week study resulted in a decrease in observed spontaneous
activity (Heppel and Neal, 1944).
5.3.3.2 Effects on CDHb Levels—Only one study in the literature has looked
at the effects of chronic low level concentrations of DCM upon COHb formation.
Haun et al. (1972) reported significantly elevated COHb levels in dogs and
monkeys exposed to 25 and 100 ppm (87 and 347 mg/m ) DCM. The 100 ppm monkeys
showed this highest COHb percentage, followed by the 100 ppm dogs, then the
25 ppm monkeys. The 25 ppm dogs did not have significantly elevated COHb levels.
Additionally, the authors reported finding no significant macro-or microscopic
changes in any organ in either monkeys or dogs after up to 100 days of chronic,
low level (25 ppm and 100 ppm) DCM exposure.
5.3.3.3 Effects on Internal Organs and Metabolism—Many of the "high-dose"
chronic animal studies with OCM have revealed a certain degree of liver and
kidney involvement as target organs. The magnitude of this involvement in-
creases in such a way that extremely high doses of DCM (depending upon dosage
and duration of exposure) can produce toxic effects upon these organs. Heppel
et al. (1944) have observed moderate centrilobar congestion and fatty degenera-
tion of the liver in dogs and guinea pigs exposed to 10,000 ppm (34,740 mg/m ),
4 hr/day, 5 days/wk for 8 weeks. Weinstein et al. (1972) have reported identical
findings after exposing mice to 5,000 ppm (17,370 mg/m ) continuously for 7
days. High mortality was observed in the studies by MacEwen et al. (1972) where
14 weeks of continuous exposures at 1,000 or 5,000 ppm (3,480 or 17,400 mg/m )
resulted in severe toxic effects and a high degree of mortality in mice, rats,
dogs, and monkeys.
005DC4/A 5-27 12-9-31
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More importantly, chronic mouse studies by Weinste1'n and Diamond C1972)
and Haun et al. (1972) have revealed that continuous exposure to even such low
levels of DCM as 100 ppm (347 mg/m ) can effect changes in both liver function
and cell architecture.
Savolainen et al. (1977) exposed male rats to 489 ppm (1700 mg/m ) DCM
for six hours per day for four days. Accumulation of DCM in perirenal fat was
found 17 hours after the last exposure. After an early uptake, the level seemed
to be maintained during the four days of the experiment. A number of spontaneous
behavior patterns were studied. The only ones that seemed to be affected were
preening frequency and preening duration; both increased. Depression in any
behavior pattern was not mentioned.
Weinstein and Diamond (1972) exposed ICR mice (17 to 25 g) continuously for
three days to 10 weeks to 100 ppm (347 mg/m ) of chemical grade DCM in a
Thomas Dome with specified dynamic characteristics and at 96.66 Pa (725 mm Hg)
pressure. Twelve groups of 16 mice each were used. Except for one instance
at week 2, body weights were comparable to controls. Liver weights followed
body weights, and in all cases liver to body weight ratios were within control
limits. Triglycerides showed an approximate threefold increase at week 2, a
nearly fourfold increase by week 3, then fell to about double at week four.
Four mice withdrawn at three days showed no abnormalities, but at 7 days,
centrilobular fat accumulation was seen accompanied by a decrease in liver
glycogen. These abnormalities persisted to the termination of the experiment
at 10 weeks. During tnis time, the nuclei enlarged. No other histopathology
was seen under the light microscope. Under the electron microscope, autophagic
vacuoles containing debris appeared in the hepatocytes. The smooth and rough
endoplasmic reticulum showed no changes.
005DC4/A 5-28 12-9-81
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Haun et al. (1972) exposed mice, rats, dogs, and monKeys to 25 ana 100 ppm
(87 and 347 mg/mJ) reagent grade DCM for continuous exposure periods up to
100 days in Thomas Domes at ambient pressure. This is the longest continuous
exposure study reported. Strain, sex and size of the test animals were not
specified. Exposure to the lower concentrations had no observable effect, but
exposure to the higher concentrations resulted in positive fat stains and
vacuolization. The rats showed nonspecific tubular degeneration and regenera-
tion in the kidneys, but no changes in organ-body weight ratios.
Heppel et al. (1944) conducted two inhalation experiments. One used
1,700 rng/m (489 ppm) commercial DCM, for 7 hours daily, 5 days per week for
six months. The other involved exposure for four hours per day, five days per
week, for 7-8 weeks at a concentration of 34,000 mg/m (9,780 ppm). Dogs,
rabbits, guinea pigs and rats were used in each experiment. Two monkeys were
added to the experiment at the higher concentration. Animals were exposed
together in a single chamber. Temperature and humidity were uncontrolled. At
the lower dose, 3 of 14 male guinea pigs died. They had fatty degeneration of
the liver and pneumonia. No other animals' deaths attributed to DCM exposure
were reported.
At the higher dose, the dog experiment was terminated after six exposures
because of the continuing Stage II excitement reactions. Three rabbits and
one rat died during the course of the experiment. Each animal had extensive
pulmonary congestion. Clinical observations in the dogs at the lower dose
showed no changes in blood pressure, blood chemistry or liver function tests,
and at autopsy, organ weights of liver, kidneys, heart, lungs and spleen were
similar to controls. .No lesions due to DCM were found. All animals showed
clinical effects after exposure to the higher concentration. Aside from the
responses in dogs noted above, the other animals showed progressive signs of
005DC4/A 5-29 12-9-81
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depression. J5uai]7 becoming prostrate at the eno of eac~ jai \, exposure period.
All animals, including dogs, recovered rapidly upon removal from the chamber,
and fed well. At autopsy, dogs and guinea pigs showed fatty degeneration of
the liver. Pulmonary congestion was found in the rabbits. Monkeys and rats
showed no lesions related to DCM exposure.
Norpoth et al. (1974), when studying the possibility of enzyme induction
in inhalation of hydrocarbon solvents, tested DCM. Male SPF Wistar rats (80-
100 g) were exposed 5 hours per day to vapors containing 0, 500, or 5,000 ppm
(0, 1,737 or 17,370 mg/m OCM) of the hydrocarbon solvents in an exposure period
of 10 days or 250 ppm (869 mg/m DCM) of the solvents in an exposure period of
28 days. There were 50 animals in the control group and six animals in each
of the exposed groups. At the end of these exposure periods, the animals were
killed and the concentration of liver cytochrome P-450 and microsomal aminopyrine
demethylase activity were determined.
Following the 10-day exposure, a significant increase in liver cytochrome
P-450 in animals exposed to 500 ppm (1,737 mg/m ), but not in animals exposed
to 5,000 ppm (17,370 mg/m ), of the compound was reported. In contrast,
aminopyrine demethylase activity was not elevated in animals exposed to 500 ppm
(1,737 mg/m ) DCM but was substantially elevated in those exposed to 5,000 ppm
(17,370 mg/m3) OCM. After 28 days of exposure to DCM at 250 ppm (869 mg/m3),
no changes were noted in liver enzymes, liver weight, or histologic appearance.
These results indicate that differential enzymatic induction can be pro-
duced by exposure to DCM. The inverse dose-response relationship noted for
cytochrome P-450 may be due to the combined effect of DCM and CO. Acclimatiza-
tion may have occurred by the 28th day of exposure to ameliorate the effects
seen at 10 days. In addition, the doses given in the second exposure period
(28 days) were much smaller than during the first period.
005DC4/A 5-30 12-9-81
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Ten aault Wistar rats were exposed to 0 or 503 x 35 opm (.1,747 z 295 Tig/m")
DCM 6 hours per day for 4 days (Savolainen et al., 1977). Biochemical analysis
of tissue from the right cerebral hemisphere showed no difference in protein,
RNA, or glutathione levels as compared with levels in control animals. Rela-
tively small increases in acid proteinase and nonspecific cholinesterase acti-
vities were reported, but the determinations for treated animals were made by
means of only two assays and therefore may be of questionable significance.
5.4 TERATOGENIC, EMBRYO-TOXIC, AND REPRODUCTIVE EFFECTS
It is not possible, on the basis of limited available data, to define the
full potential of OCM to produce adverse teratogenic or reproductive effects.
Human epidemiology studies have not been conducted to evaluate the effects of
DCM on the exposed population. The available mammalian studies were not pro-
perly designed to evaluate the-ability of DCM to produce a teratogenic response
over a wide range of doses which include doses high enough to produce signs of
maternal toxicity and lower doses which do not produce maternal toxicity. The
teratology studies in laboratory animals were performed in rats and mice using
only single doses of DCM which produced signs of maternal toxicity. Other
studies in chicken embryo have indicated that DCM disrupts embryogenesis in a
dose-related manner, however, since administration of DCM directly into the
air space of chicken embryo is not comparable to administation of dose to
animals with a placenta, it is not possible to interpret this result in rela-
tionship to the potential of OCM to cause adverse human reproductive effects.
Another preliminary study in rats indicated that adverse behavioral effects
may occur after exposure to low levels of DCM. However, additional behavioral
studies have not been conducted to more fully evaluate this effect. In summary,
although several studies have been conducted to evaluate the ability of DCM to
005DC4/A 5-31 12-9-31
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cause adverse teratogem'c, embryo toxic and reoroduct1 ve 9ft"ects. the limita-
tions of the available data do not allow for a full assessment of these effects.
A better assessment of these effects could be performed if the available studies
met criteria similar to those suggested for teratogenicity and reproductive
testing (U.S. EPA, 1978; U.S. EPA, 1979).
5.4.1 Animal Studies
The teratology studies that have been conducted on DCM are summarized
below. The teratogem'c and fetotoxic potential of DCM has been evaluated in
the avian embryo system, and in the rat and mouse. These studies were con-
ducted in accordance with current teratogenic testing methodology using reagent
or technical grade DCM. The presentation subscribes to the basic viewpoints
and definitions of the terms "teratogenic" and "fetotoxic" as summarized and
stated by Chernoff (1980):
Generally, the term "teratogenic" is defined as the tendency to produce
physical and/or functional defects in offspring i_n 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, reductions 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 Environmental Protection
Agency (EPA) 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 carcino-
genicity) which are attributable to i_n utero exposure. The third category would
005DC4/A 5-32 12-9-81
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oe emoryo or fetal toxicity as comprised of those effects «nich are potentially
reversible. This subcategory .vould therefore include sucn effects as weignt
reductions, reduction in the degree of skeletal ossification, and delays in
organ maturation.
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 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 in one species might,
under a given agent, react in another species in a more serious and irrever-
sible manner.
5.4.1.1 Chick Embryos
Elovaara et al. (1979) reported the toxicity and teratogenic potential of
OCM relative to other aliphatic chlorinated hydrocarbons. The approximate LDj--
of DCM was greater than 100 umole/egg. Malformations were produced in embryos
with doses of 5 to 100 utnole/egg injected into the air space of fertilized
chicken eggs at 2, 3 and 6 days of incubation. An estimate of embryotoxicity
was evaluated by determining the survival and death incidences after 14 days
of incubation. Although teratogenicity resulted in chicken embryos in this
study, extrapolation of these results to higher mammals is not possible at
this time. This type of study may be appropriate for screening chemicals for
comparative toxicity only (Johnson, 1981), and not for determining teratogenic
potential (Karnofsky, 1965).
5.4.1.2 Mice
Swiss-Webster mice were exposed via inhalation to 4,350 mg/m (1250 ppm)
of methylene chloride for 7 hours daily during days 6 through 15 of gestation
(Leong et al., 1975; Schwetz et al., 1975). This level was cited as twice the
maximum excursion limit for industrial exposure. Two control groups were
similarly exposed to filtered room air. Day 0 of gestation was determined when
005DC4/A 5-33 12-9-31
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a vaginal olug was observed. Caesarean sectioning of jams «as oerfcned on
day 18 of gestation.
Dams were evaluated for body weight gain 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. Following Caesarean sectioning, fetuses were weighed,
measured (crown-rump length), sexed and examined for external malformation.
One-half of the fetuses in each litter were examined for soft-tissue malforma-
tions (free-hand sectioning) and one-half were examined, following staining,
for skeletal malformations. One fetus in each litter was randomly selected
and evaluated using histological techniques following serial sectioning.
In this study, maternally toxic effects of DCM exposure were observed,
consisting of a significant increase in body weight, a significant increase in
absolute liver weight and significant increases in carboxyhemoglobin values
with return to control levels after 24 hours. On the basis of the maternal
liver weight observations, a minimal toxicity may have occurred. The cause
of the increased maternal weights is unknown. Since only the absolute liver
weight was reported, and not the increases in liver weight per animal, it is
not possible to be certain that the observed increased maternal weight gain
was a result of gains in liver weight alone.
Of the twelve litters examined, a statistically significant number of
litters contained fetuses that had a single extra center of ossification in
the sternum. This common variation in mice is thought to reflect the degree
of embryonic development. It is not known if this observation resulted from
an acceleration in development or was a chance occurrence. The litters in
exposed group were heavier than control fetuses (5.74 g vs 5.42 g); however
005DC4/A 5-34 12-9-81
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this may be due to the ^act that the average litter size //as siightly smaller
than controls (10 vs 12). The litters in this treatment group also had a lower
incidence of delayed ossification of the sternebrae (17% vs 23%), split
sternebrae (8% vs 18%), and ossification of the skull bones (25% vs 36%).
Cleft palate and "rotated kidney" were observed in two (17%) of the OCM exposed
fetuses, and not observed in any of the control litters. However, because of
the low incidence of these effects, these may reflect spontaneous malformation
rates.
5.4.1.3 Rats
A study using the same design as that used for the mice (Section 5.4.3.3),
was performed in Sprague-Oawley rats (Leong et al., 1975; Schwetz et al., 1975).
Rats were exposed by inhalation to DCM at 4,350 mg/m (1250 ppm) for 7 hours
daily on days 6 through 15 of gestation, with day 0 being the day spermatozoa
were observed in smears of vaginal contents. Dams were Caesarean sectioned on
day 21 of gestation. All other procedures were identical to those performed
in the mouse study with the exception that in the rat study, food consumption
was monitored.
No effect on maternal body weight or food consumption was observed. The
average absolute maternal liver weight was significantly increased in comparison
with the control values but there was no effect on the relative weight of the
liver. Carboxyhemoglobin values in the dams were significantly increased
during exposure but returned to control levels within 24 hours.
In the 19 litters evaluated, there was no effect on the average number of
implantation sites per litter, litter size, number of resorptions, or fetal
sex ratio and body weight. The incidence of dilated -renal pelvis was signi-
ficantly increased, but this observation is thought to indicate a slight but
005DC4/A 5-35 12-9-81
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reversible de]ay in development similar to ef£ects s^ch ss de-ays in sterna1
ossification. However, since this study evaluated only one dosage level, it
is not possible to firmly establish the significance of this effect or its re-
versibi1ity.
A study by Hardin and Manson (1980) using Long Evans rats evaluated the
effect of exposure to DCM via inhalation at 15,660 mg/m (4500 ppm) for 6 hours
daily, 7 days per week to determine whether exposures before and during gesta-
tion were more detrimental to the developing conceptus than exposures before
gestation only. Minimal maternal toxicity was observed consisting of increased
liver weight, both absolute and relative, and elevated carboxyhemoglobin levels.
The litters of rats exposed to DCM during gestation also had lower fetal body
weights than controls. No other significant deleterious effect was observed.
Bornschein et al. (1980) reported on the behavioral teratogenic effects
in the Long-Evans rats exposed to methylene chloride from the study by Hardin
and Manson (1980). Ten rats were evaluated for general activity at 5, 10, 45
and 108 days of age, avoidance learning at approximately 4 months of age and
activity following avoidance learning at approximately 5 months of age.
Fetuses delivered by Caesarean section had lower fetal body weight than those
which were naturally delivered. Treatment related effects were reported for
animals in the general activity tests as early as 10 days of age (both sexes)
and were still demonstrable in male rats at 150 days of age. No adverse
effects were observed in growth rate, long-term food and water consumption,
wheel running activity or avoidance learning.
In the Bornschein et al. (1980) study the number of rats per test group
was small, usually one male and one female per litter. Therefore, this study
should be regarded as preliminary and additional studies are needed to fully
005DC4/A 5-36 12-9-81
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confirm these effects. It should also be noted that the entire field of oe-
navioral teratology is in its early stages of development (Buelke-Sam arid Kimmel,
1979), and the significance of alterations in behavioral effects to human risk
assessment is not clearly defined.
5.5 MUTAGENICITY AND CARCINOGENICITY
Methylene chloride has been tested for its mutagenic potential in bacteria,
yeast, and Drosophila and for its ability to cause chromosome damage in rat
bone marrow cells. It is clearly mutagenic in bacteria, but it appears to be
a weak mutagen. It has also been reported to cause reverse mutations, gene
conversion, and mitotic recombination in yeast. It has not been shown to
cause mutations in Drosophila but this negative result may be due to low ex-
posures to the test organism. It has not been shown to cause chromosomal
aberrations in rats. Based on the weight of evidence, it is concluded that
methylene chloride is mutagenic in bacteria and yeast thus demonstrating the
compound has intrinsic mutagenic potential. If the metabolism and pharmaco-
kinetics of this compound in humans results in metabolic products which can
interact with DMA, as is the case for bacteria and yeast, it may cause effects
in humans as well. See evaluation of mutagenicity reports presented in section
IV of Appendix, CAG report, for details of studies and a discussion and inter-
pretation of the strengths and limitations of the data.-
Conflicting results have been reported with regard to mammalian cell trans-
forming potential of DCM. Price et al. (1978) reported that OCM transformed a
continuous line of Fischer rat F1706 embryo cells grown in culture. Normally
nontumorigenie when injected into newborn Fischer rats, F1706 embryo cells
after exposure in culture to DCM, not only exhibited morphologic evidence of
malignant transformation but also produced undifferentiated fibrosarcomas when
005DC4/A 5-37 12-9-81
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injected into newborn Pi'sche1" "ats. While QCM *as -nore potent than tr^'chToro-
ethylene or methyl chloroform in this assay, the positive control, 3-methyl-
cholanthrene, was more than a thousand times more potent than OCM. In contrast
to this positive evidence of DCM mutagenicity, DCM has been found to be nega-
tive in a BALB/C-3T3 neoplastic transformation assay (Little, 1980). This test
is designed to measure the ability of chemical agents to induce alterations in
a population of mammalian cells from a pattern of controlled monolayer growth
to one exhibiting foci of disoriented, piled-up growth against background mono-
layer. The altered cells usually produce tumors when they are injected into
the appropriate hosts.
Friedlander et al. (1978) conducted an epidemiological study of Eastman
Kodak Company (Rochester N.Y.) male workers exposed continuously to 104 to
4,176 mg/m (30 to 1,200 ppm) DCM for ap to 30 years. No increase was found in
the incidence of cancer-related deaths when compared with three control groups
consisting of other Kodak employees, and subjects taken from New York State
and United States male mortality tables.
There are no well-designed animal bioassays available that positively
support the suggestive evidence of carcinogenic potential indicated by the
bacterial mutagenic test results. Theiss et al. (1977) examined DCM in a pul-
monary tumor assay with A/St male mice. Groups of 20 mice were injected intra-
peritoneally three times per week for 24 weeks before sacrifice. The spontane-
ous occurrence of lung surface adenomas was increased in surviving animals at
all three dose levels used (160, 400, 800 mg/kg), but not significantly except
at the lowest dose with the highest animal survival rate (90 percent). The
high mortality of the mice at the higher doses (60 and 75 percent) contributed
to the equivocality of the study results. Long-term toxicological studies
005DC4/A 5-38 12-9-81
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earned out by Heppel and Meal (1944), Heppel et al. (1944) ana by MacEwen et
al. (1972), in which a variety of animal species (dogs, rabbits, guinea pigs,
and rats) were exposed to DCM by inhalation at levels of 500 to 10,000 ppm
(1,737 to 34,740 mg/m ) for periods of 14 weeks to 6 months. These studies
did not reveal any incidences of tumors; however they were not carried out for
a full lifetime. Further, they employed neither sufficient numbers of animals
nor adequate controls for a meaningful carcinogenic evaluation.
The Dow Chemical Company (1980) has recently completed a two-year chronic
toxicity and oncogem'city inhalation study of OCM in Sprague-Oawley rats and
Syrian Golden hamsters. A dose-response increase in salivary gland sarcomas
in the male rats become statistically significant at the highest dose (3,500 ppm,
12,159 mg/m ). There were also increases in benign mammary tumors in female
rats at all dose levels (500, 1500, 3,500 ppm; 1,737, 5,211, and 12,159 mg/m3)
and in male rats at the highest dose level (3,500 ppm; 12,159 mg/m ). The
response pattern of the salivary gland tumor is unusual, consisting of sarcomas
only and appearing only in males.
The National Cancer Institute has completed the exposure phase of a two-
year carcinogenicity study by gavage in rats and mice. The results of this
study are not yet available for review. The National Cancer Institute also
planned to begin an inhalation bioassay with OCM in November 1980. The data
from these studies, with results of the Dow study, should be sufficient to
evaluate the oncogenic hazard of DCM.
A final assessment of the carcinogenicity of methylene chloride will be
deferred until information on the purity of the material used in the positive
mutagenicity tests is obtained and until the results of the NTP gavage bioassay
are evaluated, perhaps by late 1981. The decision to defer a final assessment
is made because the additional information will help to clarify the unusual
005DC4/A 5-39 12-9-81
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nature of the salivary glana resoonse in the Dow study ana to c'ari'y the -'ale
of impurities in the positive mutagenic results in bacteria and yeast.
5.6 SUMMARY OF ADVERSE HEALTH EFFECTS AND LOWEST OBSERVED EFFECTS LEVELS
5.6.1 Am'mal Toxicity Studies Useful for Hazard Assessment
The preferred studies for hazard assessment are those which provide defi-
nite effect levels. Adverse affects are defined here as functional impairment
and/or pathological lesions which may affect the performance of the whole organ-
ism or which reduce an organism's ability to respond to an additional challenge.
Adverse effects which are not carcinogenic are assumed to be threshold pheno-
mena. The threshold region of toxicity is estimated by evaluating four types
of effect levels:
NOEL: No-Observed-Effect Level: That dose level at which no
statistically significant changes in effect(s) are
observed in the exposed group as compared with its
appropriate control.
NQAEL: No-Qbserved-Adverse-Effect Level: That dose producing
observable effects which do not in themselves represent
known functional impairment, behavioral abnormality and/or
pathological lesions which hinder the performance of the
whole organism.
LOAEL: Lowest-Observed-Adverse-Effect Level: The lowest dose in
a study or group of studies producing functional impairment,
behavioral abnormality and/or pathological lesions which
hinder the performance of the whole organism.
FEL: Frank-Effect Level: That dose which produces statistically
significant change in adverse effects on exposed groups as
compared with its appropriate control.
5.6.2 Inhalation Exposure
The effects of inhalation exposure to OCM in several animal species and
in humans are summarized below.
Although DCM is highly volatile and has been used extensively as an in-
dustrial solvent, reports in the scientific literature of effects on humans
are sparse. A few experimental human studies have been performed using single
005DC4/A 5-40 12-9-81
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or repeated inhalation exoosures to DCM. Altnougn these stuaies provide pre-
cise information on exposure levels, they are limited by the small numbers of
subjects and the restricted number of physiological and behavioral parameters
assessed. One human epidemiological study has been published, although this
study dealt only with mortality data. Accounts of acute or chronic overexpo-
sure of humans are compromised by uncertainties regarding the level and dura-
tion of OCM exposure and potential exposure to other chemicals. A series of
subchronic animal experiments using continuous inhalation exposure to DCM and
one chronic animal study with an intermittent exposure regimen can be used in
conjunction with the limited human data to provide a framework for human risk
assessment.
5.6.2.1 Effects of Single Exposures—Several experimental studies on the acute
effects of OCM inhalation have been conducted with human subjects- For the
most part, these studies focused on central nervous system function or carboxy-
hemoglobin levels. Exposure of volunteers to concentrations of OCM ranging
from 200 to 800 ppm (695 to 2,779 mg/m ) for 3 to 4 hours resulted in signi-
ficant decreases in visual critical flicker frequency (CFF), performance on
auditory vigilance tasks (AVT) (Fodor and Winneke, 1971; Winneke, 1974; Winneke
and Fodor, 1976), and performance on psychomotor tasks (Winneke, 1974; Putz
et al., 1976).
Exposure of subjects to approximately 1,000 ppm (3,474 mg/m ) for 2 hours
decreased the amplitude of visual evoked responses (VER), indicating CNS
depression (Stewart et al., 1972b). These subjects reported being aware of a
strong odor and a feeling of 1ightheadedness. Subjects exposed successively
to 250, 500, 750, and 1,000 ppm DCM (1 ppm = 3.474 mg/m ), 30 minutes per con-
centration, had more irregular reaction times than when unexposed, but heart
rate, numerical ability, and short-term memory appeared to be unaffected by
this exposure regimen (Gamberale et al., 1975).
005DC4/A 5-41 12-9-81
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DCM is metabolized to carbon monoxide (CO), which then combines with hemo-
globin to form carboxyhemoglobin (COHb). Some of the acute effects of dichloro-
methane on the central nervous system and the heart are similar to those of CO
(NIOSH, 1972) and are thought to be due to CO (NIOSH, 1976). Male nonsmokers
exposed to 50 ppm DCM (174 mg/m ) for 7.5 hours had blood COHb levels of 2.9
percent as compared to their baseline levels of 0.9 percent (Stewart et al.,
1973). During exposure to 50 ppm (174 mg/m ), COHb levels in the blood appeared
to reach a plateau at about 3.2 percent towards the end of a 16-hr exposure
(Fodor and Roscovanu, 1976). Smokers have been reported to have 4.6 to 5.2
percent COHb blood levels (Stewart et al., 1974; Kahn et al., 1974). Peterson
(1978) reported that regardless of whether a person smoked or not, exposure to
50 or 500 ppm (174 or 1,740 mg/m ) DCM for 7.5 hr elevated the COHb level 1.5
or 10 percent, respectively, over baseline levels. The increase in COHb levels
was proportional to the concentration of DCM inhaled and the duration of ex-
posure.
Accidental exposures of humans to high concentrations of DCM vapor have
resulted in narcosis, elevated blood COHb levels (to saturations as high as 19
percent), irritation of the eyes and respiratory tract, and sometimes death
(Moskowitz and Shapiro, 1952; Bonventre et al., 1977; Benzon et al., 1978;
Hughes, 1954; Riley et al., 1966). One 66-year-old man suffered acute myocar-
dial infarctions immediately after each of three separate exposures to a paint
stripping compound that contained 80 percent DCM (other ingredients were not
specified) (Stewart and Hake, 1976).
In none of these cases was the level of exposure measured and the duration
of exposure was usually not known. Riley et al. (1966), however, measured the
concentration of OCM in the breath of a worker who had been inadvertently
anesthetized by a 4-hr exposure to DCM. On the basis of breath concentrations
005DC4/A 5-42 12-9-31
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of 3CM. measured at intervals after removal of the Tian from the exoosure area,
and known pharmacokinetics of DCM breath excretion, Riley et al. (1966) esti-
mated that the man had been exposed to 8,000 to 10,000 ppm (27,792 to 34,740
mg/m3) DCM.
Compiling the available human data, the following relationships between
levels of acute inhalation exposure to DCM and effects may be tentatively
suggested:
Concentration
Length of Exposure
Effects
50 ppm
200-800 ppm
1,000 ppm
7.5 hours
3-4 hours
2 hours
8,000-10,000 ppm 4 hours
elevation of COHb by about
1.5% over baseline values
to about 2.9% in non-
smokers
impairment of perception
and psychomotor perform-
ance; increased COHb
levels proportional to
level and duration of
exposure; possible lignt-
headedness at high end of
exposure range
slight CNS depression;
1ightheadedness; awareness
of strong odor; no irrita-
tion
narcosis; 1ife-threatening.
with prolonged exposure
These hypothetical dose-response relationships are rough approximations and
contain several previously discussed areas of uncertainty.
Although OCM would appear to have a perceptible odor at concentrations
lower than those suspected of causing narcosis, some of the workers inadvert-
ently anesthetized by vapors of DCM had been warned to leave the worksite at
005DC4/A
5-43
12-9-31
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once if they detected the odor of the solvent. None of the men recalled smell-
ing DCM (Moskowitz and Shapiro, 1952).
Data from experiments with animals can be used to illuminate the effects
of DCM, particularly for exposures to high concentrations. Exposures of mice,
rats, and rabbits to DCM at levels of 500 to 3,000 ppm (1,737 to 10,422 mg/m3)
produced dose-related increases in total sleep time and in the intervals between
REM episodes, decreases in spontaneous running activity and increases in blood
COHb levels (Fodor and Winneke, 1971; Thomas et al. 197.1; Berger and Fodor,
1968, cited in NIOSH, 1976; Fodor et al., 1973; Roth et al., 1973). A study
with dogs indicated that repeated 2-hour exposure of the animals (through a
tracheostomy) to 1,000 to 5,000 ppm (3,474 to 17,370 mg/m ) DCM decreased the
heart rate but increased blood pressure, coronary blood flow, myocardial con-
tractility, and COHb levels, and induced cardiac arrythmias (Aviado et al. 1977).
A separate study in rabbits (Taylor et al. 1976) correlates well with the dog
data. Similar effects have been noted with CO (NIOSH, 1972). Flury and Zernik
(1931) had reported that 4,000 or 6,000 ppm (13,896 or 20,844 mg/m3) OCM pro-
duced light narcosis in. cats, rabbits, guinea pigs, and dogs in about 1 to
6 hours.
LC5Q values for 6- to 7-hr exposure were 14,100 to 16,100 ppm (48,983 to
55,931 mg/m ) for rats and about 11,500 ppm (39,951 mg/m ) for guinea pigs
(Gradiski et al., 1974; Svirbely et al., 1947; Balmer et al., 1976). Rats
entered a state of deep narcosis followed by complete cessation of brain elec-
trical activity after 6 hours of exposure to 16,000 to 18,000 ppm (55.6 to 62.5
g/m3) or 1.5 hours of exposure to 25,600 to 28,000 ppm (88.9 to 97.3 g/m )
(Berger and Fodor, 1968, cited in NIOSH, 1976). Concentrations of 25,000 or
50,000 ppm (86.8 or 173.7 g/m ) DCM given to anesthetized dogs and rabbits for
5 minutes or less produced primary depression of myocardial contractility
005DC4/A 5-44 12-9-81
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(Aviado at a!., 1977; Taylor et al.. 1975). Similar results were ootained .-nth
dogs exposed to a paint stripper containing 90 percent DCM (Aviado et al., 1977).
Sensitization of the heart to epinephrine was produced in dogs by high concen-
trations of DCM; the EC™ for this response was 24,000 ppm (83.4 g/m ) for a
5-minute exposure (Clark and Tinston, 1973).
The animal data indicate that DCM produced slight CNS effects at exposure
levels similar to those which produced minor CNS effects in humans and also
extending into a higher range than was tested on humans (up to 3,000 ppm; 10.4
g/m ). An exposure estimated to have caused unconsciousness in a human, 8,000
to 10,000 ppm (27.8 to 34.7 g/m ) for 4 hours, is bracketed by the production
of light narcosis in several species of animals at 4,000 to 6,000 ppm (13.9 to
20.8 g/m ) (1 to 6 hours) and the production of deep narcosis and death in rats
and guinea pigs at 11,500 to 16,100 ppm (39.9 to 55.9 g/m ) (6 to 7 hours).
Although marked adverse effects on the heart (i.e., primary depression of myo-
cardial contractility and sensi'tization of the heart to epinephrine) were
observed in animals only at very high exposure concentrations (about 25,000 ppm;
86.9 g/m ), some cardiovascular changes were noted in dogs exposed to 1,000 to
5,000 ppm (3,474 to 17,370 mg/m ). Hence, acute exposures lower than those
which produce narcosis may still carry an element of danger because of potential
cardiovascular effects.
5.6.2.2 Effects of Intermittent or Prolonged Inhalation Exposure—Limited in-
formation is available on the effects of subchronic or chronic continuous ex-
posures to DCM on humans or experimental animals. Most of the studies either
involve occupational exposures or are designed to mimic occupational exposures.
Consequently, while the data described below may be directly applicable to
estimating effects from occupational exposures, an additional element of un-
certainty must be considered in any attempt to estimate the effects of contin-
uous exposures that may occur from ambient air.
005DC4/A 5-45 12-9-81
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As was the case for single exoosures, the exoerimental studies of the
effects on humans of repeated exposure to DCM focused on CNS function and car-
boxy hemoglobin levels. Results from behavioral tests of subjects exposed to
50, 100, or 250 ppm (173.7, 347.4, or 868.5 mg/m ), 5 days a week for up to
5 weeks were inconclusive. These tests included the Romberg equilibrum test,
alertness and coordination tests, and arithmetic tests (Stewart et a1.,
1972a,b).
Nonsmokers exposed to 50, 100, 250, or 500 ppm (173.7, 347.4, 868.5, or
3
1,737 mg/m ) for 7.5 hours a day for 5 consecutive days had elevated blood
carboxyhemoglobin levels. After exposure to 50 ppm (173.7 mg/m ), COHb levels
returned to baseline by the start of the next day's exposure. After exposure
to 100 ppm (347 mg/m ) or greater, COHb levels were still somewhat elevated by
the start of the next day's exposure, returning to baseline over the weekend.
Although there was no consistent accumulative increase in COHb levels with
daily exposure (Figure 4-7) (Fodor and Roscovanu, 1976), peak values tended to
be higher on the fifth day (Stewart et al., 1973). Peak COHb levels, which
occurred at the end of exposure or 1 hour after the end of exposure to 50,
100, 250, or 500 ppm (173.7, 347.4, 868.5 or 1,737 mg/m3) DCM were 2.9, 5.7,
9.9, and 11.7 percent, respectively, on the fifth day (Stewart et al., 1973).
COHb levels greater than 5 percent can adversely affect patients with angina
pectoris or other cardiovascular diseases (Aronow and Isbell , 1973; Anderson
et al., 1973; Scharf et al., 1974). As detailed in Sections 4.3, 5.2.4, and
6.1.1, the elevated COHb levels derived from DCM metabolism are more sustained
than those resulting from exposure to exogenous CO and are additive to COHb
levels resulting from smoking and other exogenous CO exposure.
An epidemiological study of male Eastman Kodak workers exposed primarily
to DCM gave no indication of increased risk for death from circulatory disease
005DC4/A 5-46 12-9-81
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(including the suocategory iscnemic neart disease) or otner causes ;Fried!ander
et a]., 1978). The workers had been exposed to time-weighted average levels
of 30 to 125 ppm (104 to 434 mg/m ) OCM (estimated both from air monitoring
and from blood COHb) for up to 30 years. Two studies were done: 1) a propor-
tionate mortality study, using death certificates from 334 exposed workers who
died from 1956 to 1976, and 2) a cohort mortality study (non-concurrent pro-
spective) involving all 751 workers employed in the exposure area in 1964 and
also separate analysis of a subgroup of 252 of these workers who had a minimum
of 20 years' exposure as of 1964. This subgroup was analyzed separately to
facilitate the demonstration of effects requiring long latency periods. The
follow-up period in the cohort mortality study was 13 years. Comparisons were
made with three different control groups: other Eastman Kodak male employees
working in production activities but not exposed to DCM, New York State male
age- and cause-specific mortality rates, and United States male age-specific
mortality rates. Follow-up for workers aged 25 or more as of 1964 was greater
than 97 percent.
Another epidemiological study (Ott et al., 1980a, 1980b; Skory, 1980; Skory
et al., 1980a, 1980b), not published as of this writing but cited briefly by
Burek and coworkers (1980), has also apparently failed to reveal adverse health
effects attributable to DCM.
Reports of prolonged human exposure to higher concentrations of DCM are
anecdotal in nature and, hence, not particularly useful for risk assessment.
A 39-year-old chemist exposed occupational^ for several hours a day to about
660 to 900 ppm (2.293 to 3,127 mg/m ) DCM in the air and also dermally to
liquid DCM complained of restlessness, palpitations, forgetful ness, insomnia,
and a feeling of drunkenness after 3 years' exposure. He began to have auditory
and visual hallucinations at the end of 5 years' exposure. He was diagnosed
005DC4/A 5-47 12-9-81
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as having encephalosis. Another individual, exposed to 300 to 1.000 ppm (1.042
to 3,474 mg/m ) DCM for 3 years, developed bilateral temporal lobe degeneration.
This condition was attributed to chronic CO intoxication resulting from DCM
exposure (Barrowcliff, 1978; Barrowcliff and Knell, 1979).
The effects of continuous exposure to DCM have been studied only in experi-
mental animals exposed for relatively short periods of time (1 to 14 weeks).
This research was undertaken because of concern about the possible exposure of
astronauts to DCM emanating from construction materials in space cabins. Re-
sults, obtained with technical grade DCM and reported in a series of publica-
tions (Thomas et al., 1972; Haun et al., 1972; Weinstein et al., 1972; MacEwen
et al., 1972), can be summarized as follows. Mice exposed to 25 or 100 ppm
(87 or 347 mg/m ) DCM continuously for 14 weeks had an increase in spontaneous
activity at the lower concentration but not at the higher one. No gross or
histological lesions were found at autopsy except that "livers of the mice ex-
posed to 100 ppm (347 mg/m ) stained positive for fat. Hexobarbital sleep time
was unaffected, but hepatic levels of cyctochromes were somewhat altered. Rats
subjected to the same exposure regimens had nonspecific renal tubular degenera-
tion and regeneration and hepatic cytoplasmic vacuolization and positive fat
staining. No specific macro- or microscopic organ changes or changes in hema-
tologic or clinical chemistry values were noted in the small number of dogs or
monkeys studied. Carboxyhemoglobin levels were elevated in monkeys at both
exposure levels and in dogs only at the higher exposure but there was no cumu-
lative increase in COHb over the period of exposure. No overt signs of toxicity
or changes in body weights relative to controls were noted in any of these four
species.
Higher levels of continuous exposure were also investigated. Exposure of
the same four species to 1,000 or 5,000 ppm (3,474 or 17,370 mg/m ) resulted
005DC4/A 5-48 12-9-81
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in severe signs of toxicity at the higher dose: narcosis for the first 24 hours
and pronounced lethargy for the remainder of the exposure period, reduced food
consumption, and high rates of mortality in mice, dogs, and monkeys. Rats were
somewhat less sensitive; none died. Liver and kidney damage were commonly found
in all species. At the lower exposure level, 1,000 ppm (3,474 mg/m ), only
the dogs were severely affected and died. Mice and rats did not show overt
signs of toxicity but body weight gain was slightly depressed in the rats.
Less severe histopathological changes than had been seen at 5,000 ppm (17,370
mg/m ) were noted in the livers of all four species and in the kidneys of rats
exposed to 1,000 ppm (3,474 mg/m ). Monkeys had no significant changes in
hematologic or clinical chemistry values.
The only chronic animal study available at this time is the 2-year inhala-
tion study performed by Dow Chemical Company (Burek et al., 1980). Sprague-
Dawley rats (SPF-derived; 192/sex/exposure concentration) and Golden Syrian
hamsters (about 108/sex/exposure concentration) were exposed to 0, 500, 1,500,
or 3,500 (0, 1,737, 5,211, or 12,159 mg/m3) of OCM of greater than 99 percent
purity. Exposures were for 6 hours a day, 5 days a week for up to 2 years;
interim kills were performed.
With the rats, none of the exposure levels affected body weights, clinical
chemistry, hematologic, or urinalysis values. Carboxynemoglobin levels ranged
from 0 to 5.3% in controls and from 8.9 to 20.4% in the treated rats, but there
was no dose-response relationship and no increase with time of exposure. The
lack of dose-response is probably a reflection of the saturability of OCM
metabolism to CO (see Chapter 4). Beginning in the 18th month the high-dose
females showed a statistically significant increase in mortality. All the rats
exposed to 3,500 ppm (12,159 mg/m ) appeared to be sluggish for about the first
week of exposure.
005DC4/A 5-49 12-9-81
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In rats, the only organ that had definite, statistically significant,
exposure-related changes was the liver. An increased incidence of hepato-
cellular vacuolization consistent with fatty change was noted at all exposure
levels in both sexes. The severity tended to be slight; incidence and
severity increased in a dose-related manner. Multinucleated hepatocytes, which
according to the authors occur spontaneously in aging female rats, were found
in significantly greater percentages of treated females than in controls, but
incidence did not increase with increasing dose. Male rats had an increased
incidence of hepatocellular necrosis starting at 1,500 ppm (5,211 mg/m );
females had a higher incidence of coagulation necrosis at 3,500 ppm (12,159
mg/m ). Foci and areas of altered hepatocytes, another geriatric lesion found
in these female rats, were increased only in females exposed to 3,500 ppm
(12,159 mg/m ). The number of animals with neoplastic nodules or hepatocellular
carcinomas was, however, not increased in any exposure group. Liver weights
and liver to body weight ratios were increased for both males and females at
18 months of exposure to 3,500 ppm (12,159 mg/m ).
This Burek et al. (1980) study would appear to establish a chronic LOEL
for rats at 500 ppm (1,737 mg/m ) for exposures occurring 5 hours/day, 5 days/
week. The liver changes observed at this level were slight and no clearly
deleterious effects were observed.
Hamsters were less sensitive to DCM than rats at these exposure levels.
Although carboxyhemoglobin levels were higher than had been observed in the
rats, no clear evidence of toxicity was found in hamsters. The effects observed
in hamsters appeared to result primarily from decreased deposition of amyloid
in their tissues (amyloid deposition is a naturally occurring geriatric disease
in hamsters) and were not deleterious.
005DC4/A 5-50 12-9-81
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The data described in this section and in 5.6.1.1 concerning the effects
of DCM exposure on humans and experimental animals, taken together, give some
idea of the threshold region for effects of DCM exposure. Workers exposed
occupationally in the range of 35 to 125 ppm (121 to 434 uig/m ) did not have
an increased risk of death from circulatory diseases or other causes (Friedlander
et al., 1978). Daily 7.5-hour exposures of humans to this amount of DCM did,
however, cause an increase in blood COHb to peak levels of about 5.7 percent
that did not return to baseline within 24 hours (Stewart et al., 1973). Single
exposures of humans to levels of DCM as low as 200 ppm (695 mg/m ) (4 hours)
have been reported to produce a depression of eye-hand coordination in demand-
ing situations (Putz et al., 1976). Several species of animals tolerated con-
tinuous exposure to 100 ppm (347 mg/m ) DCM for 14 weeks (Section 6.1.2) or
intermittent exposure to 500 ppm (1,737 mg/m ) (6 hours/day, 5 days a week;
time-weighted average of 90 ppm for a week's exposure) for up to 2 years (Burek
et al., 1980) with only minimal effects. Continuous exposure to 1,000 ppm
(3,474 mg/m ) DCM for 14 weeks produced frank toxic effects in experimental
animals (supra vide) and a single 2-hour exposure to 1,000 ppm (3,474 mg/m )
produced slight CNS depression in humans (Stewart et al., 1972). Based on the
human and animal data summarized in this chapter, 200 ppm (695 mg/m ) probably
represents a low-effect level for humans, rather than a NOEL. The ACGIH has
recently lowered the TLV to 100 ppm (347 mg/m ) and NIOSH (1976) has recommended
a criterion of 75 ppm (260 mg/m ).
The study conducted by Dow Chemical Company (Burek et al., 1980) identi-
fied 500 ppm (1,737 mg/m ) (6 hours/day, 5 days/week) as a chronic LOEL for
noncarcinogenic effects in rats, the more sensitive of the two species tested.
This level of exposure produced a statistically significant increase in the
numbers of male and female rats with hepatocellular vacuolization characteristic
005DC4/A 5-51 12-9-81
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of slight fatty change and a statistically significant increase in the numbers
of females with multinucleate hepatocytes. Behavior, body weights, liver weights,
macroscopic liver appearance, gross and histological appearance of other organs
(excluding neoplastic changes), clinical chemistry (including SGPT), hematology,
urinalysis, and mortality were unaffected. As described in Section 6.1.2, the
incidence and severity of liver effects increased in a dose-related manner at
the higher exposures (1,500 and 3,500 ppm; 5,211 and 12,159 mg/m3) and frank
toxic effects (i.e., hepatocellular and coagulation necrosis, increased morta-
lity) were noted at 3,500 ppm.
The as yet unpublished epidemiological study (Ott et al., 1980a, 1980b;
Skory, 1980; Skory et al., 1980a, 1980b) cited by Burek et al. (1980) appears,
from the titles of the papers, to contain data on more sensitive biological
end-points than were studied by Friedlander et al. (1978).
5.6.3 Oral Exposure
Very little information is available concerning the toxicity of OCM by
the oral route. As summarized in Table 5-2, the oral ID™ for mice was re-
ported to be 1987 mg/kg body weight (Zakhari, 1977) and oral LOcQ values for
rats were reported to be 2,388 mg/kg (Kimura et al., 1971) and 4,368 mg/kg
(Ugazio et al., 1973). LD50 values can sometimes be used to calculate an
approximate lethal dose for humans using the cubed root of the body weight
ratios for interspecies conversions (U.S. EPA, 1980b; Friereich et al., 1966;
Rail, 1969). This extrapolation assumes that larger species are more sensi-
tive than smaller species. The LD5Q values for DCM would suggest, however,
that mice may be more sensitive than rats to this chemical; the use of this
interspecies conversion factor may therefore be inappropriate.
No data from chronic oral studies are available. The NCI has completed a
two-year carcinogenicity study with rats and mice in which the animals were
005DC4/A 5-52 12-9-81
-------
given DCM by intuoation. The histopathological pnase of the study is now in
progress. When the study is completed it may provide some data on the chronic
oral toxicity of DCM.
A NOEL of 15 mg/kg/day has been derived from a subchronic oral study with
rats (Bornmann and Loeser, 1967). Thirty male (80 g) and 30 female (75 g) Wistar
rats were given 0.125 g OCM/. in their drinking water for 3 months. No differ-
ence in behavior, appearance, body weight, or survival of the treated animals
were observed as compared to an equal number of control animals. No significant
differences in hemotologic values, urinalysis, or plasma levels of non-esterified
fatty acids were found in 8 to 10 male rats from each group. Urine from both
treated and control animals sometimes tested positive for albumin. The blood
glucose levels for 10 treated males were slightly elevated over levels for 10
control males but values for both groups, according to the authors, were within
the normal range. The estrous cycles of the females, evaluated by vaginal
smear tests, were unaffected by treatment. About 20 animals of each sex and
treatment group were autopsied. Weights of the endocrine glands were not
altered by treatment. The authors stated that histological examination of the
internal organs revealed no pathological alterations.
The U.S. Environmental Protection Agency (1981) estimated that the rats
had ingested about 15 mg DCM/kg body weight/day. This value is taken as a
no-observed-effect level.
5.6.4 Dermal Exposure
Studies on the dermal toxicity of OCM are not adequate for quantitative
risk assessment. Pharmacokinetic studies with human subjects indicate that
absorption of liquid DCM through the skin is slow enough that toxic quantities
would be unlikely to be taken into the body from direct contact of the solvent
005DC4/A 5-53 12-9-81
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with the skin of the hands and forearms (Stewart ana Oodd, 1964). Some indi-
viduals have complained of local pain from dermal contact with the liquid DCM;
others have not (Stewart and Oodd, 1964; Hughes, 1954; Weiss, 1967). Chronic
intermittent dermal contact with the solvent produced local irritation (Weiss,
1967). A workman who, narcotized by inhaling vapors of DCM, fell into a vat
of the solvent, suffered acrocyanosis, labored breathing, severe chilblains,
and repeated myocardial infarctions, and died 26 hours after this combined
massive inhalation and dermal exposure (Kuzelova et al., 1975)
The only indication of toxicity solely from dermal exposure comes from an
experiment with animals. Rats exposed dermally to 2 ml OCM for periods of up
to 20 minutes had hemoglobin in their urine and voided decreased volumes of
urine in the 3 hours following exposure (Schutz, 1958).
5.6.5 Responses of Special Concern
The potential of DCM to cause teratogenic, mutagenic, and carcinogenic
effects is an important and controversial question. The limited data available
concerning teratogenic and embryotoxic effects is discussed in Chapter 5 section
4. A detailed discussion and evaluation of the mutagenicity and carcinogenicity
of DCM is presented in the appendix.
005DC4/A 5-54 12-9-81
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drinking water standards. J. Amer. Water Works Assoc., April, 1958.
Su, G. and K. A. Wurzel. A regulatory framework for setting air emission
limits for noncriteria pollutants. J. Air Pollut. Contr. Assoc.
31(2):160-162, 1981.
Svirbely, J. L., B. Highman, W. C. Alford, and W. f. Von Oettingen. The
toxicity and narcotic action of mono-chloro-mono-bromo-methane with
special reference to inorganic and volatile bromide in blood, urine and
brain. J. Ind. Hyg. Toxicol. 29:383-389, 1947.
Taylor, G. J., R. T. Drew, E. M. Lores and T. A. Clemmer: Cardiac depression
by halo-alkane propellants, solvents, and inhalation anesthetics in
rabbits. Toxicol. Appl. Pharm. 38:379-387, 1976.
Tenhunen, R., H. S. Marver, and R. Schmid. Microsomal hemeoxygenase;
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Theiss, J. C., G. D. Stoner, M. B. Shimkiu, and E. K. Weisburger. Test for
carcinogenicity of organic contaminants of United States drinking waters
by pulmonary tumor response in strain A mice. Cancer Res. 37: 2717-2720,
1977.
Thomas, A. A., M. K. Pinkerton, and J. A. Warden. Effects of methylene
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Thomas, A. A., M. K. Pinkerton, and J. A. Warden. Effects of low level
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Q05DC2/E 6-16 12-9-81
-------
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005DC2/E 5-17 12-9-81
-------
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pharmacokinetics of 14C-methylene chloride in mice. Fed. Am. Soc. Exptl.
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343, Gentner Verlage, Stuttgart, 1975.
005DC2/E 6-18 12-9-81
-------
APPENDIX
THE CARCINOGEN ASSESSMENT GROUP'S
CARCINOGEN ASSESSMENT
OF
METHYLENE CHLORIDE
7-1
-------
EXTERNAL REVIEW DRAFT
May 29, 1981
THE CARCINOGEN ASSESSMENT GROUP'S
CARCINOGEN ASSESSMENT
OF
METHYLENE CHLORIDE
Roy E. Albert, M.D.
Chairman
PARTICIPATING MEMBERS
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.
David L. Bayliss, M.S.
Chao w. Chen, Ph.D.
John R. Fowle III, Ph.D.*
Herman J. Gibb, B.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.
Robert McGaughy, Ph.D.
Beverly Paigen, Ph.D.
Dharm V. Singh, D.Y.M., Ph.D.
Nancy A. Tanchel, B.A.
Todd W. Thorslund, Sc.D.
Peter t. Yoytek, Ph.D.*
*Reproduct1ve Effects Assessment Group
DRAFT
DO NOT QUOTE OR CITE
This document has been reviewed and approved by the Chairman and staff of the
Carcinogen Assessment Group, Office of Health and Environmental Assessment, U.S.
Environmental Protection Agency. It has not been formally released by the EPA
and should not at this stage be construed to represent Agency policy. It 1s
being circulated for comment on Its technical accuracy and policy Implication.
7-2
-------
CONTENTS
I. Summary and Conclusions 1
II. Introduction 3
III. Metabolism 3
IV. Mutagenicity and Cell Transformation 6
Cell Transformation
V. Toxldty 22
VI. Carclnogenl city 22
Human Studies
Fried!ander et al.
Animal Studies
Thelss et al. 1977
Heppell et al. 1944
McEwen et al. 1972
Dow Chemical Company (1980) Inhalation Study In Rats
Dow Chemical Company (1980) Inhalation Study 1n Hamsters
Discussion and Conclusions of the Dow Chemical Company (1980)
Inhalation Study
Other Animal Studies in Progress
VII. Unit Risk Estimate 49
VIII. References 50
7-3
-------
I. SUMMARY AND CONCLUSIONS
The data base is neither extensive nor adequate for assessing the
'cardnogenlcity of methylene chloride. There 1s a marginally positive pulmonary
adenoma response in strain A mice. Two negative animal inhalation studies were
inadequate because they were not carried out for a full lifetime (Heppel et al.
1944, McEwen et al. 1972). Two long-term animal bioassay studies are currently
in progress at the National Toxicology Program (NTP), a gavage test is nearing
completion,- and an inhalation test was recently started. The animals in the NTP
gavage study were sacrificed in December 1980. The Dow Chemical Company (1980)
has recently reported the results of chronic inhalation studies in rats and
hamsters. The rat study showed a small but statistically significant Increase
in incidence of benign mammary tumors in female rats at all doses and in male
rats at the highest dose, as well as sarcomas in male rats probably of the
salivary gland origin. The response pattern of the salivary gland tumors is
unusual, consisting of sarcomas only and appearing in males but not females.
In hamsters, there was an increased incidence of lymphosarcoma in females only,
which was not statistically significant after correction for survival.
Methylene chloride has been tested for mutageniclty in bacteria, yeast,
Drosophila. and for its ability to cause chromosome damage in rat bone marrow
cells. It is clearly mutagenlc in bacteria but only weakly so. It has also
been reported to cause reverse mutations, gene conversion, and mitotic
recombination In yeast, but did not exert a genotoxic action in Drosophila or
rat bone marrow cells. Based on the weight of evidence available at this time,
it is concluded that methylene chloride is mutagenie in bacteria and yeast and
thus, has the ability to cause genetic damage. If the metabolism and
pharmacokinetics of this compound in humans results in metabolic products which
7-4
-------
can interact with DNA, as is the case for bacteria and yeast, 1t may cause
effects In humans as well. Methy!ene chloride also showed an ability to
transform cells using the rat embryo cell line F1706. This was not confirmed
when a reportedly purer grade of methylene chloride was tested.
Only one occupational epldemlologlcal study of methylene chloride has been
reported and it showed no Increased Incidences of neoplasms at any sites which
could be related to methy!ene chloride. However, the study was Insensitive due
to the short duration of the follow-up.
In conclusion, a final assessment of the cardnogenlcity of methylene
chloride will be deferred until Information on the purity of the material used
1n the positive mutagenldty tests 1s obtained and until the results of the NTP
gavage bloassay are evaluated, perhaps by late 1981. The decision to defer a
final assessment 1s made because the additional information will help to clarify
the unusual nature of the salivary gland response in the DOM study and to
clarify the role of Impurities in the positive mutagenlc results 1n bacteria and
yeast.
7-5
-------
II. INTRODUCTION
Methylene chloride or methylene dlchloride (dichloromethane, CH2C12) is
a colorless, volatile, chlorinated hydrocarbon liquid with a penetrating
ether-like odor. It is slightly soluble in water, and soluble in alcohol and
ether. Its molecular weight is 84.9 and the boiling point is 40°C.
United States production of methylene chloride in 1976 exceeded 500 million
pounds. An estimated 367 million pounds were released into the environment.
The compound is most commonly used as a paint remover, a dispersive use with
high human exposure. Methylene chloride is also used as a propel!ant for
aerosol sprays; a blowing agent in foams; a degreasing agent; in the manufacture
of photographic film, textiles, and plastics; and as a food additive extract.
Most of the chemical is released into the home and factory; 955 of its uses are
dispersive. The number of workers exposed is 2.5 million (National Institute
for Occupational Safety and Health 1976).
Methylene chloride does not bioaccumulate and is not highly toxic to aquatic
organisms. It is persistent in the environment, but due to its low boiling
point, it disperses in the atmosphere.
III. METABOLISM
Methylene chloride is absorbed by the lungs. Some of the absorbed chemical
is metabolized but most of it is eliminated unchanged by the lungs. Radioactive
methylene chloride (412 to 930 mg/kg) administered to rats intraperitoneally was
eliminated via the lungs as 911 unchanged methylene chloride, 25 carbon
monoxide, 31 carbon dioxide, and 15 unidentified metabolite (DiVincenzo and
Hamilton 1975). Urine contained 11 of the radioactivity and the carcass 2%.
Formaldehyde levels increased in serum. Rodkey and Collison (1977a)
administered small doses 0.2 mM/kg (17 mg/kg) of radioactive methylene chloride
7-6
-------
to rats by inhalation or intraperitoneal injection. In this experiment a much
higher fraction of metabolized methylene chloride was found compared to those
discussed above; 47% of radioactivity was recovered as carbon monoxide (CO), 29%
as carbon dioxide (COg). and none remained in the carcass. The investigator
asserted that the remainder was exhaled as unchanged methy!ene chloride but this
was not measured. A second experiment by Rodkey and Collison (1977b) showed that
the conversion to CO was rate limited by enzyme saturation, and the author
suggests that this fact may explain the low percent conversion observed by
DiVincenzo and Hamilton (1975) who used much higher doses.
Peterson (1978) exposed 20 individuals to methy!ene chloride at
concentrations ranging from 50 to 500 ppra for up to 7.5 hours/day for five
successive days. As in animals, much of methylene chloride was excreted
unchanged from the lungs. Carboxyhemoglobin levels Increased in the blood of
exposed individuals up to about 101 saturation. This Is of concern In
occupational exposure due to possible increased cardloresplratory stress. No
acceleration of methylene chloride metabolism occurred during the five exposure
days. From the rate of excretion of methylene chloride after exposure stopped,
1t appears that much of the dose 1s stored In a body compartment other than the
blood (probably the llpld) and released at a slower rate as compared to the rate
of excretion by the lungs.
Studies of metabolism in rats have Indicated two pathways for methylene
chloride, one occurring in the mlcrosomes and the other occurring in the
cytosol. In the mlcrosomes, metabolism by the mixed-function oxidases leads to
carbon monoxide (Kublc and Anders 1975a, Kublc et al. 1974). An intermediate
metabolite was bound to both protein and Hplds {Kublc and Anders 1975b). In
the cytosol, metabolism yields formaldehyde, a known mutagen and carcinogen, and
requires glutathlone as a cofactor (Ahmed and Anders 1976). Addition of MAD*
decreases the formaldehyde product and causes the appearance of formic add
7-7
-------
(Ahmed and Anders 1978)
P-450
CO]
mlcrosomal
monooxygepole
nonenzymatlc
CO + HC1
Based on these studies the proposed pathways are:
CH2C12
GSH cytosol
- HC1
glutathione
transferase
GS-CH2-C1
+ H20
- HC1
nonenzymatlc
GS-CH2OH4— 'HCHO + GSH
formaldehyde dehydrogenase
0
II
GS-C-H
- GSH
HCOOH
i
C02 + H20
Figure 1. Proposed metabolism of CH2C12.
The cytosol metabolism cannot be Increased with pretreatment by
phenobarbltal or 3-methylcholanthrene as can the mlcrosomal metabolism.
It 1s this cytosol1c glutathione-dependent metabolism that gives rise to C02
as an end product. The exact mechanism Is not known.
Ahmed and Anders (1978) suggest that the chloromethyl glutathione
Intermediate would be quite reactive and may alkylate nucleophlles. Since this
Intermediate 1s an a - chloromethyl thloether, 1t may possess reactivities
similar to b1s-chloromethyl ether, a known carcinogen.
7-i
-------
IV. MUTAGENICITY* AND CELL TRANSFORMATION
Methylene chloride has been tested for its mutagenic potential in bacteria,
yeast, Orosophila. and rats. The studies presently available for review are
summarized in Table 1.
There are six reports in the literature concerning the mutagenic potential
of methylene chloride in bacteria. All were conducted using the Salmonella
histldine reversion assay (Simmon et al. 1977, Simmon and Kauhanen 1978, Kanada
and Uyeta 1978, Jongen et al. 1978, Snow et al. 1979, and Green 1980).
Methylene chloride tested positive in all six studies without and/or with
metabolic activation. Data were presented in five of the six reports and a
clear dose-related response 1s apparent for each. The doses employed and the
responses observed are summarized in section A of the first table. The purity
and/or composition of the compound tested was not reported for any study, and
the source was not given for most. The source of the material tested was given
1n the reports by Jongen et al. (1978) and Snow et al. (1979). Written
inquiries have been made as to the purity of the tested material for all tests
reviewed here (bacterial, yeast, and Drosophlla) and when replies are received
this information will be included in the document.
Although there is no doubt that methylene chloride is mutagenic in
Salmonella, questions have been raised about the applicability of these results
to predicting mutagenlclty in other species, especially mammals. Green (1980)
in an abstract, and in a poster format, has presented a summary of preliminary
results on the comparative metabolism of methylene chloride in rat liver and in
Salmonella. Although these data cannot be evaluated in detail because of their
brief presentation, they are considered at some length here because of their
*Prepared by the Reproductive Effects Assessment Group.
7-9
-------
TABLE 1. HUTAGCHtCITV TESTING Of MITIULEME CHLORIUt
A. BACTERIA
I
(—'
o
Reference
Slwuon et al.
1977
Slnuon and
Kauhanen l»78
Test System Strain
Salmonella/!) 9 TA 1S3S
vapor exposure TA 1537
IA 1538
TA 98
TA 100
Salmonella/Si TA 100
vapor exposure
Activation
Systea
None
Aroclor 1254
Induced rat
liver
•IcrosOM
S9»lx
Concentration
(Extrapolated fro*
figure 17) 0. SO.
100. 200. 400. and
800 ul/9 liter
desiccator
0 and 1 Ml/9 liter
desiccator for 6.5
and 8 hours
Result
(Extrapolated from fig. 17)
TA 100
Dose (ul) Revertants/plate
0 170
50 210
100 300
200 400
400 650
800 1350
TA 100
revertants
Net.
fh Act. Treated Control
O ~^~ "MS"" ~TJ3
* 1344 130
8 - 830 174
* 912 1&8
Coiiwents
1. Toxtclty not reported.
2. Nuaber of revertants
observed for TA 100 not
specified numerically.
3. Data not presented for
strains other than TA 100.
4. Purity and source of
coopound not provided.
1. Toxlclty not reported.
2. Purity and source of
compound not provided.
3. Used as a positive
control In the testing of
2-chloroethyl
chlorofontaU.
-------
Table 1. (continued!
A. BACTERIA
Reference
Kanada and
Uyela 1978
Jongen et al.
1978
Test Systea Strain
SalM>nella/S9 TA 98
and TA 100
B. subtil Is
rec assay
testing
Salw>nella/S9 TA 98
vapor exposure TA 100
Activation
System
PCB Induced rat
liver BlcrosoM
S9 •!»
Phenobarbltal
Induced rat
liver •Icrosone
S9 Mix
Concentration
Hot reported
(pp» x 10?)
0
5.7
11.4
M.I
22.8
57.0
Resul t
Hethylene chloride
reported negative for
both strains In B. subtllls
and positive for~6oth In
S. typhinurluni
TA 100* TA 98*
»S-9 -S9 *$9 -S9
152»l9 129«I2 21«4 I9V5
329*37 248732 5<»5 44*8
515T76 407*47 74>4 56*10
757*82 S82T56 93«9 66T12
865*82 653*89 123*10 96*11
1201*191 740794 149*42 110*42
Comnents
1. Results summarized
In abstract for*.
(•possible to Indepen-
dently evaluate.
2. Positive results
of "Ames* testing
supports reports by
other authors using
same system.
1. Testing conduct-
ed in gas tight
perspex boxes.
2. Only highest dose
exhibited less than
831 survival.
3. Purity of Muthylene
chloride not reported.
~*~ResuTts fro* three experiments, five plates7dose.
-------
TABLE 1. (continued)
A. BACTERIA
Actlvttlon
Reference Test Systea Strain Systei Dose
Snow et •). Sal»onell»/S9 TA 98 Hethylene chloride Induced tut/Chamber!
1979 vapor exposure TA 100 Syrian Golden master
liver S9 •Icrosoae •(« 0
100
300
500
1000
Green 1980 Salnonella/S9 TA 1535 Rat liver fractions Dose
vauor exposure TA 100 is in airi
"U '"
1.4
2.8
5.5
a.)
1 TA 1
»59
S<
177
463
£42
972
TA
»S9
69»3
283T|0
506*27
B2ST34
1050*88
~~
Result
iOO« TA 98
-S9 +S9 -S9
<3 38 14
142 47 31
274 69 46
468 92 61
632 39 72
100
-S9
-
267*20
462*28
872T27
997*88
Contents
1. Purity of nethylene not
reported.
2. No Information about
variability of results.
1. Preliminary results
presented In abstract form
2. Metabolic studies
conducted In rat tissue and
TA 100. Sliillar metabolism
In both system. Hadlolabel
reported to bind to
bacterial QUA but not to rat
liver DMA.
~*Hean calculated fro* three plates/dose.
-------
B. ttAST
TABLE 1. (continued!
Response/Id** Survivors
Reference
Simon .
et *1. 1977
Test Systea
Saccharoqyces
cereylslae Dl
suspension tests
ad<
Dose trp-5
Strain («Ml I Survival Conversion Recombination
Nltotlc
recombi-
nation
s-Z
Total Genetic llv-l
Alterations Revertants Conwents
1. Dili not provided,
but reported negative.
2. Cytochrome P4SU
concentration nut known.
C«))en et al. 11900)
report different yeast
strains have different
levels.
Ctllen
et tl. I960
Saccluronyces
cerevtslte
07
0
104
157
209
too
77
42
18 310 3300 2.7 I. Indicates ability of
28 190 3900 4.4 eukaryotlc P4SU
107 4490 14000 5.6 dependent nonoxygena&e
systew to nctabollcdlly
activate me thy lent-
chloride.
2. Active metabolites
produced by this system
are made Intracellularly
unlIke exoqcnously
employed activation
systems.
(continued on the following page)
-------
B. YEAST
TABLE I. (continued)
Be5ponse/I06 Survivors
Dost
Reference Test System Strain l*H) S Survival
trp-5
Conversion
ade-Z
fteconbl nation
TotaT Genetic
Alteration
llv-t
Revertants
Conuients
I
I—»
-p»
Cillen
et at. 1980
(continued)
3. Spectral analysis of
carbon tetrachlurldc,
ha)oethane, and
tricllloroethylcne
conducted. Spectrun
of nlcrosowes produced
by addition of
tricllloroethylcne to
suspensions of whole
cells similar to that
observed In wanna I Ian
nlcrosoiues. Other
•Icrosowal spectra not
slallar to uiautual Ian
counterparts.
(continued on the following payel
-------
TABLE 1. (continued)
C. OROiOPIIILA
No. of Chroaosooes
Reference
Abrahanson
Test Systea Strain
Drosophtla FHb
and Valencia sex-linked feaales.
1960
~j
i — *
01 0. RAIS
Reference
Johnston
et al.
19UO
recessive Canton
lethal test S Mies
Chealca)
EHS
Trls
Route
Fed
fed
Meg. Controls fed or Inj
Hethylene fed (1.91)
Chloride
Inj (3 ul of
0.21)
Tested
773
2442
94491
14662
8262
Ho. of Lethal s
44
35
230
34
18
Corrected
Lethal s U)
5.69
1.43
0.233
0.204
0.157
Contucnts
1. No precaution
to design exposure
taken
Clumbers to prevent
evaporation ol the
test compound for
feeding experiment
2. No concurrent
negative controls
reported for the
.
Injection cxperiwi-iit.
Dose
Test Systea Strain (ppa)
cytogenetlc Sprague- 0
evaluation Dawley
of bone albino
•arrow rats 500
cells (Spartan
Inhalation substraln, 1500
SPF-
derlved 3500
Chroaatld Break
with Frag.
0.9 * 0.99
0.5 * 0,71
0.5 * 0.97
0.7 » 0.48
without Frag.
0
0
0.1 * 0.316
0
Chroaosoae Break
with Frag.
0.2 « 0.42
0.2 * 0.42
0.1 * 0.316
0.2 * 0.42
without Frag. Olcentrlcs Rings
0 0
0 0
0 0
0 0
0
0
0
No.
Exchanges Abnormal
0 1.1 •
0 (I.e. •
0.1 » 0.31o U.ti >
0.2 *_ U.42 0 I.I i
ul
fells
1.20
U.b'J
1.22
u.uy
Contents
I. Values represent X * S£ of da*age/2000 cells. 200 cells each frow 10 animals/dose
2. HaxfnuB tolerated dose nay not have been approached In this study.
3. Under conditions of test, •ethylene chloride did not Induce chroiuosoMl aberrations.
-------
implications with respect to the significance which should be placed on the
results from bacterial tests. From previous studies, it was shown that two
metabolic pathways exist for methylene chloride in rats, both involving
potentially mutagenlc Intermediates; one Involving glutathlone producing the
Intermediate formaldehyde and the end product carbon dioxide, and the other, an
oxidative pathway, 1s thought to form formyl chloride as an intermediate and
carbon monoxide as the end product (Ahmed and Anders 1978, Kublc and Anders
1975a). In Salmonella, Green's studies using stable Isotopes indicate that one
or more mutagenlc metabolites of methylene chloride are produced 1n bacteria by
an oxldatlve pathway similar to the one existing 1n rat liver and that this
accounts for the mutagenlc activity of the compound in bacteria. Green
concludes, however, that the mutagenlc interned1ate(s) formed by bacterial
metabolism of methylene chloride 1s so unstable that 1n the Ames test 1t 1s only
effective when produced Inside the bacterial cell via action of bacterial
metabolism and not when produced outside the cell due to metabolism by the S9
mix. The bases for this conclusion are that rat liver fractions used for
metabolic activation have little effect on the mutageniclty of methylene
chloride in the Ames test and in vivo radiotracer studies using 14C-methylene
chloride Indicate that this compound or an active metabolite binds to bacterial
DMA but not to rat liver DMA (Green, unpublished). By Implication he Indicates
that the mutagenlc metabollte(s) of methy!ene chloride 1s so unstable within the
rat cell that 1t would not produce mutations. Without a more detailed
description of the methods used and results observed, one is not able to
critically evaluate the conclusions reached by Green, but it 1s important to
note that experiments performed by Call en et al. (1980) 1n yeast described below
cast doubts upon his conclusions.
Call en et al. (1980) studied the ability of methy!ene chloride obtained from
Fisher Scientific Company (purity not reported) and six other halogenated
7-16
-------
hydrocarbons to cause gene conversion, mltotic recombination, and reverse
nutations in Saccharomyces cerevisiae. They also spectrophotometrically studied
cytochrome P-450 mediated metabolism of three of these compounds (carbon
tetrachloride, halothane, and trichloroethylene). With respect to the
mutagenlclty of methylene chloride, Saccharomyces cerevisiae strain 07 log phase
cells were incubated for one hour in culture medium containing 0, 104, 157, and
209 mM methylene chloride. Due to the toxicity of this compound, the genetic
endpoints were not measured at the highest dose. (Percent survival for the
doses utilized were 100, 77, 42, and < 0.1, respectively). The responses for
the other doses (0, 104, and 157 mM) expressed per 106 survivors were: gene
conversion at the trp-5 locus (18, 28, and 107); mltotic recombination for ade-2
(310, 190, and 4490); total genetic alterations for ade-2 (3300, 3900, and
14,000); and reverse mutations for ilv-1 (2.7, 4.4, and 5.8). A greater than
twofold dose-related increase over negative controls was observed for each
endpoint measured. On this basis, one concludes methylene chloride is mutagenic
in yeast. No exogenously applied metabolic activation was used in these
experiments and with respect to the study by Green (1980), this work by Callen
et al. indicates that metabolism of methylene chloride to a mutagenic
metabollte(s) results in mutagenesis in yeast as well as in bacterial cells.
The positive test results obtained by Call en and coworkers using Saccharomyces
for compounds which had previously tested negative in nutageniclty assays using
metabolic activation (halothane, chloroform, carbon tetrachloride, and
tetrachloroethylene) strengthens this conclusion and suggests that the yeast
cells produce an active metabolite(s) much closer to the nucleus than is
possible with extracellular liver S9 in vitro metabolic activation systems.
The second part of the study reported by Callen et al. (1980) involved an
7-17
-------
invest!gation of the metabolic capabilities of yeast. The metabolism of three
compounds (chloroform, halothane, and trichloroethylene) was
spectrophotometrically analyzed and the results indicate that P-450 dependent
monooxygenases are Involved in their metabolism. Although the metabolism of
methylene chloride was not analyzed by Gallon et al., it is reasonable to assume
that P-450 dependent monooxygenases are Involved in its metabolism too, since it
is chemically and structurally related to the other chemicals which were
analyzed and since the mutagenlc response was similar for all of them.
The results of Green (1980) and Call en et al. (1980) both indicate that
methylene chloride requires metabolic activation for mutagenic activity and both
indicate that the mutagenlc metabolic intermediate(s) 1s so short-lived that it
must be formed within the cell to produce mutagenlc activity. Call en and
coworker's report of the matagenic activity of methylene chloride in yeast
raises questions about the implication of Green's work that methylene chloride
would not cause mutations in mammals. Like mammals, yeast are eukaryotes and in
distinction to bacteria and other prokaryotes, possess a nuclear membrane,
chromatln, mitochondria, etc. The ability of the metabolite(s) of methylene
chloride to produce mutations in yeast indicates its potential to produce
mutations in other eukaryotes as well.
In another genetic study employing yeast; Simmon et al. (1977) reported that
methylene chloride (source and purity not given, but stated to be the highest
available purity) did not increase mitotlc recombination in strain D3 of
Saccharomyces. However, this may be due to strain-specific differences in
cytochrome P-450 levels in 03 compared to 07, with strain 03 possessing a lower
level. Differences in these levels within yeast have been reported by Call en et
al. (1980) although no information was provided about strain 03. Clearly more
work is needed to resolve the issues raised by Green (1980) and Call en and
coworkers (1980), but at this time there is no compelling evidence to believe
7-18
-------
that the mutagenlc activity of methylene chloride observed in Salmonella 1s
unique to bacteria and not predictive of mutagenicity in other organisms.
There is one report concerning the ability of methylene chloride to mutate
Drosophila melanogaster. Methylene chloride tested negative in this system.
Abrahamson and Valencia (1980) conducted sex-linked recessive lethal tests using
two routes of administration, feeding and injection. Due to the low solubility
of methylene chloride in aqueous solutions it was not possible to utilize high
concentrations of the test substance in these experiments, and the negative
response observed may be due in part to the fact it was not possible to test
higher doses. In the feeding study, male files were.placed 1n culture vials
containing glass nricroflber paper soaked with a saturated solution of methylene
chloride in a sugar solution (1.9% methylene chloride) for 3 days. (The
methylene chloride solution was added twice daily to compensate for evaporation
of the compound). At this dose there was no evidence of toxicity. After
mating, chromosomes from the 14,682 offspring of treated parents and chromosomes
from the 12,450 offspring of concurrent control parents were assessed for
recessive lethal mutations. No evidence of mutagenicity was observed. However,
because of the volatility and insolubility of methylene chloride, the actual
dosing to the animals may have been much less than expected.
In the injection study, 0.3 ul of an isotonic solution containing 0.2%
methylene chloride was administered to male flies. This level of exposure
resulted in 30S post-injection mortality. However, the post-Injection mortality
observed for the controls was not reported. Since the mortality observed in
studies such as this is due not only to the test chemical administered, but also
to the damage caused by Injection, it is Important to have concurrent negative
controls upon which to base conclusions concerning toxiclty of the test chemical
administered. After mating, 8,262 chromosomes from the offspring of treated
7-19
-------
parents and 3,723 chromosomes from the offspring of control parents were
assessed for recessive lethal mutations. No evidence of mutagenicity was
observed by this route of administration either. Because of the low solubility
of methylene chloride and its high volatility with respect to the feeding
experiment, the negative results obtained after testing this chemical in
Drosophila do not reduce the significance of the positive results obtained in
bacteria and yeast.
There are no reports concerning the ability of methylene chloride to cause
gene mutations in mammalian cells. It would be appropriate to test methylene
chloride for its ability to mutate mammalian cells in culture in any adequately
designed and conducted experiment.
Johnston et al. (1980) assessed the ability of a technical sample of
methylene chloride (> 99% pure) to cause chromosomal aberrations in rat bone
marrow cells. Doses of 0, 500, 1500, and 3500 ppm of methylene chloride were
administered to the rats via inhalation 6 hours/day, 5 days/week for 6 months.
Ten animals (5/each sex) comprised each exposed group. The animals were
sacrificed at the end of the exposure period, bone marrow cells were collected
and analyzed for cytogenetic damage. As seen in Table 1, no increased incidence
in the frequency of chromosomal aberrations was observed (e.g., the x_+_ SE for
chromatid breaks with fragments for the four groups was 0.9 _+ 0.99, 0.5 +_ Q.71,
0.5 ^0.97, and 0.7 ^0.48). This negative result may indicate that methylene
chloride is not clastogenic. It may also indicate that the compound did not
reach the cells of the bone marrow in sufficient concentration to cause the
effects. Furthermore, it is generally recognized that chromosomal aberrations
are not as sensitive an endpolnt as are gene mutations (Vogel L976). The
negative results of the cytogenetic study by Johnston et al. (1980) are not
considered to reduce the significance of the positive responses In bacteria and
yeast.
7-20
-------
In conclusion, methylene chloride has been tested for its mutagenic
potential in bacteria, yeast, and Drosophila and for its ability to cause
chromosome damage in rat bone marrow cells. It is clearly mutagenic in
bacteria, but it appears to be a weak mutagen. It has also been reported to
cause reverse mutations, gene conversion, and mitotic recombination in yeast.
It has not been shown to cause mutations in Drosophila but this negative result
may be due to low exposures to the test organism. It has not been shown to
cause chromosomal aberrations in rats. Based on the weight of evidence, it is
concluded that methylene chloride is mutagenic in bacteria and yeast thus
demonstrating the compound has intrinsic mutagenic potential. If the metabolism
and pharmacokinetics of this compound in humans results in metabolic products
which can interact with DNA, as is the case for bacteria and yeast, it may cause
effects in humans as well.
CELL TRANSFORMATION
Price et al. (1978) exposed Fischer rat embryo cell cultures of (F1706
subculture 108) to methylene chloride liquid at concentrations of 1.6 x 10^
and 1.6 x 10^ uM for 48 hours. Methylene chloride was diluted with growth
medium to yield the appropriate doses. The methylene chloride sample, obtained
from Fisher Scientific Company, was ^ 99.91 pure. 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 nonessential amino acids, 100 ug
pencillin, and 100 ug streptomycin per ml. Quadruplicate cultures were treated
at 50% confluency with each dose. After treatment, cells were cultured in
growth medium alone at 37°C. Transformation of cells treated with either dose
level of methylene chloride was observed by 23 and 30 days of incubation and was
characterized by progressively growing foci composed of cells lacking contact
Inhibition and orientation. There was no transformation of cell grown in medium
7-21
-------
alone or in the presence of a 1:1000 acetone concentration even after a
subculture. Twenty and 27 microscopic foci per three dishes with the low and
high methylene chloride dose, respectively, were found 1n dishes Inoculated with
50,000 cells from cultures treated four subcultures earlier and held for 4 weeks
at 37°C in a humidified C02 Incubator prior to staining.
Subcutaneous Injection of cells treated with 1.6 x 102 uM methylene
chloride five subcultures earlier produced local fibrosarcomas in 5/5 newborn
Fischer 344 rats within 60 days following treatment. The ability of cells grown
in growth medium alone to Induce local fibrosarcomas was not determined;
however, negative responses were obtained with cells grown fn the presence of a
1:1000 concentration of acetone. Exposure of cells to 3.7 x 1Q-1 uM
3-methylcholanthrene produced 124 microscopic foci per three dishes in the
inoculation test described above by 37 days of incubation and local flbrosarcoma
in 12/12 rats by 27 days following subcutaneous Injection of cells. The
exposure of 3-methylcholanthrene was attained by initial dilution 1n acetone to
1 mg/ml followed by further dilution fn growth medium to 0.1 ug/ml (personal
communication, Dr. Price).
Dr. Price wrote a letter to the CAG dated Nov. 14, 1980 saying that "the
analysis of methylene chloride showed a purity of 99.9%. The original study was
done 1n quadruplicate and 1n each case the Fischer rat cells were transformed.
Since the publication, the same batch of methylene chloride was sent to Andy
Slvak at Arthur 0. Little to be run against the Kakunago clone A31 of BALB/c
3T3. It did not transform his cells. We then repeated the study 1n Fischer rat
cells and at the same time tested methylene chloride sent to us by the National
Coffee Producers Association (NCA). The test was run in triplicate. The Fisher
•nethylene chloride again transformed the cells, while the National Coffee
Producers' (supplied by Diamond Shamrock) was negative." The different
7-22
-------
responses in the two experiments may be due to the level of impurities present
in each sample. Chemical composition of different supplies are given in
Table 2.
It is understood that this cell line contains the genome of the Rausher
leukemia virus, but there is no basis for minimizing the positive results, since
the mode of action of methylene chloride is not known i.e., due to activation of
the virus.
7-23
-------
TABLE 2. METHYLENE CHLORIDE ANALYSES3
Methyl chloride
Vinyl chloride
'Ethyl chloride
Vinyl 1dtne chloride
Carbon tetrachlorfde
Chloroform
THchloroethyTene
i ,2-01 chl orethyl ene
'Methyl bromide
Cycl bhexane
Flsherb
Fisher D-123
Lot 761542 Diamond ShamrockC
(ppm) (ppm)
0.5 <1
<0.1 <1
o.a
-------
V. TOXICITY
The toxldty of methylene chloride has been reviewed recently (U.S.
Environmental Protection Agency 1979). One major effect 1s on the central
nervous system producing anesthesia, sleep alterations, disturbed central
nervous system function, and depression. Recovery 1s rapid and complete. In
fatal poisonings, death 1s due to cardiac Injury and heart failure. Methylene
chloride produces cardiac arrhythmias and tachycardia. Its metabolite, carbon
monoxide, Increases the carboxyhemoglobln levels 1n the blood. Methylene
chloride is an Irritant to mucous membranes. Chronic exposure can cause liver
damage. Methylene chloride is reportedly teratogenic to chick embryos (Elovaara
et al. 1979).
-»
VI. CARCINOGENICITY
HUMAN STUDIES
Fried!ander et al. (1978)
Friedlander et al. (1978) analyzed mortality of Eastman-Kodak male employees
exposed to low levels of methylene chloride. Measurements from 1959 to 1975
were in the range of 30 to 120 ppm. Mo increase 1n neoplasms, heart disease, or
any other cause of death was found compared to the two control groups composed
of other Kodak employees and New York State males. The population was
relatively stable and the workers were rotated throughout the work area, so
exposure was averaged among all the workers. Methylene chloride was used as
the primary solvent In its operation for 30 years.
Two separate mortality analyses were done. One approach used the
proportionate mortality ratio to.examine 334 deaths of methylene chloride-
exposed workers during 1956 to 1976. Seventy-one neoplasms were found; 73 were
expected based on other Kodak employee mortality ratios. Furthermore, no single
7-25
-------
site was over-represented.
A second approach was a cohort mortality study of all 751 employees who were
in the methylene chloride work area in 1964. Of these, 78 died during the
13-year follow-up (retrospective). Two control groups were used, other Kodak
employees or New York State males, and the expected number of deaths in the
exposed groups based upon these control groups was 69 and 115, respectively.
The differences between the observed and the expected deaths based on the
controls are not statistically significant.
Malignant neoplasms accounted for 14 of the 78 deaths in the study cohort,
which was less than the 19 or 25 expected malignancies based on the control
data. Five of these 14 deaths were from respiratory cancer (also less than
expected based on the control groups) and four were from cancers of the
digestive organs (also less than expected). Only the two deaths associated with
brain or nervous tissue represented a higher than expected total (SMR » 230 and
SMR 3 290 vs. two control groups) but these SMR's (standard mortality ratio)
were not statistically significant.
Further stratification of the cohort focused on the 252 males with 20 years
or more of exposure who were employed in 1964. In this group there were 45
deaths, 7 due to malignant neoplasms (12.5 and 16.1 expected based on the two
control groups) and 24 due to circulatory diseases (26 and 40.6 expected).
A further analysis of the 252 males shows that this cohort was fairly young
(median age approximately 54 years in 1964). With this group the more common
cancers would have to be markedly increased in order to have a reasonable
probability of detecting the increase. For example, following the cohort for 13
years, cancer mortality at this age would require more than nine deaths from
respiratory cancer to detect a significant result at the P » 0.05 level. This
represents an increase of at least 2001 over that expected, the expected
7-26
-------
probability of lung cancer death for this cohort being O.Q133 over the 13 years.
For a less common cancer, liver cancer, the expected probability of death was
6.4 x 10-5, jo that two or three deaths would be statistically significant at
the P = 0.05 level, nevertheless, this would require a SMR » 1240. Thus, only
relatively large cancer effects, at least a 100% increase would be statistically
significant. Furthermore, the remaining 499 males with less than 20 years
exposure were significantly younger (median age about 36 years) and a follow-up
of this cohort for 13 years mortality might fall to detect even a moderate
effect, since expected cancer mortality in this age group 1s so low.
The study appears to have been well conducted and analyzed. Further
follow-up of the 20-year exposure cohort 1s necessary for a definitive
statement.
ANIMAL STUDIES
Thelss et al. (1977)
Only one positive animal test for tumorfgenicity has been reported and this
was marginally positive. This involved the pulmonary tumor bloassay in mice by
Thiess et al. (1977). Groups of 20 male strain A mice were injected
intraperftoneally three times a week with 0, 160, 400, or 800 mg/kg for a total
of 16 or 17 injections. Mice were sacrificed 24 weeks after the first injection
and the lungs were examined under the dissecting microscope for surface
adenomas. Some adenomas were confirmed by histology.
Tumors were found at all three dose levels; however, due to poor survival
and the small number of animals, the increase in tumors did not reach
statistical significance at the higher two doses (Table 3). At the lowest dose,
a highly significant increase in the number of tumors was observed (P * 0.013).
7-27
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TABLE 3. PULMONARY TUMOR 3IOASSAY IN STRAIN A MICE
Dose
mg/kg
0
160
400
800
Total dose
given
0
2,720
6,800
12,800
No. mice at
begl nni ng
20
20
20
20
No. mice
examined
for tumors
15
18
5
12
Tumors /mouse
0.27
0.94
0.80
0.50
Significance*
—
< 0.013
> 0.1
> 0.1
*Tne test of significance used is the Exact test of ratio of two Poisson
parameters.
Heppell et al. (1944)
Two Inhalation studies not designed to test for carc1nogen1c1ty have been
done. Heppel et al. (1944) exposed dogs, rabbits, guinea pigs, and rats by
Inhalation at levels of 5000 pom for 7 hours/day and 10,000 ppm for 4 hours/day,
5 days weekly, for 6 months. No tumors developed 1n any animals.
McEwen et al. 1972
McEwen et al. (1972) exposed dogs to methylene chloride by Inhalation at 500
ppm for 14 weeks; no tumors were reported but edema of the menlnges of the brain
occurred. Neither of these studies could have detected a carcinogenic response
due to the short observation times.
7-28
-------
Dow Chemical Company (1980) Inhalation Study in Rats
A total of 1,032 male and female Sprague-Oawley rats (129/sex/exposure
concentration) were exposed by Inhalation to 0, 500, 1500, or 3500 ppm of
methylene chloride for 6 hours/day, 5 days/week (excluding holidays), In a
2-year toxldty and oncogenlclty study. Approximately 95 rats/sex/exposure
concentration were part of the chronic toxldty and oncogenlc portion of the
study and Included those animals dying spontaneously, killed moribund during the
study, or killed at the end of the 2-year exposure. The remaining animals were
sacrificed as part of the cytogenlc studies or for one of the interim kills at
either 6, 12, 15, or 18 months of exposure. The rats were received at 6 to 7
weeks of age, (males weighed 220 to 250 g; females weighed 170 to 200 g) from
Spartan-Research Animals, Inc., Haslett, Michigan and were individually
identified using metal ear tags. All rats were maintained on a 12-hour
light/dark cycle. Rats were observed daily, including weekends and holidays,
for general health status and signs of possible toxicity.
Methylene chloride, representative of technical grade material, was obtained
from DOM Chemical Company, Plaquemine, Louisiana, and was used throughout the
exposure. Fourteen different samples of methylene chloride were analyzed during
the 2 years of animal exposure; each sample showed 99% pure methylene chloride
with few trace chemical contaminants that varied slightly from sample to sample
as shown in Table 4. The concentration of methylene chloride vapor in chambers
was considered well within the range of expected varfbiHty. Hematologic
determinations, serum clinical chemistry, urfnalysls, bone marrow collection,
and blood carboxyhemoglobln determination were done in animals sacrificed at 6,
12, 15, and IS months (Interim kills). Plasma estradiol determination was done
at the 12- and 18-month interim kills.
7-29
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TABLE 4. ANALYTICAL ANALYSIS of HETHYLEUC CHLORIDE
(OOM Chemical Coayany 1980)
Specific Gravity
HCI, pp.
H20. pp.
Nonvolatile
•aterlal. pp.
Methyl Chloride, pp.
Chlorofor.. pp.
Vinyl Idene
Chloride, pp.
Trans 1,2-Dlchloro-
ethylene. pp.
Cyclohexane. pp.
Ethyl Chloride, pp.
Vinyl Chloride, pp.
Methyl Broalde. pp.
Carbon Tetrachlorlde.
pp.
un. u.i n.»-_rrr
1
1.320
14.2
207
HO
<1
60
60
560
365
6
<1
23
<2
JL
I 3
NO NO
i.a 2.)
560 37
<7 <7
<4 <4
<27 399
52 53
561 266
385 247
9 5
—
—
4
NO
2.9
55
<7
4.5
48
90
706
467k
11
~
—
S 6
ND NO
1.8 1.1
52 27
<7 <7
4.5 <4.5
48 48
75 65
550 550
365 374
8 11
—
—
7
NO
2.5
112
<7
4.5
32
60
487
335
5
—
—
8
ND
--
—
NO
1
52
66
—
399
2
1
1
1
9
NO
—
—
ND
1
576
72
321
266
2
1
1
15
10
ND
1
324*
NO
1
64
62
653*
426
2
1
1
1
II
NO
—
—
ND
1
562
78
323
262
2
1
1
13
12
ND
1
264*
ND
1
516
70
318
268
6
1
1
16
13
NO
1
340*
NO
1
547
77
337
288
1.5
1
1
20
14
NO
1
33
HI)
1
460
72
298
242
b
1
1
12
•Original analysis was lost; sample was subsequently reanalyzed.
--Means not detected.
-------
This included samples from six controls/sex and four high exposure animals (3500
ppm/sex) from the 12-month km which were pooled together (two animals/sample)
to give three control samples and two high exposure (3500 ppm) samples/sex. Ten
Individual samples/sex (not pooled) from the high exposure and control groups
were also sent from the 18-month km.
All animals that died spontaneously, were killed in moribund condition, or
were killed at the interim or terminal kills, were subjected to complete gross
and microscopic pathological examinations by a veterinary pathologist. Liver
samples for possible electron microscopic evaluation were collected.
In females exposed to 3500 ppm, there was a statistically significant
Increase of mortality from the 18th through 24th months which may be
exposure-related. The remaining treated groups in males or females did not
differ significantly from the controls (Table 5). There was no exposure-related
difference in body weights of either male or female rats exposed to 500, 1500,
and 3500 ppm of methylene chloride.
Although some hematologic values were Increased and others were decreased,
the mean values were within the normal range of "biological variability." Serum
glutamlc pyruvlc transamlnase (SGPT), blood urea nitrogen (BUN), and serum
alkaline phosphatase (AP) values were 1n the normal range. It is noted that the
females had significantly increased P < 0.025} plasma estradlol level at 18
months which may be related to the higher incidence of mammary tumors in the
exposed (3500 ppm) group. UHnalysis findings were 1n the normal range with the
exception of a few statistically significant values 1n specific gravity in males
exposed to 1500 ppm at 6 months and male and females exposed to 3500 ppm at 12
months. Rats exposed to 500, 1500, or 3500 ppm have elevated carboxyhemoglobin
values but with no evidence of either dose-response or Increased values with
prolonged exposure.
7-31
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TABLE 5. CUMULATIVE PERCENT MORTALITY OF RATS
2-YEAR METHYLENE CHLORIDE INHALATION STUDY
(Dow Chemical Company 1980)
Month of
Study 0 ppm
Males
500 ppm 1500 ppm
Females
3500 ppm 0 ppm 500 ppm 1500 ppm 3500 ppm
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
¥TT
0.8
0.8
0.8
0.8
0.8
0.8
0.9
0.9
1.7
1.7
1.7
2.6
2.7
3.6
10.9
15.2
23.8
32.4
44.2
48.4
56.8
63.2
80.0
85.3
0
0
0
0.8
0.8
1.6
2.6
3.4
3.4
4.3
6.0
6.3
10.9
10.9
11.8
16.2
20.0
36.2
45.3
52.6
56.3
65.3
73.7
85.3
0
0
0
0
0
0.8
0.9
0.9
0.9
1.7
3.4
4.3
5.5
10.0 '
13.6
20.0
31.4
37.1
49.5
63.2
74.7*
83.2*
89.5
93.7
0
0.8
2.3
3.9
4.7
4.7
6.0
6.0
6.0
6.0
6.8
8.5
9.8
15.2*
21.4
29.0*
33.6
43.0
54.6
58.8
72.2*
80.4*
87.6
92.8
0
0
0.8
0.8
0.8
0.8
0.9
2.6
3.4
5.1
5.1
6.0
7.2
9.0
13.5
14.2
23.6
26.4
36.5
42.7
49.0
62.5
70.8
78.1
0
0
0
0
0.8
0.8
1.7
2.6
3.4
5.1
6.0
6.8
8.2
8.2
10.9
15.2
20.0
29.5
42.1
50.5
58.9
64.2
65.3
74.7
0
0
0.8
0.8
0.8
0.8
1.7
1.7
1.7
2.6
4.3
5.1
8.2
8.2
10.0
13.3
20.0
29.5
46.3
55.8
65.3*
73.7
81.1
86.3
0
0
0
0
0.8
0.8
0.9
0.9
2.6
2.6
4.3
6.0
12.5
12.5
19.6
20.6
29.9
42.1*
57.7*
68.0*
81.4*
86.6*
90.7*
95.9*
7-32
-------
Gross and H1stolog1c Observations of Rats from the 6-, 12-, 15-, and 18-Month
Interim Kins-
Numerous gross and hlstopathologlc observations were recorded for control
and methylene chloride-exposed rats at each time period and most were typical of
spontaneous or naturally-occurring lesions normally seen 1n rats of this strain.
There were many palpable masses in males and females. Some palpable masses
appeared to be abscesses of the preputial or clltoral glands, while others were
cyst-like lesions of the skin. The total number of masses 1n the 3500 ppm group
of males were significantly Increased over the controls at 15, 18, and 21
months, but not at 23 months. Female rats exposed to 500, 1500, and 3500 ppm
showed an exposure-related Increase in total number of masses. There was also a
trend of Increased benign mammary tumors 1n females exposed to 1500 and 3500
ppm. The total numbers of animals with benign mammary gland tumors were 9/28 (0
ppm), 10/29 (500 ppm), 11/29 (1500 ppm), and 14/27 (3500 ppm), whereas the total
numbers of benign mammary gland tumors were 17/28 (0 ppm), 17/29 (500 ppm),
28/29 (1500 ppm, P - 9.23 x 1Q-4), and 37/27 (3500 ppm, P =• 2.33 x 10-10).
These observations were apparent only when the cumulative results of the 6-,
12-, 15-, and 18-month kills were evaluated.
There were a few other observations that appeared to reflect
exposure-related lesions. The liver was the only organ that exhibited definite
exposure-related non-neoplast1c effects in both males and females at all
exposure concentrations. Grossly, the effect was most prominent fn females
exposed to 3500 ppm and consisted of Increased numbers of dark or pale foci.
The control group had an Incidence of 0/28, while the 3500 ppm methylene
chloride-exposed female rats had a significantly greater number of foci (11/27).
Some rats from the 3500 ppm exposure group had mottled livers or had an
accentuated lobular pattern to the liver (0/29 control males compared to 6/27
males exposed to 3500 ppm). Because of the limited number of rats at each
7-33
-------
interim kill, this latter change may be related to exposure but may also be due
to "biological variability."
Histologlcally observed, exposure-related lesions were present 1n the livers
of both males and females exposed to 500, 1500, or 3500 ppm. In males, the
total number of animals with any degree of vacuollzatlon consistent with fatty
changes were 5/29 (0 ppm), 19/29 (500 ppm, P =• 2.05 x 1Q-4), 21/29 (1500 ppm,
P = 2.47 x 10-5), and 23/27 (3500 ppm, P - 2.81 x 10-7). The liver of female
rats also had alterations considered to be related to the exposure to methylene
chloride. The total number of females with any degree of vacuollzatlon
consistent with fatty changes were 13/28 (0 ppm), 20/29 (500 ppra, P » 7.16 x
10-2), 20/29 (1500 ppm, P * 7.16 x 10-2), and 22/27 (3500 ppm, P = 7.16 x
10-3). Because of these effects, it may be considered that this experiment
was performed at the maximum tolerated dose (MTD).
Gross and Hlstopathologlc Observations of Rats Killed Moribund or Dying
Spontaneously During The Study and Those From Terminal Sacrifice (24 months)—
Non-neoplast1c observations—The liver was affected In both males and
females exposed to 500, 1500, or 3500 ppm. The percentage of total rats with
any degree of vacuollzatlon was 171, 38%, 451, and 54% in the males of the 0,
500, 1500, and 3500 ppm exposure groups, respectively, and 34%, 52%, 59% and 65%
In the females, respectively. Also, the degree of severity tended to Increase
with the dose. The males In all exposure groups had fewer cases of
grossly-observed mottled and/or enlarged adrenals. These gross alterations
appeared to correspond to the h1stolog1ca1ly-observed decrease in the number of
cases of adrenal cortical necrosis, but the nodular nyperplasla Incidence
(unilateral or bilateral) was Increased: 18/95 (0 ppm), 30/95 (500 ppm, P * 3.07
x ID'2), 31/95 (1500 ppm, P - 2.2 x lO'2), and 24/97 (3500 ppm).
7-34
-------
Tumor or tumor-like lesions—The Sprague-Dawley rats used in this study
normally have a high incidence of benign mammary tumors. The incidence varies
slightly from study to study but normally exceeds 80% in females and about 10S
in males by the end of a 2-year study. The mammary gland tumors have been
classified, based on their predominant morphological cellular pattern, as
fibromas, fibroadenomas, or adenomas.
The benign mammary tumor response was present in males and to a lesser
extent in females. There was a non-statist!cally significant increase in the
number of rats with a benign mammary tumor in males exposed to 3500 ppm (14/95
compared to 7/95, 3/95, and 7/95 in the 0, 500, or 1500 exposure groups). There
was a slight increase in the total number of benign mammary tumors in males
exposed to 0 ppm (8/95), 500 ppm (6/95), 1500 ppm (11/95), or'3500 ppm (17/97, P
- 4.6 x 10-2).
The total number of female rats with a benign mammary tumor was not
increased in any exposure group (0, 500, 1500, or 3500 ppm groups had a total of
79/96, 81/95, 80/95, and 83/97 benign mammary tumors, respectively). However,
the total number of benign mammary tumors increased in an exposure-related
manner with 165/96 in the controls, and 218/95, 245/95, and 287/97 in the
females exposed to 500, 1500, and 3500 ppm, respectively. Expressed another
way, the average number of benign mammary tumors per tumor-bearing female rat
increased from 1.7 in the control rats, to 2.3 in rats exposed to 500 ppm, to
2.6 in those exposed to 1500 ppm, and to 3.0 in rats exposed to 3500 ppm. This
effect is exposure-related and a dose-response relationship was apparent. There
was no indication of an increased number or incidences of malignant mammary
tumors in either males or females.
The number of malignant tumors (Table 6) was increased in male rats exposed
to 3500 ppm. This Increase did not appear to clearly correlate with an
7-35
-------
TABLE 6. SUMMARY OF TOTAL TUHOR DATA FOR RATS ADMINISTERED HETHYLENE CHLORIDE FOR 2-VEARS BY INHALATION
. (Dow Chealcal Company 1980)
Hales
Spontaneous
^ Total nuaber of rats
i exaalned
u>
CT\
Total nuaber of rats
with a tuaor
Total number of rats
with a benign tuaor
Total nuaber of rats
with a aallgnant tuaor
Concentration
In ppa
0
500
1500
3500
0
500
1500
3500
0
500
1500
3500
0
500
1500
3500
Interla
Kills
29
29
29
27
9
7
9
11
6
3
8
9
6
4
S
7
Deaths and
Killed
Moribund
81
81
89
90
S3
52
67
68
33
29
42
37
39
45
48
SS
Teralnal
Kill
14
14
6
7
13
13
6
7
11
II
6
S
12
10
4
7
Cumulative
Totals
124
124
124
124
75
72
82
86
50
43
56
51
57
59
57
69
Interla
Kills
28
29
29
27
14
IS
14
14
14
15
14
14
2
3
1
2
Feaales
Spontaneous
Deaths and
Killed
Moribund
75
71
82
93
67
68
79
91
65
60
73
U4
23
28
31
32
Terminal
Kill
21
24
13
4
21
24
13
4
21
24
12
4
13
14
7
2
Cunulattvc
Totals
124
124
124
124
102
107
106
109
100
99
•J9
102
Jb
45
J9
36
-------
increased number of any one tumor type or location. However, this observation
led Dow Chemical Company to re-evaluate the gross and hlstopathologlc data on
all tumors arising 1n or around the salivary glands. Table 7 lists the specific
Individual animal data for these salivary gland area tumors showing the palpable
mass data, specific hlstopathologlc diagnoses, and the number of sarcomas with
metastases. Table 8 summarizes the Incidence of salivary gland region sarcomas
1n male rats.
Grossly, these tumors were large (several centimeters in diameter), cystic,
necrotlc, or hemorrhaglc. They appeared to Invade all adjacent tlsues In the
neck region and often completely replaced the normal salivary gland tissue.
H1sto1og1ca1ly, all were sarcomas. They were composed of cells that varied from
round to spindle-shaped, but appeared to be of mesenchymal cell origin. M1tot1c
figures were frequently observed, as were necrosis and local Invasion Into
adjacent tissues. Most tumors had remnants of normal salivary acini or ducts
that were caught-up 1n the cellular proliferation. Two were relatively small
masses and appeared to be arising In the Interstitial and capsular tissue of the
salivary glands.
One tumor of this type was found 1n the controls (1/93) compared to 0/94 1n
the 500 ppm exposure group, 5/91 1n males from the 1500 ppm exposure group, and
11/88 in males from the 3500 ppm exposure group (P 3 0.002). Historically, a
spontaneous Incidence (0 to 2%) of this tumor type has been observed in Dow's
laboratory. Therefore, the 12.5% incidence (11 of the 88 rats, Table 8) found
in the males from the 3500 ppm group was higher than the corresponding controls
of this study and was higher than expected based on historical control data for
male rats of this strain. Also, the males exposed to 1500 ppm had five of these
tumors which was also slightly higher than expected, but was not statistically
significant. Therefore, this effect appeared to be exposure-related in the
7-37
-------
TABLE 7. TIME TO TUHOR. PALPABLE MASS. AND H1STOPATHOLOGY DATA FOR SALIVARY 6LANO REGION SARCOMAS IN INDIVIDUAL HALE RATS
EXPOSED TO METHYLENE CHLORIDE BY INHALATION FOR 2 YEARS
(Dow Chealcal Coayany I960)
OJ
oo
AnlMl
Hunter
76-3301
76-3733
76-3809
76-3722
76-3743
76-3749
76-3583
76-3698
76-3580
Exposure
Croup (ppa)
0
1500
1500
1500
1500
1500
3500
3500
3500
Month of Stu4y
Tumor First
Observed
IS
16
Not Observed
24
14
18
Not Observed
14
21
Size of Tunor
Men First
Observed leal
5
3
-
1.5
2
3
-
3
2
Month of Study
for Necropsy
IS
17
19
24
14
20
24
IS
24
Slie of Tumor
it Necropsy (en)
7x6x4
S x 4 x 2
Not Detected (slightly
enlarged salivary gUnd)
4x3x2
No Size Given
16 gra«s)
S x 4 N 4
Not Detected
7x6x3
9 x 6 x 4.5
Hlstologlcal
Diagnosis
Subcutaneous - Undlf-
ferentlated Sarcoma
Salivary Gland -
Ha 1 1 gnant Sc hMannona
Salivary Gland -
Malignant Schwannoua
Salivary Gland - Undlf-
ferentlated Schuannona
Subcutaneous -
Round Cell Sarcoma
Subcutaneous -
Round Cell Sarcoma
Salivary Gland -
F IbrosarcoMia
Salivary Gland -
Carclnosarco«a
Subcutaneous -
Neurof Ibrosarcouid
(continued on the
Evidence of
Distant
Metastases
None
None
None
None
None
None
None
Yes
Vcs
fallowing |uiji')
-------
TABLE 7. (continued)
Anlnal
Number
76-3663
76-3682
76-3578
76-3608
76-3621
76-3666
76-3671
76-3597
Exposure
Group (pp>)
3500
3500
3500
3500
3500
3500
3500
3500
Month of Study
Tuaor First
Observed
20
21
16
16
17
15
Not Observed
19
Size of Tuaor
Uhen First
Observed (CM)
2
6
a
2.5
4
3
-
6
Month of Study
For Necropsy
21
21
16
16
18
16
14
19
Size of TuBor
at Necropsy (CB|
No size given
7x4x4
7 x 7 x 3.5
6x4x3
5x5x3
8x6x4
3.5
7 x 7 x 3.5
Illstologlcal
Diagnosis
Subcutaneous -
Flbrosarcowa
Subcutaneous -
Undlfferentlated
Round Cell Sarcoma
Subcutaneous - Undlf-
ferentlated Sarcoma
Subcutaneous -
PleoBorphlc Sarcoma
Subcutaneous -
Pleoaorphtc Sarcoma
Subcutaneous -
Pleonorphlc Sarcowa
Subcutaneous -
Neuroflbrosartoma
Subcutaneous -
IbrosarcoNia
Evidence of
Distant
Hutastases
Yes
None
Hone
None
None
Yes
None
None
-------
TABLE 8. SUMMARY OF SALIVARY GLAND REGION SARCOMA INCIDENCE IN MALE
RATS IN A 2-YEAR INHALATION STUDY WITH METHYLENE CHLORIDE
Dose Incidence Fisher Exact Test
0 ppm 1/93 (IS)
500 ppm 0/94 (01)
1500 ppm 5/91 (5.5S) (P - 0.10, N.S.)
3500 ppm 11/88 (12.5%) (P - 0.002)
7-40
-------
males exposed to 3500 ppm.
The total number of male rats with a malignant tumor was similar in the
control, 500 ppm, and 1500 ppm exposure groups. Males exposed to 3500 ppm had
an Increase 1n this category since 69 of the 124 rats had malignant tumors
compared to 57, 59, and 57 in the 0, 500, and 1500 ppm exposure groups,
respectively.
7-41
-------
Dow Chemical Company (1980) Inhalation Study in Hamsters
A total of 866 Golden Syrian hamsters [Ela: Eng (syr) strain; Engle
Laboratory Animals, Inc., Farmersburg, Indiana] (107 to 109/sex/exposure
concentration) were exposed by inhalation to 0, 500, 1500, and 3500 ppm of
methylene chloride. The materials and methods for experimental design are the
same as mentioned previously in the rat portion of the Dow study. The body
weights of hamsters were Initially 61 to 70 grams when they were received.
The hamsters were identified by a unique toe clip for group identification
purposes and ear punch for Individual identification within the cages.
The mortality data for males and females are presented in Table 9. Female
hamsters exposed to 3500 ppm had a statistically significantly decreased
mortality from the 13th through the 24th month. Females exposed to 1500 ppm
also had statistically significantly decreased mortality from the 20th through
the 24th month. This decreased mortality in females exposed to 3500 and 1500
ppm was considered to be exposure-related. The remaining exposure groups of
male (500, 1500, and 3500 ppm) and female (500 ppm) hamsters had no
differences in mortality that were exposure-related. Some hamsters of all
groups had alopecia at 5 1/2 months into the study, but this alopecia was
secondary to a mange mite (Deroodex species) infection. This parasite did not
result 1n an Increased mortality and morbidity. No treatment-related
differences were observed 1n the body weights of either males or females
exposed to 500, 1500, or 3500 ppm of methylene chloride.
Based on the Information available to the CA6, it is very difficult to
conclude whether the MTD was used. DOM Chemical Company has not submitted any
90-day study (dose-finding), but a 30-day inhalation study has been reported
in a letter from Dr. J. Burek to Dr. D. Singh, dated May 1, 1981. "This study
was conducted prior to the 2-year study, but results have not been reported.
7-42
-------
TABLE 9. CUMULATIVE PERCENT MORTALITY OF HAMSTERS
2-YEAR METHYLENE CHORIDE INHALATION STUDY
(Dow Chemical Company 1980)
Month of
Study 0 ppm
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0
0
0
0
0
1.0
1.9
1.9
2.9
3.8
5.1«
6.4
10.6
14.9
23.4
24.5
31.9
36.0
37.1
41.6
56.2
61.8
82.0
Males
SOO ppm 1500 ppm
0
0
0
0
0.9
1.9
1.9
3.8
5.8
5.8
5.8
6.7
10.1
14.1
17.2
18.2
19.2
24.2
29.8
43.6
51.1
61.7
63.8
78.7
0
0
0
0
0
0
1.0
1.9
1.9
2.9
2.9
3,9
10.2
13.3
14.3
17.3
19.4
24.5
31.2
41.9
54.8
68.3
75.3
88.2
3500 ppm
0
0
0
0.9
3.7
3.7
5.8
5.8
6.7
9.6
11.5
12.5
17.2*
24.2
26.3
33.3
34.3
37.4
42.6
55.3*
60.6
69.1
74.5
85.1
0 ppm
0.9
0.9
1.9
1.9
2.3
3.7
3.9
4.9
4.9
5.8
9.7
13.38
22.6
26.9
32.3
36.6
41.9
52.7
63.6
71.6
80.7
88.6
94.3
100.0
Females
500 ppm 1500 ppm
0
0
0.9
1.9
1.9
2.8
4.9
4.9
5.8
9.7
10.7
11.7
16.3
24.5
26.5
30.6
32.7
38.8
59.1
63.4
70.0
82.8
91.4
95.7
0.9
0.9
1.9
1.9
2.8
3.7
4.8
7.6
8.6
8.6
9.5
13.3
15.0
20.0
24.0
29.0
32.0
40.0
50.5
55.8*
61.1*
68.4*
75.8*
89.5*
3500 ppm
0
0
0.9
0.9
0.9
0.9
2.9
3.9
3.9
4.9
4.9
4.9
7.1*
11.2*
15.3*
23.5*
27.6*
30.6*
40.9*
45.2*
53.8*
72.0*
80.6*
90.3*
• I • «• Ml** I *. ^ 1*11W !•*•*• t %.«!•«* I >.^ Vft * \*V4 V4M« teW I W*SU W«pl I V U V I Wll • I 11C 37 (111 I Hid I 4 fff^ I ^
subsequently deleted from mortality calculations.
*S1gn1f1cantly different from controls by Fisher's Exact Probability test, P <
0.05.
7-43
-------
CD-I mice, Golden Syrian hamsters, Sprague-Dawley and CDF (F-344) rats were
exposed to 0, 2500, 5000, or 8000 ppm methylene chloride vapor 6 hours/day, 5
days/week, for a total of 20, 19, 20 or 6-2/3 exposures, respectively, in
21-29 days. Body weight data was obtained throughout the study. Clinical
chemistry parameters were measured. All animals underwent gross pathological
examination at the termination of the experiment. The weights of the liver
and kidneys were recorded from animals in the 0, 2500, and 5000 ppm groups,
and organ/body weight ratios were calculated. Animals exposed to 8000 ppm
methylene chloride exhibited anesthetic effects, increased blood urea nitrogen
levels in Sprague-Dawley male rats, and decreased body weights in rats. The
animals exposed to 5000 ppm showed slight anesthesia, decreased body weight in
male rats, increased S6PT values in female mice and Sprague-Oawley rats and
increased liver weights in female mice, hamsters and rats. Animals exposed to
2500 ppm methylene chloride appeared to scratch more than controls and
therefore appeared to be affected, but showed no other effect attributable to
exposure. The target organ in this study was the liver. Because of the
results obtained, 8000 and 5000 ppm methylene chloride were considered to be
too high a dose level for the two-year study, and 2500 ppm did not appear to
have produced a severe enough response over the 30-day period. Therefore,
concentrations of 3500 of methylene chloride was chosen as the top dose for
the two-year study."
It is interesting to note that Dow conducted only a 30-day subchronlc
(dose-finding) study rather than the usual 90-day study which is used in most
animal bioassays. Further, it should be noted that there is no decrease in
body weight or mortality rate in the experimental group of hamsters as
compared to the controls. Hematologic determinations, serum clinical
chemistry, urinalysls, bone marrow collection, blood carboxyhemoglobin
7-44
-------
determination were done 1n animals at the 6-, 12-, 18-, and 24-month interim
kills. No treatment-related effects were observed 1n any of the parameters
evaluated 1n male or female hamsters after 6, 12, 18, or 24 months of exposure
to 500, 1500, or 3500 ppm of methylene chloride, respectively.
Carboxyhemoglobln determinations were performed on the blood of male and
female hamsters following 22 months on test. Males and females exposed to
500, 1500, or 3500 ppm all had significantly elevated Carboxyhemoglobln
values. There was a slight trend 1n a dose-response relationship 1n females
since the mean carboxyhemoglobin values for those exposed to 500 ppm was 23.6%
while those for females exposed to 1500 or 3500 ppm were 30.2% and 34.6%,
respectively. However, an apparent dose-response relationship was not
observed in males. Dow Chemical Company Indicated that these data, when
compared to those In the rat study, suggested that hamsters had a greater
degree of metabolism of methylene chloride to carbon monoxide. Furthermore,
the apparent dose-response in females was surprising. As a result, additional
hamsters were exposed to a single 6-hour exposure and their carboxyhemoglobin
values were determined. There was no apparent sex difference and the
dose-response relationship observed in females after 22 months could not be
verified. Since there was no dose-response relationship in male and female
rats or male hamsters and since the female hamsters exposed to a single 6-hour
exposure did not show a dose-response relationship, the apparent trend for an
exposure-related increase in female hamsters at 22 months may be a cumulative
effect but is probably not an exposure-related effect.
7-45
-------
Gross and Histopathologic Observations of Hamsters from the 6-, 12-, and
18-Month Interim Kills—
A variety of gross and histopathologic observations were recorded for
hamsters that were sacrificed at the 6-, 12-, or 18-month interim kills.
Histopathologically, exposure-related differences were present consisting of
decreased numbers of hamsters with amyloidosis of the liver, kidney, adrenals,
thyroid, and spleen. There were a few animals in each group which may or may
not represent the trend of amyloidosis in males.
Gross and Histopathologic Observation of Hamsters Killed Moribund or Dying
Spontaneously During the Study and Those From Terminal Sacrifice (24-months)--
Neoplastic and non-neoplastic observations—A gross and histopathologlc
examination was conducted on all hamsters that died or were killed moribund
during the study, and on all surviving hamsters at the end of the study.
The histopathologlc observations for males and females are presented in the
Dow Chemical report (1980), tables 124 to 127. The observations shown include
all the neoplastlc and non-neoplastic lesions recorded for these hamsters.
Most observations were within the normal or expected range for Golden Syrian
hamsters as indicated by Dow Chemical Company. The female rats had increased
incidences of lymphosarcoma in the experimental group. The incidence was
1/96, 6/95, 3/95, and 7/97 (P « 0.033) in the 0, 500, 1500* and 3500 ppm
groups, respectively (letter from Hugh Farber, Dow Chemical Company, to EPA,
dated April 14, 1981). A re-evaluation of lymphosarcoma data of female
hamsters by the CAG resulted 1n the following incidences: 1/91, 6/92, 3/91,
and 7/91 (P * 0.032) in the 0, 500, 1500, and 3500 ppm groups, respectively.
The differences between the denominators above reflects the CAG's use of
actual numbers of animals examined (which did not include animals that were
severely cannibalized, autolyzed, or missing), whereas, the Dow denominator's
7-46
-------
included the total number of animals. It should also be noted from this table
that only a small number of mammary gland tissues were examined and no lesions
were found. Table 10 summarizes the total tumor data. Total number of
hamsters with benign tumors was significantly increased in females at 3500 ppm
and malignant tumors in males at 1500 ppm.
Discussion and Conclusions of the Dow Chemical Company (1980) Inhalation Study
Based on all the data evaluated, the following points are considered to be
major findings in the rat and hamster studies:
1) Male rats exposed to 1500 or 3500 ppm appeared to have an increased
number of sarcomas in the ventral nrfdcervical area near the salivary glands.
There were 1/93, 0/94, 5/91, and 11/88 (P * 0.002) sarcomas 'in male rats
exposed to 0, 500, 1500, or 3500 ppm, respectively. Based on routine
sections, special stains, and ultrastructural evaluations, these tumors
appeared to be of mesenchymal cell origin; however, a myoepithelial cell
origin of these cells could not be ruled out. These tumors had some areas
that morphologically resembled one cell type (i.e., neurofibrosarcoma,
flbrosarcoma), and still other tumors had cell types that were
^differentiated or pleomorphic. In some, one cell type was predominant,
while in others, areas of all of the above cell types were present depending
on the area of the tumor examined. Furthermore, the origin of each of these
tumors remains questionable. All appeared to be arising in the midcervical
region and all Involved the salivary glands. Only two tumors were small
enough to be localized within the salivary gland. The rest were larger tumors
that clearly involved the salivary glands as well as adjacent tissues and
could have been growing either into or out of the salivary glands. However,
they probably arose within the salivary glands based on the two localized
small tumors described above.
7-^7
-------
TABLE 10. SUMMARY OF TOTAL TUMOR DATA FOR HAMSTERS ADMINISTERED METHYLENE CHLORIDE UV INHALATION FOR 2 YEARS
(Dow Chemical Company I OHO)
I
4^
co
Concentration
Total number of
hamsters during
this period*
Total number of
hamsters with
a tumor
Total number Of
hamsters with a
benign tumor
Total number of
hamsters with •
malignant tumor
«PP«»
0
500
1500
3500
0
500
1500
3500
0
500
1500
3500
0
500
1500
3500
Interim
Kills
15
10
10
15
4
3
2
4
4
2
2
3
0
1
0
1
Males
Spontaneous
Deaths and
Killed
Horibund
76
74
82
7B
ia
17
17
ia
14
10
6
10
6
a
12
a
»• • • • " •
Terminal
Kill
16
20
II
14
3
9
a
7
2
7
S
6
1
4
3
1
— ' * 1 • • «• • — r"
Cumulative
Totals
107
104
103
107
25
29
27
29
20
19
13
19
,
13
15*
10
— -••*— •• 7TT — KV '
Interim
Kills
15
10
10
14
2
0
2
2
2
0
1
1
0
0
1
1
i ,L a* j * — T- _ j
Females
Spontaneous
Deaths and
Killed
Moribund
91
88
81
82
17
20
13
27
II
a
9
22
a
13
4
9
[ — H — ^ n m:
Terminal
Kill
0
4
10
9
.
1
4
3
_
1
3
3
_
0
2
0
Cumulative
Totals
lUb
ID?
101
105
19
21
19
32'
13
9
13
2b'
b
13
1
10
•Significantly different from controls when analyzed by Fisher's
-Indicates none examined or not applicable.
'Uoes not Include hamsters that escaped from their cages, or hamsters that were severely autolyzvd, or severely camiibul ut.l
Also, this total does not Include 500 ppn or 1500 pum male and female hansters fruui the 6-month Interim kill because no
hlstopathology was done on these animals except for a liver special stain (f.uwori's Prussian Illue Iron Reaction).
-------
Therefore, there was an apparent association between the Increased
Incidence of sarcomas 1n the salivary gland region of male rats and prolonged
exposure via Inhalation to 1500 or 3500 ppm methylene chloride. It 1s
interesting to note that there were no salivary gland sarcomas 1n female rats
or in hamsters of either sex. Further, it will be of interest to find out
what kind of lesions are present or absent In the National Toxicology Program
study.
2) Male and female rats exposed to methylene chloride had increased
numbers of benign mammary tumors compared to control values. Female rats
exposed to 500, 1500, or 3500 ppm of methylene chloride had increased numbers
of benign mammary tumors per tumor-bearing rat compared to the controls. The
increase was evident in the palpable mass data and the gross necropsy findings
which were confirmed by the histopathologic examination. The total number of
female rats with a benign mammary tumor was not statistically increased in any
exposure group (0, 500, 1500, or 3500 ppm groups had a total of 79, 81, 80,
and 83 animals with benign mammary tumors, respectively). It should be noted
that Sprague-Oawley rats have very high incidence of spontaneous mammary
tumors. However, the total number of benign mammary tumors have increased in
an exposure-related manner with 165/92 in the controls and 218/90, 245/92, and
287/95 in the females exposed to 500, 1500, or 3500 ppm, respectively.
Expressed another way, the average number of benign mammary tumors per female
rat increased from 1.7 in the controls, to 2.3 in those exposed to 500 ppm, to
2.6 in those exposed to 1500 ppm, and to 3.0 in those exposed to 3500 ppm.
This increase was considered to be exposure-related and dose-dependent.
A mammary tumor response was present in male rats also, but to a lesser
extent than in females. There was an Increase (not statistically significant)
in the number of rats with a benign mammary tumor in males exposed to 3500
7-49
-------
ppra. There Mas a slight increase in the total number of benign mammary tumors
in males exposed to 1500 or 3500 ppm. As was the case in females, these
effects in males exposed to 1500 or 3500 ppm were considered to be
exposure-related.
There were no mammary gland tumors In male or female hamsters. It 1s
also interesting to note that only 28/92, 44/93, 30/94, and 27/93 mammary
glands tissues were examined In the 0, 500, 1500, and 3SQQ ppm groups,
respectively. Not a single lesion was recognized in the mammary gland tissues
examined. The CAG feels that a greater number of hamster mammary gland
tissues should have been examined to better evaluate the true Incidence of
mammary tumors.
3) There was an Increased incidence of lymphosarcoma in female hamsters.
The incidence was 1/96, 6/95, 3/95, and 7/97 (P » 0.033) in the 0, 500, 1500,
and 3500 ppm groups, respectively (letter from Hugh Farber, Dow Chemical
Company, to EPA, dated April 14, 1981). A re-evaluation of lymphosarcoma data
of female hamsters by the CAG resulted in the foil owing incidences: 1/91,
6/92, 3/91, and 7/91 (P - 0.032) in the 0, 500, 1500, and 3500 ppm groups,
respectively. The differences between the denominators above reflects the
CAG's use of actual numbers of animals examined (which did not Include animals
that were severely cannibalized, autolyzed, or missing), whereas, the Dow
denominator's included the total number of animals. Dow Chemical Company
believed that the females exposed to 3500 ppm had better survival
(statistically significant) than the controls and thereby had a greater chance
to develop these tumors. After correction for survival (1/39 vs. 7/63) by the
CAG this data 1s not statistically significant (P - 0.12).
4) There appears to be a question as to whether or not the doses given the
rat and hamster were at or near the MTD. The body weights of male rats
7-50
-------
increased particularly toward the latter part of the experiment, whereas the
body weight of female rats were unaffected in any experimental group.
Exposure to 3500 ppm resulted in an increased mortality rate in female rats
during the last six months, but the male rats were unaffected at any
concentration. On the other hand, decreased mortality was observed in female
hamsters exposed to 1500 and 3500 ppm while mortality in male hamsters was
unaffected at 500, 1500, and 3500 ppm based on only a 30-day rat and hamster
inhalation (dose-finding) study (see details, pages 39 and 41). Based on this
information, it is difficult to judge whether the animals were given a dose
equal to the MTD.
Other Animal Studies in Progress'
Another chronic study in rats and mice started in September 1979 at
Hazleton Laboratories of America, Inc. sponsored by the National Coffee
Association. Methylene chloride in this experiment is being used in drinking
water. Details of this study are not available. The CAG will review this
experiment when the reports are available.
A bioassay for carcinogenicity has been undertaken by the National Cancer
Institute in a 2-year chronic gavage study with rats and mice. The sacrifice
date for this study was December 1980. The NTP has also scheduled an
inhalation test in rats and mice to start in April 1981.
7-51
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VII. UNIT RISK ESTIMATE
Insufficient data exist on which to base a unit risk assessment.
7-52
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