EPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-600/8-82-004B
December 1983
External Review Draft
Research and Development
Health Assessment Review
Document for Draft
Dichloromethane
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EPA-600/8-82-004B
December 1983
Review Draft
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
Region V, Library
230 South Dearborn Street
Chicago, Illinois 60604
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC 27711
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DISCLAIMER
This report 1s an external 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.S. Envtr6iimsntal PrtWctlOT
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PREFACE
The Office of Health and Environmental Assessment, 1n consultation with
an Agency work group, has prepared this health assessment to serve as a "source
document" for EPA use. Originally the health assessment was developed for use
by the Office of A1r Quality Planning and Standards, however, at the request
of the Agency Work Group on Solvents, the Assessment scope was expanded to
address multimedia aspects.
In the development of the assessment document, the scientific literature
has been Inventoried, key studies have been evaluated, and summary/conclusions
have been prepared so that the chemical's toxicity and related characteristics
are qualitatively Identified. Observed effect levels and dose-response rela-
tionships 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 (continued)
5. HEALTH EFFECTS OF DICHLOROMETHANE 5-1
5.1 HUMAN HEALTH EFFECTS 5-1
5.1.1 Acute Exposures 5-1
5.1.2 Chronic Effects 5-6
5.2 EFFECTS ON LABORATORY ANIMALS 5-7
5.2.1 Acute Effects 5-7
5.2.2 Chronic Effects 5-17
5.3 TERATOGENICITY, MUTAGENICITY, AND CARCINOGENICITY 5-22
5.3.1 Teratogenicity, Embryotoxicity, and Reproductive
Effects 5-22
5.3.2 Mutageni city 5-27
5.3.3 Evaluation of the Carcinogenicity of Methylene
Chloride 5-53
5.4 REFERENCES 5-101
APPENDIX A-l
vi
005DC1/E 12/22/83
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LIST OF TABLES
Table Page
3-1 Synonyms and identifiers for dichloromethane 3-2
3-2 Selected properties of dichloromethane 3-3
3-3 Producers of dichloromethane 3-5
3-4 Consumption of dichloromethane 3-6
3-5 Reaction rate data for OH + CH2C12 3-8
3-6 Ambient air levels of dichloromethane 3-13
3-7 Effects of dichloromethane on freshwater species in acute
tests 3-25
4-1 Pulmonary absorption of DCM by human subjects (sedentary
conditions) 4-5
4-2 Effect of exercise on physiological parameters for volunteers
exposed to DCM 4-7
4-3 Body burdens of rats after inhalation exposure to 14C-DCM for
6 hours 4-9
4-4 DCM concentrations in rat whole blood and plasma at apparent
steady-state conditions of a 6-hour inhalation exposure 4-11
4-5 Tissue concentrations of DCM in rats exposed to 200 ppm DCM for
4 days for 6 hours dai ly 4-12
4-6 Tissue distribution of 14C-activity 48 hours after 6-hour
inhalation exposure or oral dosage of rats to 14C-DCM 4-14
4-7 Comparison of postexposure pulmonary elimination half-times
of DCM for humans and rats 4-16
4-8 Blood carboxyhemoglobin concentrations of rats exposed
to CO and DCM by inhalation 4-22
4-9 Fate and disposition of 14C-DCM in rats (412-430 mg/kg),
injected intraperitoneally 4-27
4-10 Fate of 14C-DCM in rats after a single 6-hour inhalation
exposure 4-30
4-11 Body burdens and metabolized 14C-DCM in rats after inhalation
exposure to 14C-DCM 4-30
4-12 Fate of DCM in rats 48 hours after single oral doses 4-32
4-13 lr\ vitro covalent binding of 14C-DCM to microsomal protein
and lipid 4-42
4-14 Comparative covalent binding of DCM, carbon tetrachloride, and
trichloroethylene to lipid and protein in rat hepatocytes 4-43
4-15 Blood COHb and Hb concentrations in rats exposed to DCM 4-52
5-1 COHb concentrations in nonsmokers exposed to DCM at
250 ppm (869 mg/m3) for 5 days 5-6
5-2 Acute lethal toxicity of DCM 5-8
5-3 Summary of cardiotoxic action of 5% dichloromethane 5-11
5-4 Mutagenicity testing of DCM in bacteria 5-28
5-5 Gene mutations and mitotic recombination of yeast 5-38
5-6 Gene mutations in multicel lular eukaryotes jj} vivo 5-40
5-7 Gene mutations in mammalian cells in culture 5-44
5-8 Tests for chromosomal aberrations 5-46
5-9 Tests for sister-chromatid exchange 5-49
5-10 Analytical analysis of methylene chloride 5-54
5-11 Cumulative percent mortality of rats 2-year methylene chloride
inhalation study 5-56
5-12 Summary of total tumor data for rats administered methylene
chloride for 2 years by inhalation 5-60
VI I
005DC1/E 12/22/83
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LIST OF TABLES (continued)
Table Page
5-13 Time-to-tumor, palpable mass, and histopathology data for
salivary gland region sarcomas in individual male rats exposed
to methylene chloride by inhalation for 2 years 5-61
5-14 Summary of salivary gland region sarcoma incidence in male
rats in a 2-year inhalation study with methylene chloride 5-63
5-15 Cumulative percent mortality of hamsters, 2-year methylene
chloride i nhalati on study 5-64
5-16 Summary of total tumor data for hamsters administered methylene
chloride by inhalation for 2 years 5-68
5-17 Monthly mortality data for male rats on a 2-year inhalation
toxicity and oncogenicity study 5-73
5-18 Monthly ,nortality data for female rats on a 2-year inhalation
toxicity and oncogenicity study 5-74
5-19 Non-neoplastic liver lesions in male rats 5-75
5-20 Non-neoplastic liver lesions in female rats 5-76
5-21 Summary of mammary gland tumors in female rats 5-77
5-22 Group assignment of Fischer 344 rats administered monthly
chloride in deionized drinking water for 24 months 5-78
5-23 Mean daily consumption of methylene chloride in 24-month
chronic toxicity and oncogenicity study in Fischer 344 rats ... 5-79
5-24 Incidencp of hepatocellular tumors in male and female Fischer
344 rats administered methylene chloride in deionized drinking
water for 104 weeks 5-81
5-25 Historical control data of liver neoplasia in female
Fischer 344 rats at Hazelton Laboratories America, Inc 5-82
5-26 Pulmonary tumor bioassay in Strain A mice 5-83
5-27 Methylene chloride analyses 5-86
5-28 Observed and expected deaths, 1964-1980, 1964 hourly male
methylene chloride cohorts from Kodak 5-88
5-29 Malignant neoplasms, observed and expected deaths, 1964-1980,
1964 hourly male methylene chloride cohorts from Kodak 5-89
5-30 Selected methylene chloride chronic animal studies 5-104
5-31 Incidence rates of salivary gland region sarcomas in male
Sprague-Dawley rats in the Dow Chemical Company (1980)
i nhalati on study 5-105
5-32 Relative carcinogenic potencies among 54 chemicals evaluated
by the Carcinogen Assessment Group as suspect human
carci nogens 5-110
005DC1/E Vl" 12/22/83
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LIST OF FIGURES
Page
3-1 The effect of oxygen doping of the carrier gas on the ECD
response to several halogenated methanes at a detector
temperature of 300°C 3-21
4-1 Inspired and expired air concentrations during a 2-hour, 100-ppm
inhalation exposure to DCM for a 70-kg man, and the kinetics
of the subsequent pulmonary excretion 4-4
4-2 Plasma levels of DCM in rats during and after DCM exposure
for 6 hours 4-8
4-3 DCM venous blood levels in rats immediately after a single
6-hour inhalation exposure to various concentrations of DCM ... 4-10
4-4 Pulmonary elimination of 14C-DCM following oral administration
to rats of a single dose of 1 or 50 mg/kg (squares) 4-18
4-5 Carboxyhemoglobin concentrations in male nonsmokers exposed to
increasing concentrations of DCM for 1, 3, or 5 h day
for 5 days 4-23
4-6 Carboxyhemoglobin concentrations in rats after inhalation exposure
to increasing concentrations of DCM for single exposures
of 3 hours 4-24
4-7 Blood CO content of rats after 3-hour inhalation exposure with
1000 ppm dichloromethane, dibromomethane, and diiodomethane,
respectively 4-25
4-8 Rates of production of CO from DCM given to rats 4-28
4-9 Enzyme pathways of the hepatic biotransformation of
dihalomethanes 4-35
4-10 Proposed reaction mechanisms for the metabolism of
dihalomethanes to CO, formaldehyde, formic acid, and inorganic
halide 4-36
4-11 Blood COHb level in men during an 8-hour exposure for 5 conse-
cutive days to 500 ppm and 100 ppm DCM 4-46
4-12 Blood COHb concentrations in rats during and after a 6-hour
inhalation exposure to DCM 4-48
5-1 Histogram representing the frequency distribution of the
potency indices of 54 suspect carcinogens evaluated by the
Carcinogen Assessment Group 5-109
005DC1/E 12/22/83
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AUTHORS AND REVIEWERS
Stephen P. Bayard, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
David L. Bayliss, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
I.W.F. Davidson, Department of Physiology and Pharmacology, Bowman Gray
School of Medicine, Winston-Salem, N.C.
John R. Fowle, III, Reproductive Effects Assessment Group, U.S. Environmental
Protection Agency, Washington, D.C.
Mark Greenberg, Environmental Criteria and Assessment Office, U.S.
Environmental Protection Agency, Research Triangle Park, N.C.
Bernard H. Haberman, Carcinogen Assessment Group, U.S. Environmental
Protection Agency, Washington, D.C.
Jean C. Parker, Office of Solid Waste, U.S. Environmental Protection Agency,
Washington, D.C.
Dharm Singh, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
005DC1/E 12/22/83
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The following individuals were asked to review an early draft of this document
and submit comments:
Dr. Joseph Borzelleca
Dept. of Pharmacology
The Medical College of Virginia
Virginia Commonwealth University
Richmond, VA 23298
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center
New York, NY 10016
Dr. Herbert Cornish
Dept. of Environmental and Industrial Health
University of Michigan
Ypsilanti, MI 48197
Dr. I. W. F. Davidson
Dept. of Physiology/Pharmacology
The Bowman Gray School of Medicine
Winston-Salem, NC 27103
Dr. Lawrence Fishbein
National Center for Toxicological Research
Jefferson, AR 72079
Dr. John G. Keller
P. 0. Box 10763
Research Triangle Park, NC 27709
Dr. John L. Laseter
Director, Environmental Affairs, Inc.
New Orleans, LA 70122
All Members of the
Interagency Regulatory Liaison Group
Subcommittee on Organic Solvents
XI
005DC1/E 12/22/83
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Participating Members of the Carcinogen Assessment Group
Roy E. Albert, M.D., Chairman
Elizabeth L. Anderson, Ph.D.
larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.
David L. Bayliss, Ph.D.
Chao W. Chen, Ph.D.
Margaret M.L. Chu, Ph.D.
Herman J. Gibb, B.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.
Robert E. McGaughy, Ph.D.
Dharm V. Singh, D.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
Participating Members of the Reproductive Effects Assessment Group
John R. Fowle III, Ph.D.
Ernest R. Jackson, M.S.
Casey Jason, M.D.
K.S. Lavappa, Ph.D.
Sheila L. Rosenthal, Ph.D.
Carol N. Sakai, Ph.D.
Daniel S. Straus, Ph.D., Consultant
Vicki Vaughan-Dellarco, Ph.D.
Peter E. Voytek, Ph.D.
Gary M. Williams, M.D., Consultant
The Environmental Mutagen Information Center (EMIC) in Oak Ridge, Tennessee,
kindly identified literature bearing on the mutagenicity of DCM. Their initial
report and subsequent updates were used to obtain papers from which the
mutagenicity assessment was written.
Members of the Agency Work Group on Solvents
Elizabeth L. Anderson
Charles H.' Ris
Jean C. Parker
Mark M. Greenberg
Cynthia Sonich
Steve Lutkerihoff
James A. Stewart
Paul Price
William Lappenbush
High Spitzer
David R. Patrick
Lois Jacob
Arnold Edelman
Josephine Brecher
Mick Ruggiero
Jan Jablonski
Charles Delos
Richard Johnson
Priscilla Holtzclaw
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Toxic Substances
Office of Toxic Substances
Office of Drinking Water
Consumer Product Safety Commission
Office of Air Quality Planning and Standards
Office of General Enforcement
Office of Toxics 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
005DC1/E
XI I
12/22/83
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The following individuals attended a review workshop to discuss draft EPA
documents on organic compounds which included an early draft of this
document:
Dr. Mildred Christian
Argus Laboratories
Perkasie, PA 18944
Dr. Rudolf Jaeger
Institute of Environmental Medicine
New York, NY 10016
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center
New York, NY 10016
Dr. Herbert Cornish
School of Public Health
University of Michigan
Ann Arbor, MI 48197
Dr. I. W. F. Davidson
Dept. of Physiology/Pharmacology
The Bowman Gray School of Medicine
Winston-Sal em, NC 27103
Dr. John Egle
Dept. of Pharmacology
Virginia Commonwealth University
Richmond, VA 23298
Dr. John G. Keller
P. 0. Box 10763
Research Triangle Park, NC 27709
Dr. Norman Trieff
Dept. of Preventive Medicine
University of Texas Medical Branch
Galveston, TX 77550
Dr. Thomas Haley
National Center for Toxicology Research
Jefferson, AK 72079
Dr. James Withey
Food Directorate
Bureau of Food Chemistry
Ottawa, Canada
XIII
005DC1/E 12/22/83
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1. SUMMARY AND CONCLUSIONS
DichVoromethane (methylene chloride, DCM) is a solvent that is widely
used for a variety of purposes. Annual production of DCM in the United States
is about 269,000 metric tons. Approximately 85 percent of the DCM 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 DCM but none is believed to contribute significantly to ambient concentra-
tions. Although ambient air and water measurements are rather scarce, they
indicate that DCM 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 DCM in
urban areas could be one or two orders of magnitude higher than background.
DCM is not expected to accumulate in the atmosphere; estimates of half-life
vary from 20 days to 1 year. Hydroxyl radical attack is probably sufficiently
rapid to prevent most, if not all, DCM from reaching the stratosphere.
DCM has been detected in both natural (fresh and seawater) and municipal
waters in various geographical areas of the United States. Concentrations of
dichloromethane in the low parts-per-billion range have been measured in
surface water and drinking water. DCM does not appear to be formed to any
large extent during the chlorination process. DCM's short evaporation half-life
from moving water probably allows most of the compound dissolved in water to
be transported into the atmosphere. DCM also is readily degraded by bacteria
in concentrations up to 400 parts-per-million (ppm). The extent to which DCM
enters groundwater from surface waters is unknown. Some DCM is deposited in
landfills. Where leaching is possible, the compound may enter groundwater
systems because it does not easily adsorb to clay, limestone, and/or peat
moss, and retention in the soil is unlikely. There is no evidence for signi-
ficant bioaccumulation of DCM in the food chain. Data concerning the ecological
consequences of DCM in the environment indicate that it is biodegradable under
both aerobic and anaerobic conditions. DCM appears to have low toxicity to
aquatic organisms.
Ambient is a term used in this and other documents of this nature to
refer to the environment and should not be construed to include indoor and
occupational settings.
005DC1/A 1-1 12/22/83
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As with other solvents of this class, inhalation of DCM in air followed
by lung absorption is the most rapid route of entrance into the body. DCM
also is well absorbed into the body after oral ingestion. Absorption through
the intact skin occurs to some extent, but is relatively a much slower process.
DCM is appreciably more water-soluble and less 1ipid-soluble than its congeners,
carbon tetrachloride and chloroform. Because of DCM's high solubility in
water and lipids, it is probably distributed throughout all body fluids and
tissues. DCM's half-time of elimination from adipose tissue (6 to 6% hours)
is consistent with reports that DCM can be found in such tissue 24 hours after
both single and chronic exposures. DCM readily crosses the blood-brain barrier,
as evidenced by its narcotic effect at higher exposure levels. DCM also
probably crosses the placenta and distributes into the developing fetus, but
studies in experimental animals indicate that effects on the fetus are unlikely
at levels commonly experienced by humans.
Following ingestion, the absorption of DCM is virtually complete. When
ambient air exposure occurs, the amount of DCM absorbed increases in direct
proportion to its concentration in inspired air, the duration of exposure, and
physical activity. Absorbed DCM is eliminated primarily by pulmonary excretion
of the unaltered parent compound (about 85 percent). About 2 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. DCM is known to be metabolized by the liver to
carbon monoxide (CO). Carboxyhemoglobin (COHb) is formed from the interaction
of CO and hemoglobin; CO dissociates at the lung and is eliminated. Blood
normally contains about 0.5 COHb at all times; therefore, CO formed from a few
ppt or ppb of DCM vapor is of no practical consequence. However, at higher
exposure levels (up to 500 ppm and above), the COHb level would be expected to
reach a maximum between 12 and 15 percent in man. This level is below that
considered hazardous for normally healthy individuals, but could place addi-
tional stress on people with compromised cardiovascular systems.
The effects of COHb from DCM metabolism are additive to COHb formed from
exogenous CO. The toxicities of CO and DCM can differ markedly. However,
persons exposed to levels of DCM that do not exceed the U.S. Occupational
Safety and Health Administration (OSHA) standard of 500 ppm (1737 mg/m3) may
have blood COHb levels that exceed those allowable from direct CO exposure.
005DC1/A 1-2 12/22/83
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The results of animal experimentation by several investigators indicate
that carbon dioxide (C02), formaldehyde, and formic acid are additional meta-
bolites of DCM. At least two pathways exist in rat liver for the metabolism of
DCM. Together, the microsome oxidative dehalogenation and cytosol glutathione
transferase dehalogenation systems could account for the CO and C02 generated
from the metabolism of DCM. Neither the microsomal system nor the cytosol
system is inducible by microsomal inducers, such as phenobarbital or DCM.
The weight of evidence from the available literature indicates that
adverse toxicologic effects (other than carcinogenicity and mutagenicity) in
humans are unlikely to occur at ambient air and water levels found or expected
in the general environment or even at higher levels sometimes observed in
urban areas. In fact, available experimental data do not indicate that any
adverse toxicologic effects are induced in humans at a threshold limit value
(TLV®) of 100 ppm (347 mg/m3) DCM. The potential direct adverse health effect
associated with exposure levels that greatly exceed 100 ppm (347 mg/m3) is
primarily neurological. The lowest concentration reported to affect eye-hand
coordination was 200 ppm (694 mg/m3).
Liver and kidney damage is unlikely to occur as a result of DCM exposure
at environmental levels. Hepatotoxicity has not been reported in any human
case report, even following fatal overexposure. Only minimal hepatic changes
were observed in animal studies, even at doses ranging from the average LD50
(about 2 g/kg) to near-lethal levels. Animal studies also indicate that DCM
has low nephrotoxicity.
Direct cardiac effects of DCM in humans also are unlikely because of the
low levels of DCM found in the environment. Animal studies have shown that
acute exposure levels exceeding 20,000 ppm (69,480 mg/m3) are required before
a decrease in myocardial contractility and other effects on cardiac performance
are observed.
Available evidence hints that the teratogenic potential of DCM in experi-
mental animals is minimal. However, a definitive assessment of such potential
in humans can be made only after further testing in appropriate rodent species,
in accordance with current teratologic and reproductive testing methodologies.
The weight of evidence with respect to mutagenic potential shows that DCM
is capable of causing gene mutations and has the potential to cause such
effects in exposed human cells. Positive responses to DCM exposure were
observed in four different microorganisms. However, additional studies are
needed to determine the strength of evidence for mammalian systems.
005DC1/A 1-3 12/22/83
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The evidence for the carcinogenic potential of DCM is based upon six
chronic (lifetime) studies in which DCM was administered to rodent species.
Four of the studies involved rats, one involved mice, and one involved hamsters.
Chronic inhalation studies with rats and hamsters were conducted by the Dow
Chemical Company. One rat study (Dow Chemical Company, 1980) showed a small
increase in the number of benign mammary tumors compared to controls in female
rats at all doses and in male rats at the highest dose, as well as a statisti-
cally significant increased incidence of ventral cervical sarcomas, probably
of salivary gland origin in male rats. In hamsters, there was an increased
incidence of lymphosarcoma in females only; this increase was not statistically
significant after correction for survival. In the second inhalation study in
rats by Dow Chemical Company (1982) there were no compound-related increased
incidences of any tumor type, but the highest dose was appreciably lower than
previously employed. A borderline hepatocellular neoplastic nodule response
in female Fischer 344 rats was observed only in the chronic drinking water
study, which was conducted under the auspices of the National Coffee Association.
However, while the response was significant with respect to matched controls,
the incidence was within the range of historical control values at the per-
forming laboratory.
Two epidemiological studies that focused on DCM have been reported.
Although neither study showed excessive risk to subjects, there were sufficient
deficiencies to prevent the studies from being judged as showing no effect.
The weight of evidence for carcinogenicity in animals is limited, according
to the criteria of the International Agency for Research on Cancer (IARC).
This conclusion is based upon the statistically positive salivary gland sarcoma
response in male rats (Dow Chemical, 1980) and the borderline hepatocellular
neoplastic nodule response in the rat (National Coffee Association, 1982).
The occurrence of limited carcinogenic activity is reason to suggest that
additional testing is warranted to adequately clarify the carcinogenic potential.
When the absence of epidemiological evidence is considered with the limited
animal evidence, the overall evaluation of DCM, according to IARC criteria, is
a Group 3 chemical in that it cannot be classified as to its carcinogenic
potential for humans.
However, note must be made of the aggregate findings for both mutagenicity
and carcinogenicity as a singular adverse response. A positive finding of
gene mutations is usually taken as support for the likelihood of a chemical
having a carcinogenic potential. In this case, the direct carcinogenic evidence
005DC1/A 1-4 12/22/83
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for animals is limited using IARC terminology; however, the positive mutageni-
city findings should be viewed as increasing the concern about the adequacy of
carcinogenicity testing to date. The mutagenicity findings, on the other
hand, when considered in their own right, are a qualitative finding for potential
human adverse effect although further studies are required to clarify the
potential to damage genetic material in man.
RECOMMENDATIONS FOR FURTHER STUDIES
It is apparent that further research is needed in several areas. This
list does not indicate research priorities.
(1) Teratogenicity and Reproductive Effects. To conclusively determine
the teratogenic potential of DCM with respect to humans, it is
desirable to conduct more testing on appropriate rodent species at
various exposure levels.
(2) Mutagenicity. Additional tests for chromosomal aberrations should
be conducted.
(3) Pharmacokinetics. To more fully define the pharmacokinetics so as
to explain the carcinogenic responses or lack thereof and to clarify questions
and hypothesis regarding mutagenicity.
005DC1/A 1-5 12/22/83
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2. INTRODUCTION
Dichloromethane (DCM) is a high-volume industrial chemical widely used
to remove paint, clean metal, and propel aerosol sprays. This document provides
an evaluation of the health effects of DCM and a review of the relevant available
scientific literature. To provide a perspective in evaluating health effects
associated with DCM, this document contains background chapters relating to ana-
lytical methodologies, production, sources and emissions, and ambient air con-
centrations.
DCM is released into the ambient air as a result of evaporation during
its production, storage, and manufacturing or during general consumer use. It
is believed to be derived from natural sources, but such formation is not believed
to contribute significantly to global concentrations.
Information on the effects of DCM has been derived primarily from studies
involving individuals exposed occupationally or accidentally to DCM. In such
exposures, the concentrations of DCM associated with adverse effects to human
health were either unknown or greatly exceeded the concentrations measured in
ambient air. Controlled exposure studies have established that vapor inhalation
is the principal route by which DCM enters the body. DCM is eliminated from the
body primarily as the parent compound via the lungs. DCM is metabolized in ani-
mals and in humans to carbon monoxide (CO), which elevates the carboxyhemoglobin
content of blood. The distribution, storage, and metabolism of DCM to CO and
carbon dioxide (CO;,) help explain its effects upon humans. Epidemiological
studies provide some information about the impact of DCM on human health, but it
is necessary to rely on animal studies to assess any indications of potentially
harmful effects for chronic low-level exposures.
The primary concerns regarding the potential impact of DCM on human health
are narcosis effects associated with acute high-level exposures and any mutagenic
or carcinogenic effects potentially associated with chronic
low-level exposures.
Ambient is used in this and other documents of this nature to refer to the
environment and should not be construed to include indoor and occupational
settings.
005DC1/B 2-1 11-10-83
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The U.S. Occupational Safety and Health Administration (OSHA) health stan-
dard requires that a worker's exposure to DCM at no time exceed a TWAa of 500 ppm
(R)
in the workplace air in any work shift of a 40-hour week. The TLV for an 8-hour
TWA concentration is 100 ppm with a 15-minute allowable excursion for an average
concentration of 500 ppm.
TWA (time-weighted average) is defined as the time-weighted average concen-
tration for a normal 8-hour workday and a 40-hour workweek to which nearly
all workers may be exposed repeatedly, day after day, without adverse effect.
005DC1/B 2-2 11-10-83
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3. DICHLOROMETHANE: BACKGROUND INFORMATION
3.1 PHYSICAL AND CHEMICAL PROPERTIES
Dichloromethane (methylene chloride, DCM) is one member of a family of
saturated aliphatic halogenated compounds. Other common names or synonyms are
shown in 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). The impor-
tant physical properties of DCM are shown in Table 3-2. For example, DCM is
highly volatile (vapor pressure of 350 torr at room temperature). Hence, the
most common mode of entry into the body is by inhalation. The ambient air con-
centration for compounds of this nature is often expressed in parts-per-million
(ppm), parts-per-billion (ppb), and parts-per-trillion (ppt). At standard tem-
perature and pressure, 1 ppm is equivalent to 3.474 mg/m3.
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), DCM tends to carbonize when its vapor
contacts steel and metal chlorides. Moisture initiates hydrolysis of DCM, pre-
dominantly to hydrogen chloride (HC1), with trace amounts of phosgene (Anthony,
1979). However, even trace amounts of phosgene detract from the paint-stripping
qualities of DCM (De Forest, 1979). To retard production of phosgene via hydro-
lysis, inhibitors are generally added to commercial preparations of DCM (De Forest,
1979). Protection against hydrolysis also is attained by the addition of phenolic
compounds (Anthony, 1979).
Although anhydrous DCM 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
against this corrosion (Anthony, 1979). To minimize the decomposition of DCM,
storage containers should be galvanized or lined with a phenolic coating (Anthony,
1979). Commercial 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 as stabilizers (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).
005DC1/C 3-1 12/7/83
-------
TABLE 3-1. SYNONYMS AND IDENTIFIERS FOR DICHLOROMETHANE
Chemical Abstracts Service registry number: 000075092
Chemical formula: CH2C12
Structural formula: Cl
H - C - H
Cl
Synonyms:
Dichloromethane
Methylene dichloride
Methylene bichloride
Methylene chloride
The experimentally determined average evaporative half-life of DCM from
water is 18 to 25 minutes (Oil ling, 1977). In three separate experiments, Dill ing
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 fol-
lowing formula:
t, _ 0.06391d (3-1)
*- ki
where d is the solution depth and kx 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. The calculated evaporative half-
life may not be accurate for DCM in natural aquatic systems.
005DC1/C 3-2 12/7/83
-------
TABLE 3-2. SELECTED PROPERTIES OF DICHLOROMETHANE
Molecular formula
Formula weight
Boiling point (760 mm Hg)
Melting point
Vapor density
Density of saturated vapor
Density
Solubility
Explosive limits in oxygen
Flash point
Autoignition temperature
Relative evaporation rate
Vapor pressure
Conversion factors
(25°C; 760 mm Hg)
Concentration in saturated air
Log octane/water partition
CH2C12
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 to 67% by vol
None
624° to 662°C
14 (water = 1)
71 (ether = 100)
Temp °F Temp °C
50 10
68 20
77 25
86 30
95 35
ume
mm Hg
230
349
436
511
600
1 mg/1 = 1 g/m3 = 288 ppm
1 ppm = 3.474 mg/m3 = 3.474 ng/1
550,000 ppm (25°C)
1.25
Hardie, 1969.
Anthony, 1979.
Weast, 1969.
Dilling (1977) reported values from literature ranging from 19,400 ppm at 25°C
to 22,700 ppm at 1.5°C.
American National Standards Institute Inc., 1970.
Christensen and Luginbyhl, 1974.
bDe Forest, 1979.
005DC1/C
3-3
11-10-83
-------
3.2 ENVIRONMENTAL FATE AND TRANSPORT
Dichloromethane is principally used as an aerosol propellant, degreasing
solvent, paint stripper, and thinner in paints and lacquers. Because of its
volatility and dispersive use pattern, much of the DCM produced worldwide is
emitted into the atmosphere. Almost all of the emissions are from anthropogenic
sources. DCM also is formed from natural sources, but natural sources are not
believed to contribute significantly to global concentration (National Academy
of Sciences, 1978).
3.2.1 Production
Dichloromethane is produced commercially in the United States, predominantly
via the following reaction (De Forest, 1979; Anthony, 1979):
FeCl3 or ZnCl
HC1 + CH.OH 13Qu , 18Qoc * CH3C1 + H20
\3~c.)
CHaCl + Clz » CHi;Clz + HC1.
In this vapor phase reaction sequence, yields of 95 percent are usual.
Dimethyl ether is the secondary byproduct of the hydrochlorination.
A less common method used to obtain DCM is direct reaction of methane with
chlorine at 485^ to 510«*C (Anthony, 1979). Methyl chloride, chloroform, carbon
tetrachloride, and HC1 are coproducts. 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. However,
this method 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 Toxic
Substances Control Act Public Inventory showed that in 1977 there were six manu-
facturers and 13 importers (U.S. EPA, 1980b).
According to statistics gathered by the U.S. International Trade Commission,
the United States annual production of DCM was estimated to be 256,000 metric tons
in 1980 (U.S. International Trade Commission, 1980) and 269,000 metric tons in
1981 (U.S. International Trade Commission, 1982).
Edwards et al. (1982) estimated 1981 world production at 825,000 metric tons.
005DC1/C 3-4 12/7/83
-------
TABLE 3-3. PRODUCERS OF DICHLOROMETHANE
Annual capacity as of
January 1, 1979
Company
Allied Chemical
Diamond Shamrock
Dow Chemical
Dow Chemical
Stauffer Chemical
Vulcan Materials
Vulcan Materials
Location
Moundsville, WV
Belle, WV
Freeport, TX
Plaquemine, LA
Louisville, KY
Le Moyne, AL
Geismar, LA
Wichita, KA
(metric tons x 103)
23
50
92
88
28
37
60
Source: Chemical Marketing Reporter, August 6, 1979, p. 9.
3.2.2 Use
Dichloromethane is used as a paint remover, a urethane foam-blowing agent,
a vapor degreasing and dip solvent for metal cleaning, a solvent for aerosol
products, a solvent in the pharmaceutical industry, a solvent in the manufacture
of polycarbonates by polymerization, and an extractant for caffeine, spices, and
hops. DCM is used in the manufacture of plastics, textiles, photographic film,
and photoresistant coatings, as a solvent carrier in the manufacture of herbi-
cides and insecticides, and as a component of rapid-drying paints and adhe-
sives, 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 by major use is shown in Table 3-4. One of the fastest growing segments of
the DCM market is the aerosol sector because DCM is being substituted for some
chlorofluorocarbons as a solvent, vapor pressure depressant, and flame retardant.
Consumption by the aerosol industry is expected to grow (Chemical and Engineering
News, 1982).
005DC1/C 3-5 12/7/83
-------
TABLE 3-4. CONSUMPTION OF DICHLOROMETHANE
Use
Paint remover
Metal degreasing agent
Aerosol propel! ant
Blowing agent for foams
(urethane)
Exports
Other
1980 metric tons, 103
1980
73
49
46
20
44
12
Total
1977
30
20
19
8
18
5
metric tons, %
1978
29
18
21
9
15
8
Source: SRI International, Chemical Economics Handbook, 1980.
Dichloromethane is expected to retain popularity as a paint remover.
Although DCM competes with trichloroethylene and perchloroethylene as a solvent,
it is preferred as a paint remover because of better performance (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. 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 manufacture and use. Most of the losses in production,
transportation, and storage are fugitive (that is, transient) releases from
leaky pump seals, valves, and joints. The dispersive uses of DCM are varied
and widespread and are distributed geographically approximately with the industri-
alized population in the United States. Although most of the losses are to the
atmosphere, DCM is relatively soluble in water. The most effective means of
removal of DCM from water is air stripping, which transfers the chemical from
water to the atmosphere (National Academy of Sciences, 1978).
005DC1/C 3-6 12/7/83
-------
3.2.4 Persistence of PCM
3.2.4.1 Atmospheric Degradation—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 by irradiating ozone (03), and the resultant
singlet oxygen atoms then react with water vapor. The tropospheric lifetime
of a compound can be related to the OH- concentration by the expression:
1 lifetime = l
k [OH] (3.3)
where k is the rate constant of reaction.
Available evidence indicates that the lifetime of DCM in the troposphere
is less than 1 year. The modelling approaches of Crutzen and Fishman (1977)
and Singh (1977) indicate that the range of the average OH concentration is
between 2 x 10? and 6 x 10? molecules/cm3. Using an average concentration of
3 x 10? molecules cm3, Altshuller (1980) calculated a tropospheric lifetime
for DCM of 1.4 years. The rate constant expression of Davis et al. (1976) and
a tropospheric temperature of 265K were used.
Singh et al. (1979) computed a 1-year lifetime for DCM using the rate
data reported by the National Aeroneutics and Space Administration (NASA,
1977) and the National Bureau of Standards (1978) and a temperature of 265K.
An average OH concentration of 4 x 10? molecules/cm3 was employed in the
computation. More recently, Singh et al. (1983) calculated an atmospheric
residence time of 0.9 ± 0.3 year, based upon a comparison of atmospheric
budget data with available emissions data. Since the mean concentrations in
the northern hemisphere are 1.8 times the southern hemisphere values, a short
lifetime for DCM is indicated.
However, Cox et al. (1976) calculated a 0.3-year lifetime in photokinetic
studies in which DCM competed with nitrous acid as the target of OH attack.
The lifetime was derived from a rate constant of 10.4 x 10 14 cnrVmolecule •
sec at 298K. An average OH concentration of 1 x 10e molecules/cm3 was assumed.
Use of 4 x 10s molecules/cm3 for the OH concentration would have resulted in a
calculated lifetime of 0.76 year; this value is closer to those of Singh
(1977) and Altshuller (1980). Davis et al. (1976) calculated a lifetime of
005DC1/C 3-7 12/7/83
-------
0.39 year from a rate constant of 8.7 x 1014 at 265K and an average OH concen-
tration of 9 x 105 molecules/cm3.
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; Howard and Evenson, 1976; Perry et al., 1976). The values are in general
agreement (Table 3-5) with the exception of Butler et al. (1978).
3.2.4.2 Aquatic Biodegradation—Recent evidence indicates that DCM is bio-
degradable under both aerobic and anaerobic conditions. Brunner and Leisinger
(1978) first reported the isolation of facultative methylotroph with the ability
to utilize DCM as a sole carbon source for growth. The organism was tentatively
identified as a Pseudomonas species. Brunner et al. (1980) observed that utili-
zation of DCM is caused by an inducible enzyme, the activity of which is only
partially inhibited under anaerobic conditions.
Rittman and McCarty (1980) investigated sewage microorganisms for their
ability to biodegrade DCM. Bacterial cultures were enriched from a seed of
primary sewage effluent over a 12-month period. DCM supported bacterial growth
when the mineral salts culture medium was supplemented with sodium bicarbonate.
Klecka (1982) investigated the fate and effects of DCM in a system simula-
ting a municipal waste water treatment plant. After acclimation, the sludge
was used in closed-bottle respirometer studies. Respirometer studies demon-
strated the disappearance of 10 mg DCM/1 (14C-label led) within 4 hours, with
49 percent of the parent compound recovered as 14C~labelled C02 after 50 hours.
At 1 mg DCM/1, disappearance resulted within 3 hours, with a 66-percent conver-
sion of the parent compound to C02 after 50 hours. Rate constants, calculated
for the biodegradation of 1, 10, and 100 mg/1 by activated sludge, were 1.42,
1.61, and 0.35/hour, respectively.
In inhibition studies on the effect of DCM on oxygen consumption and
organic carbon degradation by activated sludge, Klecka (1982) found no signifi-
cant effect over a 24-hour period. Modelling of a continuously mixed activated
sludge reactor indicated that the rate of biodegradation was about 12 times
greater than the rate of volatilization.
Wood et al. (1981) demonstrated the degradation of DCM under anaerobic
conditions, using sediment-water samples spiked with 200 ug DCM. Degradation
was observed to proceed via methyl chloride, although accumulation in the sam-
ples was not observed.
005DC1/C 3-8 12/7/83
-------
TABLE 3-5. REACTION RATE DATA FOR OH +
(k x 10 14cmVmolecule • sec) Temperature, K
Arrhenius expression
Reference
CO
14.5 ±2.0
15.5 ± 3.4
11.6 ± 0.5
8.7
10.4
2.7 ± 1
298.5
296
245 to 375
265
298
302
4.27 ± 0.63 x 10"12
exp [-(1094 ± 81/T)]
5.2 x 10~1*exp(-1094/T)
Perry et al., 1976
Howard and Evenson, 1976
Davis et al., 1976
Cox et al., 1976
Butler et al., 1978
NASA,3 1977; National
Bureau of Standards,
1978
NASA preferred value; reliability of log k judged to be ± 0.2 at 230K.
-------
3.2.5 Products of PCM
3.2.5.1 Atmospheric Simulation Studies—Pilling et al. (1976) observed that DCM
was not very reactive in a chamber atmosphere containing nitric oxide (NO) or
nitrogen dioxide (N02). Ozone-air mixtures containing 10 ppm (34.7 mg/m ) DCM
and 5 ppm NO or 16.8 ppm NO^ were exposed to ultraviolet (UV) radiation 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 DCM had disappeared. Similarly, less than 5 percent dis-
appeared in an N02 atmosphere after a 7.5-hour exposure. The effect of varying
UV intensity or concentration on the rate of photodecomposition was not investi-
gated. Relative humidity in the photolysis reactor was 35 to 40 percent.
Further investigation of this simulated trospheric reaction showed anomalous
behavior, possibly occurring on the chamber walls (Dilling and Goersch, 1980).
Dichloromethane was judged to contribute to oxidant formation less than other
halogenated compounds investigated, e.g., tetrachloroethylene, trichloroethylene,
and vinyl chloride.
Butler et al. (1978) proposed that the OH attack on DCM in the presence of
02 may result in formation of phosgene (COC12) via the reaction sequence
CH2C12 + OH -> -CHCl^ + H20
•CHC12 + 02 •* -CHC1202 (3-4)
•CHC1202 •* COC12 + OH.
This pathway was suggested to account for a low rate constant of the reaction of
OH with DCM in the presence and absence of CO in the test atmosphere. Production
of C02 was followed.
Chlorine-sensitized photooxidation of DCM in the presence of C12 in dry air
resulted in CO and CO^ as the major carbon-containing products (Spence et al . ,
1976). After 5 minutes of irradiation of 20 ppm DCM and 5 ppm C12 in air, 19
ppm CHi-Cl^, was consumed. The product distribution was CO (5 ppm); HC1 (38
ppm); phosgene (2 ppm); formyl chloride (0 ppm); and C02 (12 ppm). The product
distribution is illustrative of a chain reaction:
•CHC1202 •* CIO + HCOC1
HCOC1 + Cl -» HC1 + COC1
005DC1/C 3-10 12/7/83
-------
COC1 + 02 -» C02 + CIO (3-5)
COC1 •* CO + Cl
Chlorine-sensitized photooxidation of DCM is not expected to be significant
under real atmospheric conditions because 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 DCM is 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).
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 solution
containing DCM. However, the DCM that leaches from landfills adsorbs very little
to clay, limestone, and/or peat moss, so retention in the soil is unlikely.
These authors consider evaporation of DCM from water a more important process
than adsorption.
3.3 LEVELS OF EXPOSURE
Dichloromethane has been detected in ambient air and in surface and
drinking waters at numerous locations throughout the United States.
Dispersion models have been used to estimate population exposure to
ambient DCM. The predicted maximum annual average to which people may be
2
exposed is 14.3 ppb (0.050 mg/m ) from living near DCM production facilities.
People living near other DCM sources such as organic solvent cleaning and
paint and varnish removal operations are expected to be exposed to
concentrations that do not exceed 7.1 to 14.3 ppb (0.025 to 0.050 mg/m )
averaged over a year's time (Systems Applications, Inc., 1983).
Average background mixing ratios are approximately 30 to 50 ppt. In
their review of ambient air data, Brodzinsky and Singh (1983) concluded that
background levels are about 50 ppt, with many urban levels one or two orders of
magnitude higher. Ambient air levels of DCM at various locations are shown in
Table 3-6. Singh et al. (1983) reported that the average northern hemisphere
005DC1/C 3-11 12/7/83
-------
PRELIMINARY DRAFT
TABLE 3-6. AMBIENT AIR LEVELS OF DICHLOROMETHANE
Location Type of site
Date of measurement/
analytical method
Mixing ratio
(ppb)
Reference
Arizona
Phoenix
California
Mill Valley
Riverside
Urban
April 23 - May 6, 1979/
GC-EC
ro
Point Arena
San Jose
Point Arena
Background subject Jan. 11 -27, 1977/
to urban trans- GC-EC
port
Urban
Badger Pass High altitude
Marine coastal
Urban
Marine coastal
Los Angeles Urban
April 25 - May 4, 1977/
GC-EC
May 5-13, 1977/
GC-EC
May 23-30, 1977/
GC-EC
Aug. 21-27, 1978/
GC-EC
Aug. 30
GC-EC
April 9
GC-EC
- 21, 1979/
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
Min 0.033
Avg 0.111 ± 0.094
Max 0.126
Min 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
Singh et al., 1981
- Sept. 5, 1978/ 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., 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1981
-------
PRELIMINARY DRAFT
TABLE 3-6. (continued)
Location
Oakland
Upland*
Kansas
Jetmar
to
,L Louisiana
CJ
Baton Rouge
Geismar
Missouri3
St. Louis
Nevada
Reese River
Date of measurement/
Type of site analytical method
Urban June 28 - July 10, 1979/
GC-EC
Urban Aug. 17 - Sept. 23, 1977/
GC-MS
Remote June 1-7, 1978/
GC-EC
Urban March 3 - May 20, 1977/
GC-MS
Urban March 1, 1977/
GC-MS
Urban May 30 - June 8, 1980/
GC-EC
High altitude May 14-20, 1977/
GC-EC
Mixing ratio
(ppb) Reference
Max 2.4058 Sinqh et al.. 1981
Min 0.0859
Avg 0.4155 ± 0.3146
Max 13 Pellizzari and Bunch ,
Min 0.54 1979
Avg 8.5 ± 4.5
Max 0.105 Singh et al., 1979
Min 0.033
Avg 0.054 ± 0.015
Max 0.55 Pellizzari et al.. 1979
Min 0.046
Ayj 0.25 ± 0.17
Max 0.33 Pellizzari, 1978a
Min 0.33
Avg 0.33 ± 0.062
Max 0.62 Singh et al., 1980
Min 0.16
Avg 0.39 ± 0.14
Max 0.099 Singh et al., 1979
Min 0.015
Avg 0.052 ± 0.022
-------
PRELIMINARY DRAFT
TABLE 3-6. (continued)
CO
I
Location
New Jersey3
Bridgeport
Edison
North Pacific
37°N
0°N - 33°N
Panama
Canal Zone
Southern Hemi
0° to 42°S
77° to 90°S
32° to 55°S
77° to 90°S
Type of site
Urban
Urban
Ocean
Marine
Marine
sphere
Marine
Remote
Marine
Remote
Date of measurement/
analytical method
Sept 22, 1977/
GC-MS
March 24 - July 1, 1977/
GC-MS
April 1977/GC-MS
Oct. 1976/GC-MS
July 1977/GC-MS
Oct. 1976/GC-MS
Jan. 1977/GC-MS
Oct. 1977/GC-MS
Nov. 1977/GC-MS
Mixing ratio
(ppb) Reference
Max 0.26 Pellizzari and Bunch, 1979
Min 0
Avg 0.13 ± 0.19
Max 69 Pellizzari, 1978a; 1977
Min 0
Avg 26 ± 30
Avg 0.030 ± 0.008 Cronn et al. , 1977
Avg 0.033 ± 0.046 Robinson, 1978
Max - Cronn and Robinson,
Min - 1979
Avg 0.034
Avg 0.035 ± 0.003 Robinson, 1978
Avg 0.034 ± 0.004
Avg 0.040 ± 0.002
Avg 0.033 ± 0.001
-------
PRELIMINARY DRAFT
TABLE 3-6. (continued)
Location Type of site
Texas
Aldine* Suburban
Houston* Urban
Virginia*
Front Royal Urban
Washington
Pullman Rural
Pullman Rural
Date of measurement/
analytical method
June 22 - Oct. 20, 1977/
GC-MS
June 28, 1977, -
May 24, 1970/GC-MS, GC-EC
Sept. 29 - Nov 16, 1977/
GC-MS
Dec. 1974 - Feb. 1975/
GC-MS
Nov. 1975/GC-MS
Mixing ratio
(ppb) Reference
Max 1.30 Pellizzari et al . , 1979
Min 0.29
Ayj 0.84 ± 0.50
Max 1.30 Pellizzari et al., 1979
Min 0 Singh et al. , 1980
Ayg 0.57 ± 0.32
Max 21 Pellizzari, 1978b
Min 0.49
Avg 8.5 ± 4.5
Max - Grimsrud and Rasmussen,
Min - 1975
Avg <0.005
Avg 0.035 Rasmussen et al., 1979
GC-EC = gas chromatography-electron capture.
GC-MS = gas chromatography-mass spectrometry.
*Data obtained from summary report of Brodzinsky and Singh, 1983.
-------
background mixing ratio is approximately 38 ppt and the global average is 29 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 coupled with analysis by high resolution
gas chromatography-mass spectrometry (GC-MS). The highest DCM concentration
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 (November 1977), 55 ± 0.1 ppb DCM 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 7.25-hour period, about 70 ppb DCM were detected at a site in Front
Royal, Virginia (October 1977). In the southwest, a level of about 1 ppb was
reported at sites in Houston, Texas, during a 3-hour sampling period (October
1977). During considerably longer sampling periods (up to 24 hours), lower
concentrations were reported for sites in Louisiana, e.g., Baton Rouge and
Geismar. Sampling during a 48-hour period in Upland, California (August 1977)
indicated concentrations of about 12 ± 9 ppb DCM.
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.
(2) Gas chromatography-mass spectrometry.
(3) Long-path infrared absorption spectroscopy, usually with preconcen-
tration of whole air and then separation of the compounds by gas
chromatography.
(4) Infrared solar spectroscopy, using the solar spectrum at large
zenith angles to obtain greatest 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.
005DC1/C 3-16 12/7/83
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The two most widely used systems for identifying and measuring trace amounts
of DCM that occur in ambient air are gas chromatography-mass spectrometry (GC-MS)
and gas chromatography-electron capture detection (GC-EC). Both systems have a
limit of detection below 30 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 hydrocarbons 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 concentra-
tion 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 concentration. 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
Contamination, absorption, and adsorption are common problems of the methods
used to analyze air and water for DCM content. Four general approaches are used
to collect samples of air for analysis of trace gas concentration: (1) cryogenic
sampling in which liquid helium or liquid nitrogen is used to cool a container
to extremely low temperatures; (2) pump-pressured samples, in which a mechanical
pump is used without cryogenic assistance to fill a sampler to a positive pres-
sure relative to the surrounding atmosphere; (3) 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; and (4) adsorption
of selected gases on adsorbants such 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. DCM aqueous samples must be care-
fully sampled, transported, and stored because of its volatility and the com-
plexity of the samples, especially those containing chlorine or other oxidants.
A technique that is often used involves filling and sealing a serum bottle with-
out air space and storing it just above freezing (Kopfler et al., 1976). Water
samples generally require additional preparation before analysis. Normally, such
samples may be concentrated by various water analysis techniques, but direct
aqueous injection is used occasionally in GC analysis.
005DC1/C 3-17 12/7/83
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3.3.2.1 Sampling and Detection in Ambient Air—Several common approaches are
used to sample ambient air for trace gas analysis, including the following
approaches (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
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 and liquefied, and
the partial vacuum that is created allows more air to enter. This method allows
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
®
preconcentrated by freezeout on 100/120 mesh glass beads (use of Tenax monomer
was discontinued because oxygen oxidized the monomer and interfered 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 before 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 et al. (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. For mass spectrometry, the detection limit for DCM
was 9 ppt with a percent standard deviation of 13. The detection limit with
temperature-programmed GC-EC was 4 ppt and the percent standard deviation was 7.3.
005DC1/C 3-18 12/7/83
-------
Pellizzari and Bunch (1979) reported an estimated detection limit of 200
ppt using a high-resolution GC-MS system in which DCM was first adsorbed onto a
®
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 DCM
from the sampling cartridge after a period of storage; and (4) the reproduci-
®
bility of thermal desorption from the Tenax cartridge and its introduction
(R)
into the analytical system. Oxidation of the Tenax monomer was not reported.
Singh et al. (1982) have cautioned that the adsorption of halogenated compounds
®
on Tenax may not reliably reflect ambient air levels because measurements by some
investigators have been reported as less than background (100 to 200 ppt).
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
because of a greater-than-coulometric response. Dichloromethane was reported to
have a very low ionization efficiency. The observed response might be attributed
to the products of ionization having greater electron affinities than the reac-
tants.
Cox et al. (1976) reported that polyglycol stationary-phase chemically
bonded to porous glass (Durasil Low Kl) was the only material found to separate
DCM 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 the percent standard deviation was 26.2. The GC column
contained 10 percent SF-96 on 100/120 mesh Chromosorb W.
The National Institute for Occupational Safety and Health (NIOSH) method
P & CAM 127 (NIOSH, 1974) is recommended for measurement of DCM in samples where
the concentration is greater than 0.05 mg/sample. This method uses 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 02 in the carrier
005DC1/C 3-19 12/7/83
-------
gas. At the highest 02 doping (5 ppth) the response of the detector to DCM
(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 Water—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
®
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 pur-
pose column is an 8-foot by 0.1-inch inside diameter (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/1.
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 results in
an increase in the concentration of certain halomethanes (not including DCM)
upon storage.
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 (1977), DCM formation resulting from chlo-
rination treatment of water was reported.
In a survey for volatile organics in five drinking water supplies, Coleman
et al. (1976) found that DCM was common to all the cities evaluated (i.e.,
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 (EPA) (1975a),
DCM was detected in 9 of 10 water supplies. Lawrence, Massachusetts, had the
highest concentration (1.6 ug/1). A mean concentration of less than 1 ug/1 in
finished water was reported in a survey of Region V water supplies (U.S. EPA,
1975b). This survey indicated 8 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), and it was also found in Mississippi River clarifier effluent. The
005DC1/C 3-20 12/7/83
-------
/ 300°
12345
O2 CONCENTRATION, ppth
Figure 3-1. The effect of oxygen doping of the carrier gas on the ECD response
to several halogenated methanes at a detector temperature of 300°C.
Source: Grimsrud and Miller, 1979.
005DC1/C
3-21
11-10-83
-------
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 DCM 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 ug/1 DCM resulted
after a 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 DCM (8.2 ug/1).
Before chlorination and after preliminary treatment, the water contained 0.8 ppb
DCM (2.9 ug/1). After chlorination, the effluent contained 1 ppb DCM (3.4 ug/1).
These tests show that DCM and other chlorocarbons may have formed as a result of
the chlorination treatment. The most notable chlorocarbon was chloroform, for
which an increase of 7.1 ug/1 to 12.1 ug/1 was observed after chlorination.
GC-MS analysis was performed using a headspace preconcentration technique.
Dichloromethane 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 near major rivers and
57 were in tidal areas and estuaries. Samples (125 ml) were held at 60°C and
(R)
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
Hites (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/1.
005DC1/C 3-22 12/7/83
-------
Data for DCM concentrations in general ambient waters in EPA's Storet files,
covering the period January 1978 to April 15, 1981, indicate that levels ranged
from 0 to 120 (jg/1 (U.S. Environmental Protection Agency, 1981). Sediment sam-
pling detected DCM in 60 of 118 cases. Concentrations were from 427 to 433 ppb.
Ambient soil concentrations of DCM are unknown (U.S. Environmental Protection
Agency, 1981).
Singh et al. (1983) have detected DCM in seawater samples from the eastern
Pacific Ocean. Mean surface concentrations of 2 ng/1 were measured.
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 in this chapter focuses upon observed DCM con-
centrations that were reported to result in adverse effects under laboratory
conditions. Such parameters of toxicity are not easily extrapolated to environ-
mental situations. Test populations may not be representative of the entire
species because susceptibility to the test substance at different lifestages
may vary considerably. Guidelines for the use 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. Environmental Protection Agency, 1980b).
The toxicity of DCM 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 exposes the organism(s) to the added initial concentration 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 3-7.
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 EPA procedures described by the Committee on Methods for
Toxicity Tests with Aquatic Organisms (1975). Alexander et al. reported that
the fish were observed for the following effects: loss of equilibrium, mela-
nization, narcosis, and swollen, hemorrhaging gills. The DCM concentration that
005DC1/C 3-23 12/7/83
-------
PRELIMINARY DRAFT
TABLE 3-7. EFFECTS OF DICHLOROMETHANE ON FRESHWATER SPECIES IN ACUTE TESTS
Species Type of test LC5oa EC5o Reference
Bluegill (Lepomis macrochirus) static 224,000 pg/1 - U.S. EPA, 1980a
Fathead minnow (Pimephales static 310,000 pg/1 - Alexander et al., 1978
promelas)
Fathead minnow (Pimephales flow-through 193,000 jjg/1 - Alexander et al., 1978
promelas)
Daphnia magna static - 224,000 M9/1 U.S. EPA, 1980a
a96-hour test.
b48-hour test.
-------
produced one or more of these observable effects in 50 percent of the fish (EC50)
was 99.0 mg/1 for DCM. Fish affected during the exposure were transferred to
static freshwater aquaria at the end of the 96-hour exposure period. Only the
fish that were severely affected by high concentrations of the chemical did not
recover. However, short exposures to these compounds at the sublethal level seem
to produce only reversible effects.
Chronic test data concerning life cycle or embryo-larval tests are not
available nor were data found on the chronic toxicity to invertebrates.
3.4.1.2 Effects on Saltwater Species—Static tests with mysid shrimp resulted
in an LC50 value of 256,000 ug/1. No data exist on the chronic effects of DCM.
In a 96-hour static test with Sheepshead minnow (Cyprinodon variegatus)
the LC50 value was 331,000 ug/1 (U.S. Environmental Protection Agency, 1980a).
3.4.2 Effects on Plants
The 96-hour EC50 values for DCM, based on chlorophyll a and cell numbers
of the freshwater alga, Selenastrum capricornutum, were greater than 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 greater than
the highest test concentration (662,000 ug/1) (U.S. Environmental Protection
Agency, 1980a).
Few studies on the effects on vascular plants are available (U.S. Environ-
mental Protection Agency, 1981). Lehman and Paech (1972) tested the effect of
DCM vapors on the photosynthetic fixation of 14C02 by alfalfa seedlings. At a
very high concentration (21 percent), DCM reduced photosynthesis by 82 percent.
3.4.3 Bioconcentration Potential
Bioconcentration refers to increased concentration of a substance in the
tissue of an organism (e.g., fish) relative to the ambient water concentration
under steady-state conditions. A measure of the potential for organic chemicals
to bioconcentrate in the fatty tissues of aquatic organisms is given by the
octanol/water partition coefficient (Neely et al., 1974). The log octanol/
water partition coefficient for DCM has been measured at 1.25 (Hansch et al. ,
1975), but no steady-state bioconcentration factor (BCF) has been measured.
The BCF represents the ratio of the chemical concentration in the organism to
that in the water. An approximate BCF for DCM of 5.2 has been calculated
using the relationship Log BCF = 0.76 Log P - 0.23 (Veith et al., 1979). This
estimated BCF places DCM in the low range of bioconcentration values, suggesting
that the potential for bioconcentration in lipids is low. However, no infor-
005DC1/C 3-25 12/7/83
-------
(nation is available concerning DCM levels in living organisms (Pearson and
McConnell, 1975) or the rate of transport of DCM through food chains.
However, the BCF alone may not be the most useful measure of the overall
fate of a substance in water or its potential for producing toxic effects.
Chemical and biological degradation of the substance, volatilization, desorption,
and the depuration rate are among the key determinants of toxicity.
3.5 CRITERIA, REGULATIONS, AND STANDARDS
Permissible levels of DCM in the working environment have been established
in various countries. The OSHA health standard requires that a worker's exposure
to DCM at no time exceed 500 ppm (1,737 mg/m3) TWA in any 8-hour work day of a
40-hr week, with an acceptable ceiling concentration of 1000 ppm (3,474 mg/m3),
that should not exceed 2000 ppm (6,948 mg/m3) for more than 5 minutes in any
2-hour period. The American Conference of Government Industrial Hygiene (ACGIH)
TL\r for inhalation exposure [200 ppm (695 mg/m3)], proposed for prevention of
narcotic effects or liver injury and for protection against excessive carboxy-
hemoglobin formation, has recently been lowered to 100 ppm (347 mg/m3). The
8-hour TWA value in the Federal Republic of Germany is 100 ppm; in the German
Democratic Republic and Czechoslovakia it is 144 ppm; and in Sweden it is 100 ppm.
The acceptable ceiling concentration in the USSR is 14 ppm (49 mg/m3). NIOSH has
recommended that occupational exposure to DCM not exceed 75 ppm (261 mg/m3),
determined as a TWA for up to a 10-hour work day of a 40-hour week, in the
absence of exposure to carbon monoxide above a TWA of 9 ppm for up to a 10-hour
work day.
The Ambient Water Quality Criteria for Halomethanes (U.S. EPA, 1980a) indi-
cates that for the maximum protection of human health from the potential car-
cinogenic effects caused by exposure to any of several halomethanes, including
DCM, chloromethane, bromomethane, bromodichloromethane, tribromomethane,
dichlorodifluoromethane, and/or trichlorofluoromethane, through ingestion of
contaminated water and contaminated aquatic organisms, the ambient water
concentration should be zero based on the assumption of no threshold for these
chemicals. However, zero level may not be attainable; therefore, the levels that
may result in incremental increase of cancer risk over the lifetime are esti-
mated at 10 5, 10 6, and 10 7. The corresponding recommended criteria, based
on 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.
005DC1/C 3-26 12/7/83
-------
For halomethanes where one criterion is derived for an entire class of com-
pounds, 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 5
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. In most cases where halo-
methanes 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 non-carcinogenic risks and
assuming a daily water intake of 2 liters and consumption of 6.5 grams of fish
and shellfish per day (bioconcentration factor 0.91), would be 12.4 mg/1.
005DC1/C 3-27 12/7/83
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Lovelock, J. E. The electron capture detector: Theory and practice. J.
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Lowenheim, F. A., and M. K. Moran. Methylene Chloride. Jji: Faith, Keyes,
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McKetta, J. J. and W. A. Cunningham, eds. , Encyclopedia of Chemical Processing
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National Aeronautics and Space Administration. Chlorofluoromethanes and the
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Council, 1977. pp. 743-745.
National Academy of Sciences. Non-fluorinated halomethanes in the environment.
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Committee for Scientific and Technical Assessments of Environmental
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National Bureau of Standards, Special Publication 513 "Reaction rate and
photochemical data for atmospheric chemistry," 1977, R. F. Hampson, Jr.
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National Institute for Occupational Safety and Health (NIOSH). NIOSH Manual
of Analytical Methods, HEW Publication No. (NIOSH) 75-121, 1974.
Neely, W.B., D.B. Branson, and G.E. Blan. Partition coefficient to measure
biconcentration potential of organic chemicals in fish. Environ. Sci.
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Pearson, C.R. and G. McConnell. Chlorinated Cx and C^ hydrocarbons in the
marine environment. Proc. Soc. London 189:305-332, 1975.
Pellizzari, E. D. Electron capture detection in gas chromatography. J.
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Pellizzari, E. D. Analysis of Organic Air Pollutants by Gas Chromatography and
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Pellizzari, E. D. Measurement of Carcinogenic Vapors in Ambient Atmospheres.
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005DC1/C 3-33 12/7/83
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4. METABOLISM AND PHARMACOKINETICS OF DICHLOROMETHANE
Dichloromethane (DCM) is a colorless liquid with a pleasant smell. The odor
threshold is about 100 ppm (347 mg/m3) (May, 1966; Leonardos, 1969). Because of
its relatively high vapor pressure at room temperatures (350 to 500 torr), DCM is
readily absorbed into the body following inhalation, and the great majority of
severe poisoning from this solvent occurs from inhalation exposures. Reports of
poisoning in man by oral ingestion are rare (Llewellyn, 1966; Stewart and Hake,
1976; Friedlander et al., 1978), and total recovery has followed the swallowing
of quite large doses (Roberts et al., 1976).
Dichloromethane has been used as a general industrial solvent for at least
6 decades (Lehmann and Schmidt-Kehl, 1936), and its narcotic and anesthetic
properties have been known to clinical medicine for over 50 years (Bourne and
Stekle, 1923). However, most of the available information on DCM's metabolism
and pharmacokinetics is derived from studies that were conducted within the last
10 years because of the resurgence of interest in DCM following the demonstration
of its metabolism to carbon monoxide (CO) (Stewart et al., 1972a,b). These stu-
dies in animals and humans have been carried out at relatively high oral or in-
halation concentrations (100 to 1000 ppm) of DCM. Studies of the pharma-
cokinetics of DCM for the low chronic exposure circumstances expected of the
general ambient environment are lacking.
4.1 ABSORPTION, DISTRIBUTION, AND PULMONARY ELIMINATION
4.1.1 Oral Absorption
Absorption of DCM through the intestinal mucosa after oral ingestion appears
to be rapid and complete. McKenna and Zempel (1981) obtained virtually complete
recovery (92 to 96 percent) of 14C-DCM radioactivity in urine, feces, and exhaled
air of rats after single oral (gavage) closes of 1 or 50 mg/kg DCM in water.
Pritchard and Angelo (1982) observed a peak blood concentration occurrence at
less than 10 minutes in B6C3F, mice following an oral gavage dose of 50 mg/kg DCM
in either water or corn oil.
4.1.2 Dermal Absorption
Absorption of DCM through the skin from direct liquid contact or by the
immersion of hands or arms is a slow process. Early animal studies by Schutz
(1958) showed that DCM penetrates the skin and can be absorbed into the body by
this route. Schutz exposed the shaved skin of rats to direct liquid contact
005DC5/B 4-1 11-14-83
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for periods up to 20 minutes and found that kidney damage occurred after only
2 minutes of contact exposure. In contrast, Stewart and Dodd (1964) attempted to
quantify the rate of absorption through human skin by immersing the thumbs of
volunteers into liquid DCM and then determining the appearance and concentration
of DCM in the breath. An estimate of the amount of DCM entering the body was
made by comparison of these measurements with breath concentrations obtained
following controlled inhalation exposures. From these breath analyses, Stewart
and Dodd concluded that DCM is very slowly absorbed. They also suggested that
although the amount of DCM absorbed depends on the area of the exposed skin, the
slow rate of absorption would prevent toxic quantities of DCM from being taken
into the body from direct contact with the skin of the hands and forearms. Immer-
sion in DCM was found to be accompanied by excruciating pain within a few minutes,
which would serve as an effective deterrent.
4.1.3 Pulmonary Uptake
Inhaled DCM rapidly equilibrates across the alveolar endothelium because
of its water and lipid solubility and the very large lung alveolar surface area.
DCM is appreciably more water soluble (2 grams per 100 ml), but less lipid
soluble than its congeners, chloroform and carbon tetrachloride; its lipid
partition coefficient (olive oil/water) is 180 at 37°C with a blood/air co-
efficient of 7.9 (Lehmann and Schmidt-Kehl, 1936; Morgan et al., 1972; Lindqvist,
1978).
4.1.3.1 Studies in Human—The magnitude of DCM uptake into the body (dose, burden)
primarily depends on several parameters: inspired air concentration, pulmonary
ventilation, duration of exposure, the rates of diffusion into blood and tissues
and solubility in blood and 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 in-
spiratory 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 (4-1)
x v insp alvy
005DC5/B 4-2 11-14-83
-------
where Q is the quantity absorbed, C is the concentration (inspired and alveolar)
in mg/1, V is the alveolar ventilation rate in 1/min, and T is the duration of
exposure in minutes. The percent retention is defined as (C. - C , )/C. x
insp alv' insp
100, and percent retention x quantity inspired (V • T -C. ) is equal to uptake.
Figure 4-1 (from Riley et al., 1966) illustrates the overall time-course
of absorption and elimination during 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/m3) alveolar air], and finally a very slow rate occurs as
equilibrium is approached at 70 ppm (243 mg/rn^) 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 con-
centration 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 exponential
alveolar curve correspond to equilibrium attained by first-order passive dif-
fusion of DCM from blood. First, DCM diffuses through a vessel-rich group (VRG)
of tissues with high blood flow (VRG: brain, heart, kidneys, liver, and endo-
crine and digestive systems), then it diffuses more slowly through the lean body
mass (muscle group, MG: muscle and skin) and last through adipose tissues (fat
group, FG). With the termination of exposure, blood and alveolar air DCM concen-
trations 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.
DiVincenzo and Kaplan (1981a) obtained serial breath excretion curves from
four to six volunteers experimentally exposed to 50, 100, 150, and 200 ppm (173,
347, 520, and 694 mg/m^) DCM for 7.5 hours under sedentary conditions. Pul-
monary uptake was rapid during the first hour (exposure was interrupted at
4 hours, for 30 minutes). During post-exposure, DCM levels decreased rapidly
in an exponential manner. Less than 0.1 ppm (0.347 mg/m3) DCM was detected
in the end tidal air of the individuals exposed to 50, 100, or 150 ppm (173,
005DC5/B 4-3 11-14-83
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DURING
EXPOSURE
AFTER EXPOSURE
100
ui
a
E
o
o
ui
z
ui
ROOM CONCENTRATION
RESPIRATORY
ABSORPTION!
ABSORPTION AND EXCRETION
OF METHYLENE CHLORIDE
EXHALED AIR CONCENTRATIONS
.
RESPIRATORY EXC~REtT6N
TIME, hours.
Figure 4-1. Inspired and expired air concentrations during a 2-hour, 100-ppm
inhalation exposure to DCM for a 70-kg man, and the kinetics of
the subsequent pulmonary excretion.
Source: Riley et al. , 1966.
005DC5/B
4-4
11-14-83
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347, or 520 mg/m3) DCM 7 hours after exposure was terminated. For the 200 ppm
(694 mg/m3) exposures, the mean post-exposure end tidal air concentration of DCM
decreased to 1 ppm (3.47 mg/m3) at 16 hours. Post-exposure elimination of DCM
was less than 5 percent of the amount absorbed. A related exposure study in
which volunteers were exposed to 100, 150, and 200 ppm (347, 520, and 694 mg/m3)
DCM for 7.5 hours daily, for 5 consecutive days, also indicated that uptake and
elimination was directly proportional to the magnitude of exposure, thus confirm-
ing previous findings by DiVincenzo et al. (1972) and Stewart et al. (1973).
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 the duration of exposure. Variation in the values
TABLE 4-1. PULMONARY ABSORPTION OF DCM BY HUMAN SUBJECTS
(SEDENTARY CONDITIONS)
Inhalation
concentration,
Investigator ppm
DiVincenzo and Kaplan, 1981a
Lehmann and
Schmidt-Kehl, 1936
Riley et al. , 1966
DiVincenzo et al. , 1972
Astrand et al . , 1975
50
100
150
200
662
806
1,152
1,181
44-680
100
100
200
250
500
Exposure,
hr
7.5
7.5
7.5
7.5
0.3
0.5
0.5
0.5
2
2
4
2
0.5
0.5
Retention,
%
70
60
63
60
74
75
72
72
31
53
41
51
55
55
Engstrom and
Bjurstrom, 1977
750
34
005DC5/B
4-5
11-14-83
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also is caused by differences in body weights of the subjects and differences in
body composition (proportion of adipose tissue to lean mass). For exposures
greater than 1 hour, the mean retention approximates 42 percent of uptake of
DCM or approximately 125 mg/hr for an exposure of 100 ppm (347 mg/m3), assuming
a resting ventilation rate of 6 1/min.
For short exposures, the quantity (dose) of DCM absorbed into the body is
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; DiVincenzo and Kaplan, 1981b). Astrand et al. (1975)
found that physical activity during exposure to 250 and 500 ppm DCM (869 and
1737 mg/m3) for 0.5 hour decreased retention from 55 percent in a resting stage
to 40 percent during activity, but doubled the amount of DCM absorbed because
of a three-fold increase of ventilation rate (6.9 to 11 I/minute). DiVincenzo
and Kaplan (1981b) also found that physical exercise during exposure increased
pulmonary uptake. Exposure of three males to 100 ppm DCM (347 mg/m3) for 7.5
hours, during which they exercised on a treadmill for 5 minutes of each 15-min
period, resulted also in an estimated doubling of average cardiac output and as
much as an eight-fold increase in the average alveolar ventilation rate (Table 4-2).
Effects upon blood carboxyhemoglobin (COHb) and conversion of DCM to CO are dis-
cussed elsewhere in this chapter.
The quantity of DCM absorbed is dependent also on body weight and fat con-
tent of the body. Engstrom and Bjurstrom (1977) showed that for an exposure to
750 ppm DCM (2606 mg/m3) 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 (average body fat is 25 percent
of body weight) absorbed 30 percent more DCM than lean subjects (average body fat
is 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 post-exposure, respectively. These concentrations, although
lower than those found in adipose tissue of lean subjects, represented a great
total storage amount in obese subjects because their total fat 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
005DC5/B 4-6 11-14-83
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TABLE 4-2. EFFECT OF EXERCISE ON PHYSIOLOGICAL PARAMETERS
FOR VOLUNTEERS EXPOSED TO DCM
Work
intensity
(ml 02 min
Volunteer (kg-1)
1
2
3
Source:
4
14
4
15
19
4
16
28
DiVincenzo and
Aerobic
capacity, %
25
25
45
45
70
Kaplan (1981b).
, .
Heart
rate,
beats/mi n
56
84
68
96
119
68
123
145
Estimated
average
cardiac
output,
1/min
5
11
5
12
14
5
15
21
Estimated
average
alveolar
venti lation,
1/min
8
18
6
34
45
6
28
46
* •
to equivalent Og consumption measured during pre-exposure aerobic capacity
testing. Oxygen consumption at rest was taken as (4 ml 02/min • kg) body
weight, based on preexposure testing.
from the FG compartment proceeds at a slow rate. Furthermore, the residual
DCM in adipose tissue is additive to the next day exposure, particularly in
obese people. However, with multiple daily exposure, a total body equilibrium
and a constant adipose tissue concentration are eventually achieved with a
given concentration of DCM in the inspired air.
4.1.3.2 Studies in Animals--McKenna et al. (1982) used 250-gram male Sprague-
Dawley rats to estimate the total body burden of DCM resulting from a 6-hour
inhalation exposure of 50, 500, and 1500 ppm (173, 1737, and 5211 mg/m^) of
14C-DCM. After 6 hours, the animals were in apparent total body equilibrium
with the inhaled concentrations of radioactive DCM as suggested by "plateau"
blood concentrations (Figure 4-2). The body burden was calculated from the
total radioactivity recovered from exhaled air, urine and feces, and carcass
analysis, during the first 48 hours after exposure. Table 4-3 shows the body
burden associated with each exposure. The increase in body burden of 14C-acti-
vity was less than proportionate to the increments of DCM inhaled concentration.
005DC5/B
4-7
11-14-83
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10.0
o
O
M
O
<
A 50 ppm
• 500 ppm
• 1500 ppm
I I I I I I I
0.01 —
0.001
Figure 4-2. Plasma levels of DCM in rats during and after DCM exposure
for 6 hours. Data points represent mean ± standard deviation
for two to four rats.
Source: McKenna et al., 1982.
005DC5/B
4-8
11-14-83
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TABLE 4-3. BODY BURDENS OF RATS AFTER INHALATION
EXPOSURE TO 14C-DCM FOR 6 HOURS
Exposure
concentration,
ppm
50
500
1500
Number of
rats
3
3
3
Total body burden
mg Eq 14ODCM/kg
± S.D.
5.53 ± 0.18
48.41 ± 4.33
109.14 ± 3.15
Source: McKenna et a!., 1982.
S.D. = standard deviation.
4.1.3.3 Blood/Air Relationship—The blood concentration of DCM during inhal-
ation 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 750 ppm DCM
(869 and 2606 mg/m^) for 1.5 hours, the arterial blood (mg/1) to alveolar
air (mg/1) concentration ratios were constant and averaged 10.3 and 11.1,
respectively, over three-fold changes in alveolar concentrations. These
jjn vivo Ostwald coefficients agree with the value found for blood/air (7.9)
and for water/air (7.2) (Lindqvist, 1978; Morgan et al., 1972) at 37°C in vitro.
The high water/air coefficient suggests that DCM is dissolved in plasma water
as well as in lipid components of blood.
In the exposure study of DiVincenzo and Kaplan (1981a) described previously
(Section 4.1.3.1), exposure and post-exposure blood concentrations of DCM were
directly proportional to the magnitude of exposure. Blood/air coefficients
calculated from the data suggest that the 200 ppm (694 mg/m^) exposure concen-
tration may be approaching that level at which saturation of metabolism occurs,
as evidenced by an increased level of DCM in venous blood.
MacEwen et al. (1972) determined the blood DCM concentration in dogs that
were continuously exposed for 16 days to 1000 and 5000 ppm DCM (3470 and 17,370
mg/ma). The blood DCM concentrations 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
005DC5/B 4-9 11-14-83
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two exposure concentrations agree with the above values noted in man. Similar
values can be calculated from the data of Latham and Potvin (1976); Figure 4-3
shows the proportional relationship they found in rats between DCM blood and
inspired air concentrations over a range of 1,000 to 8,000 ppm (3,474 to
27,792 mg/m3) during a 6-hour exposure.
In contrast to these findings in man and other animals of a direct pro-
portional relationship between inspired air concentration of DCM and blood
level, McKenna et al. (1982) reported a greater than proportionate increase of
blood concentration with inhalation exposure concentration in rats. Table 4-4
gives the apparent steady-state concentration of DCM in plasma and whole blood
of Sprague-Dawley rats exposed for 6 hours each to 50, 500, and 1500 ppm DCM (173,
o>
fc 0.60
o
CM
U
O 0.40
ui
_i
O
O
O
_J
CO
0.20
INHALATION
1000 2000 4000 8000
EXPOSURE CONCENTRATION, ppm
Figure 4-3. DCM venous blood levels in rats immediately after a single
6-hour inhalation exposure to various concentrations of DCM.
Source: Latham and Potvin, 1976.
005DC5/B
4-10
11-14-83
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TABLE 4-4. DCM CONCENTRATIONS IN RAT WHOLE BLOOD AND PLASMA
AT APPARENT STEADY-STATE CONDITIONS OF A 6-HOUR INHALATION EXPOSURE
Exposure DCM concentration Plasma/blood
concentration, Number of pg/ml ± S.D. distribution
ppm
50
500
1500
rats
3
3
3
Whole blood
0.22 ± 0.04
-
39.53 ± 3.71
Plasma
0.05 ± 0.01
2.38 ± 0.42
8.94 ± 0.39
coefficient
0.23
-
0.23
Source: McKenna et al., 1982.
S.D. = standard deviation.
1737, and 5211 mg/m3). The data indicate that the whole blood and plasma levels
of DCM increase disproportionately with an increase in inspired air concentra-
tion. Furthermore, calculation of the blood/air ratio for these data provide
increasing values of 5.75, 5.97, and 7.59 for 50, 500, and 1,500 ppm (173,
1737, and 5211 mg/m3), respectively. McKenna et al. suggest that the resultant
increase in blood DCM concentration is greater than that predicted by increments
in the inspired air because of a rate-limited metabolism of DCM in the rat. Thus,
at low inspired air concentrations (below saturation of metabolism), the blood/air
ratio is less (because of rapid metabolism) than that at high inspired air con-
centrations (above saturation, of metabolism); and indeed, the blood/air coeffi-
cient (7.59) observed at 1,500 ppm DCM (5,211 mg/m3) agrees with coefficients
observed by others.
4.1.4 Tissue Distribution
Because of DCM's water solubility, it probably distributes throughout the
body water, and its lipid solubility allows its distribution into all body
tissues and cellular lipids and particularly into adipose tissue. Engstrom and
Bjurstrom (1977) determined that the Ostwald coefficient for subcutaneous
adipose tissue from human buttocks is 51 at 60°C; this value indicates that
the tissue/blood partition coefficient may be about 7 at body temperature
(37°C).
DCM 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/m3) (Winneke and Fodor, 1976; Winneke, 1981).
005DC5/B 4-11 11-14-83
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DCM also crosses the placenta and may affect fetal development (Schwetz et al.,
1975; Anders and Sunram, 1982).
4.1.4.1 Animal Studies—Tissue concentrations of DCM increase with exposure
concentrations and duration and, for any given tissue, are dependent on the
largely unknown tissue partition coefficients. Savolainen et al. (1977)
exposed rats chronically to DCM [200 ppm (695 mg/m3), 6 hours daily, for 5
days] and determined DCM concentrations in peri renal fat and other tissues.
The resulting data, shown in Table 4-5, indicate that significant amounts of DCM
remained in peri renal fat 18 hours after the previous exposure of day 4, and
markedly increased further with a 6-hour exposure on day 5. At termination of
the last 6-hour exposure (fifth daily exposure), the ratio of tissue to blood
concentrations was about one for brain and liver, but 6.6 for perirenal fat.
Previous work by DiVincenzo et al. (1972) in man and Carlsson and Hultengren
(1975) in rats indicated that little uptake by adipose tissue occurs. However,
single, short exposure periods of 2 hours were used in their studies, and of the
smaller amount absorbed, 95 percent was probably accomodated in VRG and MG com-
partments because the FG compartment receives only 5 percent of the cardiac out-
put.
TABLE 4-5. TISSUE CONCENTRATIONS OF DCM IN RATS EXPOSED TO
200 ppm DCM FOR 4 DAYS FOR 6 HOURS DAILY3
Exposure
DCM concentrations
nmoles/g tissue wet weight ± S.D.
on the fifth
day, hr
0
2
3
4
6
Tissue to
Cerebrum Cerebellum
-
73 ±20 57 ± 20
119 ± 33 86
57 ± 8 95 ± 8
83 90
blood coefficient after the fi
Blood Liver
-
90 ± 10 85 ± 2
79 ± 3 82 ± 1
120 ± 10 101 ± 13
100 ±1 83 ± 10
fth daily exposure
Perirenal
fat
113 ± 29
526 ± 94
537 ± 33
608 ± 58
659 ± 77
0.83:1 0.90:1 1.0:1 0.83:1 6.59:1
aSavolainen et al., 1977.
S.D. = standard deviation
005DC5/B 4-12 11-14-83
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McKenna and coworkers (1981; 1982) have studied DCM distribution in tissues
of rats by observing 14C-activity 48 hours after a single 6-hour inhalation of
50, 500, or 1500 ppm DCM (173, 1737, and 5211 mg/rn^) and after single oral doses
of 1 or 50 mg/kg. The results are shown in Table 4-6. Following the inhalation
exposures and the oral dosage, the highest concentrations of 14C-activity were
found in the liver, kidney, and lung. The observed 14C-activity in epididymal
fat was consistently lower than that observed in either whole blood or the
remaining carcass. The tissue 14C-activity increased with dose, but it did not
increase in direct proportion with increasing dose. Intact DCM was not detected
in any of the tissues assayed; therefore, the observed radioactivity is presumed
to represent nonvolatile metabolites of DCM. These results contrast with those
of Savolainen et al. (1977), who found that in perirenal adipose tissue, signifi-
cant amounts of intake DCM remained 18 hours after the last of four daily 6-hour
inhalation exposures to 200 ppm DCM (895 mg/m3) (Table 4-5).
The results of the experiments of Savolainen et al. and McKenna et al.
indicate that total body equilibrium of DCM to inspired air concentration is not
achieved in the rat within a single 6-hour inhalation period even though this
response is suggested by the achievement of a "plateau" blood concentration as
shown in Figure 4-2. Of interest also is the evident difference between the
amount of tissue 14C-metabolites associated with inhalation and oral dosage.
A close correspondence exists for tissue metabolites of DCM for an oral dose
of 50 mg/kg and a 6-hour inhalation exposure of 50 ppm, although 50 ppm
(173 mg/m^) provides a terminal body burden of only 5.5 mg/kg (Table 4-3).
4.1.5 Pulmonary Elimination
Pulmonary excretion is the mechanism of elimination of virtually all
unchanged DCM from the body. Less than 2 percent of estimated body doses of
DCM have been detected as unchanged compound in the urine of human subjects
exposed to 100 ppm and 200 ppm (347 and 695 mg/m^) for 2 hours (DiVincenzo et
al., 1972) and in the urine of dogs exposed to 5000 ppm (17,370 mg/m^)
(MacEwen et al., 1972).
4.1.5.1 Studies in Humans—Figure 4-1 shows schematically the time-course of pul-
monary elimination of DCM after inhalation exposure. The parameters of elimin-
ation equilibration of the body are the same as those of assimilation equili-
bration. After termination of exposure, DCM immediately begins to be eliminated
005DC5/B 4-13 11-14-83
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from the body via the lungs. Alveolar air equilibrates with pulmonary venous
blood whose concentration becomes a function of the first-order diffusion of
DCM from tissues, the arterial blood flow/tissue mass, and the relative solu-
bilities of DCM in tissues. Figure 4-1 shows that alveolar DCM concentration
follows an exponential decay curve with three major components reflecting
desaturation of the VRG, MG, and FG compartments, respectively. The half-
times of elimination of DCM from these compartments have not been firmly
established. Riley et al. (1966) measured expired air concentration after
termination of exposure and found half-times of 5 to 10 minutes for the
VRG compartment, 50 to 60 minutes for the MG compartment, and 400 minutes
for the FG compartment. 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
TABLE 4-6. DISTRIBUTION OF 14C-ACTIVITY IN TISSUE 48 HOURS AFTER 6-HOUR
INHALATION EXPOSURE OR ORAL DOSAGE OF RATS TO 14C-DCM
Exposure method
mgEq 14C-Activity by exposure
concentrations, tissue ± S.D.
Inhalation
Liver
Kidney
Lung
Brain
Epididymal fat
Skeletal muscle
Testes
Whole blood
Remaining carcass
Oral
Liver
Kidney
Lung
Brain
Epididymal fat
Skeletal muscle
Testes
Whole blood
Remaining carcass
50 ppm
8.4 ± 1.5
3.3 ± 0.1
1.9 ± 0.2
0.8 ± 0.3
. 0.5 ± 0.2
1.1 ± 0.1
1.1 ± 0.2
1.1 ± 0.2
1.3 ± 0.2
1 mg/kg
0.40 ± 0.04
0.15 ± 0.01
0.99 ± 0.01
0.04 ± 0.06
0.02 ± 0.002
0.08 ± 0.02
0.04 ± 0.002
0.06 ± 0.003
0.05 ± 0.002
500 ppm
35.6 ± 7.5
16.2 ± 2.4
11.0 ±1.3
4.2 ± 1.3
6.5 ± 0.5
4.4 ± 1.9
5.5 ± 1.3
8.1 ± 1.9
5.6 ± 0.9
50 mg/kg
6.67 ± 0.69
2.92 ± 0.21
1.67 ± 0.16
0.63 ± 0.10
0.33 ± 0.0006
0.86 ± 0.04
0.92 ± 0.10
1.41 ± 0.11
0.99 ± 0.60
1500 ppm
44.2 ±3.5
30.5 ± 0.2
16.5 ±1.6
6.7 ± 0.2
4.1 ± 0.9
7.7 ± 0.7
8.1 ± 0.5
8.9 ± 1.7
8.6 ± 1.4
Source: McKenna et al., 1982; McKenna and Zempel, 1981.
S.D. = standard deviation.
t
Number of animals in each group = 3
005DC5/B
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little vapor" reached the fat stores and muscle tissues under these conditions.
DiVincenzo et al. found DCM to have a half-time value in blood of 40 minutes
following 2 hours of exposure and prolonging exposure to 4 hours had no signi-
ficant effect on the half-time. DiVincenzo and Kaplan (1981a,b) have recently
extended previous studies by following the course of pulmonary elimination in
individuals exposed for 7.5 hours to 50, 100, 150, and 200 ppm DCM (173, 347,
520, and 694 mg/m3) and those exposed for 7.5 hours daily for 5 consecutive days
to the three highest concentrations. The authors concluded that post-exposure
elimination of DCM in breath is a minor route of elimination over the concentra-
tion range used; post-exposure elimination was less than 5 percent of the amount
absorbed. Pulmonary elimination of DCM was rapid at all exposure concentrations.
Less than 0.1 ppm (0.347 mg/m3) was detected in post-exposure breath samples at
7 hours. Die-away of DCM in blood paralleled that observed with pulmonary eli-
minations. Breath elimination times were prolonged by physical exercise. 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 was about 60 minutes, and
they obtained a longer time value for obese subjects. For both lean and obese
subjects, biopsies indicated that residual concentrations of DCM existed in adi-
pose tissue nearly 24 hours after exposure, suggesting that the half-time of
elimination from the FG compartment is fairly long. From the post-exposure
alveolar concentration curves prepared by Stewart et al. (1976a) for subjects
exposed to inhalation concentrations of 50 to 500 ppm DCM (173 to 1,736 mg/m3)
for 1 to 7.5 hr/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 guesses from these studies for the half-times of elimination
from the VRG, MG, and FG are 8 to 23 minutes, 40 to 80 minutes, and 6 to 6.5
hours, respectively. The long half-time from elimination of the adipose tissue
compartment and reports that DCM remains in this compartment 24 hours after sin-
gle and chronic exposures indicate that the concentration of DCM in adipose
tissue may only slowly, over a period of days, achieve equilibrium with inspired
air concentration, particularly of daily exposures of short duration.
4.1.5.2 Studies in Animals--The estimates of half-times of pulmonary excretion
of DCM in animals are derived from postexposure alveolar concentration curves.
Blood DCM decay concentration curves are a less reliable parameter of pulmonary
005DC5/B 4-15 11-14-83
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excretion because these curves are influenced by metabolism, the other major
route of elimination of DCM. McKenna et al. (1982) determined the pharmaco-
kinetic half-times for elimination of DCM from plasma of rats following a single
inhalation exposure to 500 or 1500 ppm (1737 or 5211 mg/m3) for 6 hours. Figure
4-2 shows that two first-order processes governed the disappearance of DCM from
plasma with apparent half-lives of 2 and 15 minutes for the rapid and slow com-
ponents of elimination, respectively. The kinetic parameters calculated from
these curves are summarized in Table 4-7.
The kinetic parameters for rats differ greatly from those of man. Presumably,
the half-times for the fast and slow components correspond to the half-times for
VRG and MG compartments of man. The half-time for the FG or adipose tissue com-
partment was not established in the rat because plasma decay was only followed
for 1.5 hours following inhalation exposure (Figure 4-2). The rates of elimi-
nation of DCM from plasma in the rat are first order and independent of dose,
although there is considerable evidence of extensive metabolism in these animals
(Section 4.2).
TABLE 4-7. COMPARISON OF POST-EXPOSURE PULMONARY ELIMINATION
HALF-TIMES OF DCM FOR HUMANS AND RATS
Subject
Exposure DCM
method concentration
^ first-order components (minutes)
Alpha Beta Gamma
Human
Rat
Inhalation 50-500 ppm
8-23
Inhalation
500 ppm (6 hr)
40-80
360-390
Rat Gavage
1500 ppm (6 hr) 2.4
1.06
1 mg/kg
50 mg/kg
14.8
15.5
12.6
12.6
-
~"
46.5
46.6
McKenna and Zempel (1981) also have determined the kinetics of pulmonary
excretion of DCM in rats after a single oral dose (via gavage) of 1 or 50 mg
14C-DCM per body weight. Figure 4-4 shows the time course (for 5 hours) of DCM
005DC5/B
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11-14-83
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in exhaled air of these rats. Pulmonary elimination of DCM of rats receiving 1
mg/kg dose is characterized by a two-component decay curve representing two
first-order processes with half-times of elimination of 12.6 minutes and 45.6
minutes, respectively. The pulmonary excretion of DCM of rats given the 50
mg/kg dose approximates zero-order kinetics for the first hour after admini-
stration (possibly rate-limited by absorption) and then describes the same
first-order kinetics of the 1 mg/kg dose. These results suggest that the
half-time of pulmonary elimination from the FG compartment for rats approxi-
mates 45 minutes, versus 6 to 6.5 hours for man. However, as noted for rats,
single inhalation or oral exposures in man probably do not result in total body
equilibrium, particularly with the fat compartment, and elimination from this
compartment may be longer than indicated from these experiments.
Withey and Collins (1980) determined the kinetics of elimination of DCM
from the blood of Wistar rats after intravenous administration of 3, 6, 9, 12,
or 15 mg/kg of DCM given intrajugularly in 1 ml of water. For the four lower
doses, the blood decay curves best fitted a first-order two-compartment model,
but the highest dose fitted a first-order three-compartment model. Withey and
Collins suggested that the apparent change from a two- to three-compartment
system occurred as a consequence of a "difference in biological response to
the dose either as a consequence of the magnitude of the dose or, in some
cases, due to the varying response of different animals to the same dose."
This interpretation is probably true because the k (rate constant for elimi-
nation from blood out of body, principally via pulmonary excretion and/or meta-
bolism) was relatively high for the lower doses and consistent with a half-time
of body elimination of only 4.5 minutes. Any significant equilibration of blood
DCM with the fat compartment was very unlikely during these experimental circum-
stances. Further support for limited distribution of DCM is the reported kinetics
for the highest dose (15 mg/kg) that fit a three-compartment system. In this
case, k was 0.09/min, that is, a half-time of body elimination of 7.7 minutes.
This result reflects that more time was required for equilibration into fat to
occur. The half-time of elimination from the fat compartment was given as 31.5
minutes, although the rate constant k5l [movement of DCM from the fat compartment
to the central compartment (blood)] was given as 0.006/min, indicating a half-
time of 115 minutes. The volume of distribution (Vd) of DCM was calculated as
48.8 ml or about 12 percent of body weight, which was surprisingly low for both
a water- and lipid-soluble compound that is known to diffuse into all the major
organ systems.
005DC5/B 4-17 11-14-83
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100.0
10.0
i
s>
3 1.0
(0
o
o
0.1 —
0
234
TIME, hours
Figure 4-4. Pulmonary elimination of 14C-DCM following oral administration
to rats of a single dose of 1 or 50 mg/kg 14C-DCM (squares).
Ordinate is percent dose administered during 0.5-hour collection
periods with means ± SEM for groups of three rats. SEM is the
standard error of the mean.
005DC5/B
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4.2 METABOLISM OF DCM
DCM is known to be metabolized in man to CO and in experimental animals to
CO, carbon dioxide (C02), formaldehyde, and formic acid. The CO production results
in an elevation of blood COHb content, from which CO dissociates in the lung,
followed by elimination of CO. Experiments in man and animals have shown that
the metabolism of DCM is dose-dependent and limited by hepatic enzyme saturation.
4.2.1 Evidence for Metabolism to Carbon Monoxide
Before 1972, most of the absorbed dose of DCM in man was thought to be
excreted unaltered in exhaled air, while a small amount was found in the urine
(MacEwen et al., 1972; DiVincenzo et al., 1972; Heppel et al., 1944). Metabolism
of DCM to CO was not known to occur. However, in 1972, Stewart et al. discovered
that the COHb concentration in blood increased in persons exposed to 200 to
1000 ppm DCM (695 to 3474 mg/m3) from 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 associates
(1972a,b) 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, thus indicating that these dihalomethanes also are metabolized
to CO. At first, this unique halocarbon metabolism was not generally accepted,
because the increased COHb levels might reflect a change in the rate of endo-
genous 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). However, Stewart et al. (1972a,b, 1973) observed no evidence
of an enhanced metabolism of hemoglobin in their subjects exposed to DCM 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, which increased CO
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) used X-ray diffraction to demonstrate 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
005DC5/B 4-19 11-14-83
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(Hb). He observed that CO binding to human Hb in vitro at 37°C is increased in
the presence of DCM (1000 ppm, 3474 mg/m3). The amount of COHb formed at a
given CO concentration is doubled. Also, measurement of the P50 values of Hb
(partial pressure of 02 or CO at which 50 percent saturation occurs at 37°C) in
the presence and absence of DCM (1000 ppm) indicated a six-fold increase in CO
affinity. From these observations, Settle (1975) suggested that the increased
COHb seen in vivo may be caused by an increase in CO affinity and not to meta-
bolism. More recently, Collison et al. (1977) determined the Haldane affinity
constant for CO for both human and rat blood equilibrated at 37°C with air con-
taining only CO and CO plus DCM (10,000 ppm). No difference in the Haldane con-
stants for human blood (mean value, 227) or rat blood (mean value, 179) was
found. Negative results also were obtained when the absolute affinity of CO was
measured in a nitrogen-DCM atmosphere. Dill et al. (1978) redetermined the P50
value for human and rat blood in the presence and absence of CO (2500 ppm) and
DCM (800 ppm). DCM was found to have no effect on the P50 values; therefore,
Dill concluded that DCM does not significantly increase the affinity of CO for
hemoglobin.
The biotransformation of DCM to CO and CO^ has now been confirmed by many
metabolism studies. The hepatic metabolism of DCM has been unequivocally
shown to be the origin of the CO responsible for increased COHb blood con-
centrations. Independently, several groups have shown by administering 13C-DCM
or 14C-DCM to rats that labeled CO subsequently appears in COHb with essentially
the same specific activity (Carlsson and Hultengren, 1975; Miller et al.,
1973; Kubic et al. , 1974; Zorn, 1975). Furthermore, Fodor and coworkers
(1973, 1976) and Kurppa et al. (1981) have demonstrated that rats exposed to
CO and DCM, singly and in combination, effected an additive increase in
blood COHb levels (Table 4-8). In addition, many investigators have shown a
dose-response relationship between injected or inhaled DCM and increased blood
COHb levels in both experimental animals and men (Figures 4-5 and 4-6) (DiVincenzo
et al., 1972; Astrand et al. , 1975; Fodor et al., 1973; Ratney et al. , 1974;
Stewart et al., 1973, 1976a,b; Forster et al., 1974; Roth et al., 1975; Ciuchta
et al., 1979; Hake et al., 1974).
The extensive metabolism of halogenated hydrocarbons to CO is apparently
unique to the dihalomethanes. This metabolism is not observed to any significant
extent with chloroform, carbon tetrachloride, methyl chloride, methyl iodide,
trichlorofluoromethane, dichlorodifluoromethane, carbon disulfide, formaldehyde,
005DC5/B 4-20 11-14-83
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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, bromoform, and iodoform are meta-
bolized to CO in the rat, thus increasing blood COHb levels; this has not been
the observation of other investigators. According to Fodor and Roscovanu (1976),
of the dihalomethanes, the bromo-iodo-halides are more extensively metabolized
to CO than is DCM (Figure 4-7), 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).
4.2.2 Evidence for Dose-Dependent Metabolism of DCM: Michaelis-Menten Kinetics
4.2.2.1 Studies in Humans—McKenna et al. (1980) have reported in abstract form
kinetic studies carried out in six healthy male volunteers exposed to 200 and
350 ppm DCM (694 and 1214 mg/m3) during each of two 6-hour exposure periods.
These investigators report that the metabolism of DCM was dose-dependent, based
on comparison of the kinetics of the two doses during and following exposure.
Comparison was made of the DCM blood levels and of the concentrations of DCM
in expired air. Blood COHb levels and exhaled CO concentrations were less
than those expected for the 350 ppm (1214 mg/m3) exposure. Calculations made
of the rate of DCM metabolism during exposures were consistent with Michaelis-
Menten kinetics for DCM metabolism.
More recently, DiVincenzo and Kaplan (1981a) evaluated the conversion of
DCM to COHb and CO in sedentary, nonsmoking individuals (11 males and 3 females)
exposed to DCM levels of 50, 100, 150, or 200 ppm (173, 347, 520, or 694 mg/m3),
for 7.5h, or for 7.5 h daily at 100, 150, or 200 ppm (347, 520, or 694 mg/m3)
for 5 consecutive days. Between 25 and 34 percent of the absorbed DCM was
excreted in expired air as CO, during and after exposure. The authors estimated
that as much as 70 percent of the inhaled vapor was absorbed; less than 5% was
expired as unchanged DCM. Materials balance indicates that as much as 70% of
the amount absorbed may be converted to C02. Blood COHb levels increased
directly with the magnitude of exposure and did not reach a plateau after 7.5
hours of exposure. At 200 ppm (684 mg/m3), the peak blood COHb level was 6.8
percent. At the recommended TLV for DCM for 7.5 h, COHb levels were about
3 percent less than the increase in blood COHb levels produced by an exposure
to CO at its recommended TLV of 35 ppm. DiVincenzo and Kaplan (1981b) also
observed that physical exercise resulted in higher blood levels than those found
with sedentary individuals. Physical exercise (Table 4-2) increased absorption
of DCM, the biotransformation of DCM to CO, blood COHb levels, and pulmonary
005DC5/B 4-21 11-14-83
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TABLE 4-8. BLOOD CARBOXYHEMOGLOBIN CONCENTRATIONS OF RATS
EXPOSED TO CO AND DCM BY INHALATION
Exposure concentration j>pm
DCM CO
100 (0.5 - 2)a
1000 (0.5 - 2)a
0 100
100 100
1000 100
None None
100
1000
1000 100
COHb
saturation
%
6.2
12.5
10.9
16.4
19.0
0.7 ± 0.2b
8.8 ± 1.9b
6.2 ± 0.9b
14.6 ± 1.3b
aAmbient air CO concentration.
Mean ± standard deviation, N= 5/group.
Source: Fodor et al., 1973; Kruppa et al., 1981.
excretion of CO. Individuals were exposed to 100 ppm DCM (347 mg/m3) for 7.5
hours. The authors concluded that workers performing physical exercise while
exposed to DCM at the recommended TLV of 100 ppm (347 mg/m3) are unlikely to
/a
exceed the COHb biological TLV suggested by the National Institute of Occupational
Safety and Health (NIOSH).
4.2.2.2 Studies in Animals--DiVincenzo and Hamilton (1975) provided the first
information on the extent to which DCM is metabolized in rats. These investi-
gators injected rats intraperitoneally with 14C-DCM in corn oil and determined
fate and disposition of radioactivity in exhaled air, urine, feces, and carcass
1, 8, and 24 hours after single doses ranging from 412 to 930 mg/kg. Volatile
005DC5/B 4-22 11-14-83
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100 200 300 400
INHALED AIR, ppm.
500
Figure 4-5. Carboxyhemoglobin concentrations in male nonsmokers exposed to
increasing concentrations of DCM for 1, 3, or 5 h/day for 5 days.
Pre-exposure values averaged 0.8%, but with 3- and 7.5-hour expo-
sures the pre-exposure values were above this baseline value on
the mornings following exposure.
Source: Stewart and associates (1972, 1973, 1974).
005DC5/B
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200
400 600
INHALED AIR, ppm
1000
Figure 4-6. Carboxyhemoglobin concentrations in rats after inhalation exposure
to increasing concentrations of DCM for single exposures of 3 hours.
The values are corrected for pre-exposure COHb concentration and
calculated from the data of Fodor et al., 1973.
005DC5/B
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Figure 4-7. Blood CO content of rats after 3-hour inhalation exposure with
1000 ppm dichloromethane, dibromomethane, and diiodomethane,
respectively.
Source: Fodor and Roscovanu, 1976.
005DC5/B
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compounds in exhaled air were collected, identified, and quantified by gas chro-
matography (GC) and radiotracer assay. Recovery of radioactivity was essentially
100 percent 24 hours after administration. About 98 percent of total radio-
activity was eliminated in exhaled air, and less than 2 percent was eliminated
in urine or feces (Table 4-9). Some 90 percent of injected DCM was eliminated un-
metabolized in exhaled air. Most of this elimination (85 percent) occurred within
2 hours. Only 2 percent of the dose was metabolized to CO, 3 percent to C02, and
1.5 percent to an unidentified volatile compound (Table 4-9). 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 meta-
bolized DCM found by DiVincenzo and Hamilton (1975) could be caused by the
high dose of DCM used; i.e., metabolic transformation of DCM may be limited by
DCM excretion being more rapid than metabolism. Rodkey and Collison carried
out balance studies with small doses of 14C-DCM administered to rats (17
mg/kg) by either inhalation or intraperitoneal injection. The animals were
placed in a closed rebreathing system that trapped C02 and CO after conversion
®
to C02 by passing through a catalyst bed of Hopcalite . About 76 percent of
14C-radioactivity was recovered as 14CO (46.9 percent) and 14C02 (28.9 percent).
The remaining 24 percent could have been exhaled as unchanged 14C-DCM, because
no radioactivity was recovered in carcass tissues. Their results also showed
that DCM is directly metabolized to CO without isotopic dilution. The extent of
the conversion was surprisingly great at this low dose, and it is independent of
the mode of administration. For each mole of DCM metabolized, about 0.5 mole of
CO and 0.3 mole of C02 were produced.
In a second experiment, Rodkey and Collison (1977) investigated the rela-
tionship 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 with a CO trap. DCM 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 4-8 shows the rates
of CO production for various doses of DCM. The initial rates (about 25 umole/
005DC5/B 4-26 11-14-83
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TABLE 4-9. FATE AND DISPOSITION OF 14ODCM IN RATS
(412-430 mg/kg) INJECTED INTRAPERITONEALLY
Percent of dose (averages)
2 hr 8 hr 24 hr
Breath
Unchanged 14C-DCM 84.5 94.0 91.5
14CO 0.14 1.43 2.15
14C02 0.55 1.53 3.04
Unidentified 14C 0.40 0.80 1.49
Total 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)
Source: Divincenzo and Hamilton (1975).
hr/kg body weight) are similar for all doses, but for lower doses they progres-
sively decrease after 1 to 2 hours to the endogenous rate as the DCM dose is
metabolized. For a very high dose of DCM (69 mg/kg), CO was produced at a nearly
constant rate over a 6-hour period (COHb, 44 percent). These observations sug-
gest a saturation of the metabolizing 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. For lower doses, the moles
of CO produced per mole of DCM were similar and averaged 0.48; at a high DCM dose
(69 mg/kg), the ratio was 0.62 after 10.5 hours of exposure, suggesting that sub-
strate-induced enzyme formation may occur with long exposure to high doses of DCM.
Similar results were obtained with germ-free rats obviating intestinal bacteria
005DC5/B 4-27 11-14-83
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200
150
8
§
1
o
o
I
100
50
793
I
I
I
246
TIME AFTER CH2d2 ADDITION, hours
Figure 4-8. Rates of production of CO from DCM given to rats. Each curve
represents changes above endogenous CO rate after the dose
(in umoles/kg body weight) was given by inhalation.
Source: Rodkey and Collison, 1977.
005DC5/B
4-28
11-14-83
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as a source of CO. In normal rats, inhibited methane production by intestinal
bacteria. The same results also were obtained with dibromomethane, dichloro-
methane, bromochloromethane, and diiodomethane in respective order of magnitude
and rate of CO production.
Rodkey and Collison's (1977) important finding that the metabolism of DCM
to CO in rats is rate-limited by enzyme saturation to about 25 pmole/hr/kg body
weight (b.w.) explains the seemingly low conversion observed by DiVincenzo and
Hamilton (1975) in this same species. Recalculation of their data (Table 4-5)
gives a DCM metabolism to CO of about 19 pmole/hr/kg b.w. Comparable results
for mice were also obtained by Yesair et al. (1977). These investigators ad-
ministered 1.0 and 100 mg/kg 14C-DCM in corn oil by intraperitoneal injection.
Exhaled 14CO, 14C02, and unmetabolized 14C-DCM were trapped (14CO after oxida-
®
tion with Hopcalite to 14C02 in aqueous potassium hydroxide, and 14C-DCM on
coconut charcoal) and quantified by GC and radiotracer assay. The 1 mg/kg dose
(11.76 pmole/kg) was quantitatively metabolized to CO (0.45 mole/mole DCM) and
C02 (>0.50 mole/mole DCM). In the exhaled air collected for 12 hours, the
larger dose (11.76 pmole/kg) yielded 470 (jmole/kg of unmetabolized 14C-DCM
(40 percent dose), 0.20 mole 14CO, and 0.25 mole 14C02. Hence, in mice under
these experimental conditions, a 12 umole/kg dose of DCM (1 mg/kg) does not
saturate the metabolizing enzymes, whereas 1200 pmole (100 mg) DCM/kg saturates
the enzymes and is metabolized at a constant rate of about 20 pmole/hr/kg b.w.
The remainder of the dose is excreted unchanged in the exhaled air.
More recently, McKenna et al. (1982) exposed rats to 50, 500, and 1500 ppm
14C-DCM (174, 1737, and 5211 mg/m3) for 6 hours. They also found that the net
uptake and metabolism of DCM to CO and C02 did not increase in direct proportion
to the incremental increase of DCM exposure concentration (Table 4-10). Further-
more, increasing amounts of unchanged DCM in exhaled air were found with in-
creasing exposure concentration (Table 4-10). At the end of the 6-hour exposure,
the body burden of DCM was calculated from total radioactivity recovered during
the first 48 hours following exposure, and the body burdens had not increased to
the incremental increase of DCM exposure concentration (Table 4-11).
McKenna et al. estimated the amount of DCM metabolized during each in-
halation exposure by subtracting the unchanged DCM recovered in expired air
(Table 4-10) from the calculated body burdens. The results are summarized in
Table 4-11.
005DC5/B 4-29 11-14-83
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TABLE 4-10. FATE OF 14ODCM IN RATS AFTER A SINGLE 6-HOUR
INHALATION EXPOSURE
% body burden (x ± S.D., n=3)
i a i ainc uc i
measured
Expired CH2C12
Expired C02
Expired CO
Urine
Feces
Carcass
Skin
Cage wash
50 ppm
5.42 ± 0.73
26.20 ± 1.21
26.67 ± 3.00
8.90 ± 0.39
1.94 ± 0.19
23.26 ± 1.62
6.85 ± 1.62
0.75 ± 0.33
500 ppm
30.40 ± 7.10
22.53 ± 4.57
18.09 ± 0.81
8.41 ± 0.90
1.85 ± 0.68
11.65 ± 1.87
6.72 ± 0.13
0.24 ± 0.23
1500 ppm
55.00 ± 1.92
13.61 ± 1.20
10.23 ± 1.68
7.20 ± 0.74
2.33 ± 0.05
7.24 ± 0.65
3.97 ± 0.15
0.43 ± 0.15
Source: McKenna et al., 1982.
Mean ± standard deviation, number of animals in each group = 3.
TABLE 4-11. BODY BURDENS AND METABOLIZED 14C-DCM
IN RATS AFTER INHALATION EXPOSURE TO 14C-DCM
Exposure
concentration
50 ppm
500 ppm
1500 ppm
Total body burden,3
mgEq 14CH2CI2/kg
5.53 ± 0.18
48.41 ± 4.33
109.14 ± 3.15
Metabolized 14CH2CI^,a
mgEq 14CH2CI2/kg
5.23 ± 0.32
33.49 ± 0.33
49.08 ± 1.37
Metabolized
14C-DCM, %
94.6
69.2
45.0
Values are mean standard deviation
Source: McKenna et al., 1982.
Number of animals in each group = 3
Of particular interest is the finding that the percent of body burden meta-
bolized decreased with the increase of body burden or increase of the level of
DCM exposure. McKenna et al. plotted the data in Table 4-11 in accordance with
the following linear form of the Michaelis-Menten equation:
005DC5/C 4-30 11-14-83
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dC = _K . dC/ , + v (4-2)
dt m dt/ 3 vmax
where dC/dt is the (jg/kg DCM metabolized during the fixed time of the experiment
and S is the exposure concentration in parts per million. Estimates of V and
ITlaX
K , as determined from the y-intercept and slope, were used with a computer pro-
gram of the Michaelis-Menten equation to derive the values V = 65.55 ± 2.54
^ max
mg/kg DCM metabolized, and K = 493 ± 57 ppm DCM. Deviation from first-order
3
kinetics occurred at about 250 ppm (867 mg/m ) or one-half k . Therefore,
zero-order kinetics with saturation of DCM metabolizing capacity can be ex-
pected at two to three times Km> or between 1000 and 1500 ppm of inhaled DCM
(3,470 and 5,211 mg/m3). These values correspond to a body burden of about
100 mg/kg for the rat (Table 4-11); therefore, they are in general accord with
the observations of Rodkey and Coll i son (1977) for the rat and Yesair et al.
(1977) for the mouse.
Methods of determining the kinetic behavior of DCM metabolism during or
after inhalation exposure are subject to unknown error from the indirect
methods of estimating both body burden and amount or rates of metabolism.
McKenna and Zempel (1981) investigated the kinetics of DCM metabolism in the
rat after doses of 1 or 50 mg/kg 14C-DCM. Table 4-12 gives the disposition
of DCM after single oral doses in terms of 14C-equivalent.
The results of this study clearly indicate that the metabolism of DCM in
rats after oral dosing is dose-dependent. Rats given a 1 mg/kg dose metabolized
a greater percentage of the oral dose (88 percent) than those given a 50 mg/kg
dose (28 percent). Moreover, the rates of pulmonary elimination of unmetabolized
DCM (Figure 4-4) and of the metabolites C02 and CO were first-order and were
essentially unaffected by the dose despite large differences in the amounts of
DCM and of metabolites excreted. Therefore, the dose-dependent fate of DCM is
caused by the saturation of metabolic pathways. Comparison of the near-saturating
inhalation body burden (100 mg/kg) (Table 4-11) shows a fair correspondence.
In summary, the metabolism of DCM is dose-dependent and follows Michaelis-
Menten kinetics in the rat and mouse. In these species, saturation of metabolic
capacity with zero-order kinetics (about 25 mg/kg/hr) occurs at about 50 to
100 mg/kg orally or 1000 ppm DCM (3,470 mg/m3) in inspired air for an inhalation
period of 6 hours. At doses of 1 mg/kg orally or 50 ppm (173 mg/m3) inhalation,
90 percent is metabolized, and at enzyme saturation doses 30 to 45 percent is
005DC5/C 4-31 11-14-83
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TABLE 4-12. FATE OF DCM IN RATS 48 HOURS AFTER SINGLE ORAL DOSES
Percent 14C-DCM dose by dose
Parameter concentration3
Measured 1 mg/kg50 mg/kg
Expired CH2C12 12.33 ± 1.43 72.09 ± 0.07
Expired C02 35.01 ± 0.85 6.33 ± 0.39
Expired CO 30.92 ± 1.67 11.87 ± 0.07
Urine metabolites 4.52 ± 0.05 1.96 ± 0.05
Feces 0.93 ± 0.02 0.25 ± 0.02
Carcass 5.84 ± 0.24 2.40 ± 0.24
Skin 1.56 ± 0.05 1.15 ± 0.06
Cage wash 0.53 ± 0.04 0.08 ± 0.01
Source: McKenna and Zempel, 1981.
aValues are mean ± standard deviation. Number of animals in each group = 3.
metabolized. Dose-dependent metabolism of DCM occurs also in man; at 100 to
200 ppm (347 to 694 mg/m3) inhalation concentration, 50 to 60 percent of the
body burden is metabolized, as judged from retention values of DCM determined
in man (Table 4-1). Experimental studies in man suggest that as much as 95 per-
cent of the absorbed dose may be metabolized at these exposure levels.
4.2.2.3 Effect of Dosing Vehic1e--Pritchard and Angelo (1982) have described
a physiologically-based pharmacokinetic model (Bishoff model) for mice and
used it to simulate the distribution, metabolism, and elimination of DCM after
both acute and chronic dosing. Preliminary results indicate that the kinetics
are dependent on the route and vehicle used for administration. Adminis-
tration of DCM in water by oral gavage or by intravenous injection yields
similar blood and tissue profiles, but administration in 50 percent polyethylene
glycol/water shows a rapid blood elimination and a slow liver elimination, while
oral gavage with corn oil as a vehicle shows a slower rate of tissue clearance
than that found with a water vehicle.
Withey et al. (1982) have investigated the absorption of DCM in fasting
,rats following oral gavage of equivalent doses (125 mg/kg) in 4 ml of water or
005DC5/C 4-32 11-14-83
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corn oil. The post-absorptive peak blood concentration averaged three times
higher for a water vehicle than for corn oil (121 ug/ml vs. 44 (jg/ml), while the
time to peak blood concentration averaged three times longer for corn oil than
for the water vehicle (16.3 minutes vs. 5.2 minutes). Aside from these differ-
ences in vehicle-mediated absorption, absorption was apparently rapid with both
vehicles and occurred to comparable extent because the ratio of the areas under
the blood concentration curves averaged 1.25 for water:oil. The post-absorption
kinetics of blood elimination of DCM associated with the corn oil vehicle also
was extended (tj for oil was 49 minutes vs. 32 minutes for water). These vehicle-
's
related differences in absorption, kinetics of elimination, and tissue clearance
are probably related to the differences of absorption and diffusion of water and
corn oil across the gastrointestinal (GI) tract mucosa. In contrast to aqueous
absorption into the portal system and then to the liver, corn oil and other lipids
are extensively transported via the mucosal lymphatic system, which slowly drains
by way of the left lymphatic thoracic duct at the junction of the internal jugu-
lar and subclavian veins and hence into the systemic circulation via the superior
vena cava. The absorption, first-pass metabolism, tissue distribution, and eli-
mination kinetics probably are greatly affected by the volume of lipid vehicle
as well as its nature when introduced into the stomach of a rat. While these
considerations are unlikely to affect the pharmacokinetics of DCM in man in any
practical way, they are of importance in relation to the modes of dosing employed
in long-term carcinogenicity tests of DCM and other lipophilic compounds.
4.2.3 Enzyme Pathways of DCM Metabolism
Figures 4-9 and 4-10 summarize current knowledge of the enzyme pathways
involved in the biotransformation of DCM and other dihalomethanes. The scheme
is based on studies in vivo and in vitro with hepatocytes and with microsomal
and cytosol preparations. The preponderance of evidence from in vivo experiments
indicates that these enzyme pathways are unique to the dihalomethanes and give
rise to both CO and C02 in nearly equimolar amounts. However, CO is an end pro-
duct of microsomal oxidation, while C02 is an end product of cytosolic enzyme
systems via the metabolism of formaldehyde and formic acid (Figure 4-9).
4.2.3.1 Microsomal Oxidation—The primary reaction of the dihalomethanes appears
to be an oxidative 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 NADPH and molecular 02 for maximal activity. These experiments
005DC5/C 4-33 11-14-83
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MICROSOMAL
MIXED
FUNCTION
OXIDASE
NADPH
CYTOSOL
GLUTATHIONE
TRANSFERASE
BINDING TO CELLULAR
MACROMOLECULES
HCHO
COHb
CO
PULMONARY
ELIMINATION
Figure 4-9. Enzyme pathways of the hepatic biotransformation of dihalomethanes.
005DC5/C
4-34
11-14-83
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Microsomal Pathway
MFO
NADPH
-H
-X
>
Covalent
OH
Nonenzymatic
rearrangement
t
.sH Spontaneous
f ^ ^
binding < - ^»
lipid ^0 Decomposition
protein ~H, -X
Cytosolic Pathway
formyl haTide
CO
X—
GS - C
/*CU «^ r*C ^ /M i V
bon "* uo ^*LUpA
transferase S-halomethy1gluthathi one
HOH Nonenzymatic
.. hydrolysis
GS CH2OH
S-hydroxymethyl gluthathione
NAD
formaldehyde
dehydrogenase
\
H2C=0 + GSH
formaldehyde
S-formyl gluthathione
HOH
S-formyl gluthathione
hydrolase
+ GSH
formic acid
Figure 4-10.
005DC5/C
Proposed reaction mechanisms for the metabolism of dihalomethanes
to CO, formaldehyde, formic acid, and inorganic halide.
Source: Ahmed et al., 1980.
4-35
11-14-83
-------
were carried out in a closed vessel, substrates were added without carrier sol-
vent, and CO was determined by the gas chromatographic headspace method. With
dibromomethane as the substrate, 3.6 moles of bromide were produced per mole of
CO. In the absence of NADPH, microsomal fractions dehalogenated the methanes
without CO formation. 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 di-
chloromethane, while diiodomethane yielded the greatest amount (seven times DCM).
Liver microsomes were 5 times more active than lung microsomes and 30 times more
active than kidney microsomes. Hogan et al. (1976) also found DCM to be con-
verted to CO by rat liver microsomes requiring aerobic conditions and a NADPH
generating system. These workers noted a high correlation between in vitro CO
production and microsomal cytochrome P450 content.
Further evidence of the participation of the P45d mixed-function oxidase
system in the metabolism of dihalomethanes is the observation that dibromo-
methane and dichloromethane added to microsomal cytochrome P450 preparations
produce type 1 binding spectra (Kubic and Anders, 1975; Cox et al., 1976).
However, Cox et al. (1976) found that the affinity for P456 is less for DCM
(K,, 10 mM) than for chloroform (1C, 3 mM) or carbon tetrachloride (K<-, 1.5
mM) although carbon tetrachloride and chloroform do not give rise to signifi-
cant amounts of CO in 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 in vivo pretreatment induced additional
in vitro CO production, while cobaltous chloride, which depletes microsomal
cytochrome P456» reduced CO microsomal production. Furthermore, SKF 525A,
ethylmorphine and hexobarbital (type 1 substrates) inhibited in vitro micro-
somal conversion of dibromomethane to CO (Kubic and Anders, 1975).
Kubic and Anders and coworkers (Kubic and Anders, 1978; Ahmed and Anders,
1978; Stevens and Anders, 1978, 1979) have studied the mechanism of the reaction
of DCM with deuterium and with 1802. The reaction shows a prominent deuterium
isotope effect with a comparative rate of about 12 percent of that of the hydrogen
isotope, indicating that carbon-hydrogen bond breakage is the rate-limiting step.
Studies with 1802 showed that the oxygen appearing in CO is derived from molecular
oxygen rather than from water. On the basis of these studies, Kubic and
Anders and coworkers proposed the reaction mechanism shown in Figure 4-10.
005DC5/C 4-36 11-14-83
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P45o~roediated hydroxylation of dihalomethanes yields the intermediate, hy-
droxydihalomethane (XgCHOH), which rearranges spontaneously to a formyl halide
(XCHO) with the loss of one halogen atom. The resulting formyl halide is known
to readily decompose to yield CO.
4.2.3.2 Cytosolic Pathways—In addition to being metabolized to CO, DCM is
also converted to formaldehyde, formic acid, inorganic halide, and CO^. 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 9000-gram supernatant fraction of rat liver and by liver slices and
homogenates. The system did not require 0% Dut was glutathione-dependent. Kubic
and Anders (1975) more recently confirmed these findings and localized this
metabolic pathway to the cytosol. Ahmed and Anders (1976, 1978) extended these
findings and showed that the cytosol system is a glutathione transferase that is
found only in the liver and it requires no cof actor other than glutathi one
(cysteine is not a substitute) and is not inducible by phenobarbital or by
repeated exposure to DCM or dibromomethane. Furthermore, the reaction was
inhibited by reagents that react with sulfhydryl groups, such as diethyl maleate
and parachloromercuribenzoate, as well as known substrates for glutathione trans-
ferases. The substrate order of activity is diiodo > dibromo = bromochloro >
dichloromethane; this order is the same as that found for oxidative dehalo-
genation by Kubic and Anders (1975). This pathway probably does not contribute
to CO production via a metabolism of formaldehyde to CO because formaldehyde
administration does not produce an increase of COHb in animals or humans (Kubic
et al., 1974; Rodkey and Collison, 1977; Kasselbart and Angerer, 1974).
The cytosolic pathway has been investigated in detail by Ahmed and Anders
(1976, 1978), who have proposed the reaction sequence shown in Figure 4-9.
These workers observed that both dibromomethane and bromochloromethane have
identical kinetic constants, suggesting that the initial and rate-limiting step
is a displacement of halide, with glutathione (GSH) interaction, to give the
conjugate S-halomethylglutathione. This conjugate is postulated to undergo rapid
nonenzymatic hydrolysis to produce S-hydroxymethylglutathione, and thus yield
formaldehyde and regenerate glutathione. Because the addition of nicotinamide
005DC5/C 4-37 11-14-83
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adenine dinucleotide (NAD ) to incubation mixtures decreased yields of formalde-
hyde (Ahmed and Anders, 1976), the postulated intermediate, S-halomethylgluta-
thione, a known substrate of hepatic cytosolic enzyme formaldehyde dehydrogenase,
also undergoes conversion of S-formylglutathione, and with enzymatic hydrolysis
(S-formyl glutathione hydrolase) yields formic acid and regenerated GSH. Ahmed
and Anders (1978) found that removal of formaldehyde dehydrogenase from cytosolic
fraction by precipitation with ammonium hydroxide fractionation resulted in pro-
duction of only formaldehyde. Thus, the proposed reaction sequence for the meta-
bolism of dihalomethanes accounts for the experimentally observed stoichiometric
ratio of two inorganic halides formed to one formaldehyde and the requirement
(but nonconsumption) of GSH. Furthermore, the sequence serves as a detoxifica-
tion mechanism for dihalomethanes that is dependent on cellular availability of
glutathione, and the cellular availability of NAD determines the ratio of the
products (formaldehyde/formic acid) formed.
4.2.3.3 Carbon Dioxide Formation—The cytosolic glutathione transferase system
for dehalogenation of dihalomethanes with the production of formaldehyde and for-
mic acid appears to be the major source of C02 as an end product of the metabolism
of DCM in the intact rodent (Rodkey and Collison, 1977; DiVincenzo and Hamilton,
1975; Yesair et al., 1977; McKenna et al., 1982). Neely (1964) has demonstrated
that formaldehyde is almost quantitatively metabolized to C02. When Neely
injected 14C-formaldehyde (14CHO) intraperitoneally into rats at dose levels
of 0.25 and 2.5 gmole/kg b.w., he recovered 82 percent of the dose as C02 in
exhaled air collected over a 24-hour period. Peak concentrations of 14C02 in
exhaled air occurred 1 hour after administration. Neely suggested that the
formation of C02 occurred from formaldehyde entering the 1-C metabolic pool with
transfer to glycine by the folic acid cycle to give serine. Transamination of
serine to pyruvate provides entry to the tricarboxylic acid cycle and completes
the oxidation to C02. In support of this metabolic route, small amounts of
14Olabeled serine and methionine were found in the urine. However, when 14C-DCM
was administered to rats, no 14CHO was detected in the breath, serum, or tissues
2 hours later, although substantial changes in tissue formaldehyde content were
noted (Rodkey and Collison, 1977; DiVincenzo and Hamilton, 1975).
An additional source of C02 production from the metabolism of dihalo-
methanes is the in vivo oxidation of CO to C02. Carbon monoxide is metabolized
to C02 by various animal tissues (Fenn, 1970). Luomanmaki and Coburn (1969)
also have demonstrated that 14C02 exists in the expired air of humans breathing
005DC5/C 4-38 11-14-83
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14CO during a 4-hour period in a closed rebreathing system. Carbon monoxide is
metabolized by combining with the reduced form of tissue cytochrome oxidase in
the presence of a low 02 tension, being released as C02 (Coburn, 1970). However,
the amount of C02 generated by oxidation of CO is very small, because 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 C02 appears to increase as a function of body stores of CO and thus as a
function of blood COHb concentration. 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 that results from
catabolism of DCM may stimulate the metabolic production of C02 from CO, thus
contributing to the total C02 produced by DCM metabolism.
4.2.3.4 Pathway Utilization Ratio—The microsome oxidative dehalogenation and
cytosol glutathione transferase dehalogenation systems (Figure 4-9) account for
the CO and C02 generated from the metabolism of the dihalomethanes. Because the
microsomal system is apparently saturated and rate-limiting at low doses (Section
4.2.2), the relative molar amounts of CO and C02 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 C02 with both low and high saturating doses of DCM
in mice. For low doses in rats, Rodkey and Collison (1977) found 1.6 times as
much CO produced as C02, suggesting greater metabolism (at low doses) by the
microsomal oxidative pathway. McKenna and Zempel (1981) observed a CO:C02 ratio
of 0.9 for a low oral dose of DCM (1 mg/kg) to rats and a ratio of 1.9 for high
saturating dose (50 mg/kg), indicating a greater utilization of the microsomal
oxidative pathway at "saturating doses" (Table 4-12). However, McKenna et al.
(1982) found for a low inhalation dose of DCM (50 ppm or 174 mg/m3) and a meta-
bolic saturating inhalation dose (1500 ppm or 5211 mg/m3), no significant pre-
ference for either pathway (Table 4-10). Clearly, the important factors of
hepatic content of glutathione and of P45o plaY major roles in the relative
utilization of the two pathways of metabolism.
Though it has been observed that equimolar doses of dibromomethane and
diiodomethane produce COHb concentrations greater than those produced by DCM
in rats (Fodor and Roscovanu, 1976; Roth et al., 1975; Rodkey and Collison, 1977),
in microsomal preparations (Kubic and Anders, 1975) and in cytosol preparations
(Ahmed and Anders, 1976) no information is available on the ratio of CO to C02
produced by these compounds. The use of isolated hepatocytes may prove useful
and avoid many of the difficulties inherent with whole animal experiments.
005DC5/C 4-39 11-14-83
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4.3 DCM-INDUCED CHANGES IN HEPATIC ENZYMES
The metabolism of the dihalomethanes by the microsomal oxidative dehalo-
genation pathway (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 CO (Kubic et al., 1974; Kubic and Anders, 1975; Hogan
et al., 1976; Stevens et al., 1980), 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 pheno-
barbital, but the resulting increase in local microsomal levels of CO may be
sufficient to inhibit cytochromic P450 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 bromochloro-
methane to rats led to an increased rate of dehalogenation. However, Haun et
al. (1972) found that continuous exposure of mice to 100 ppm DCM (347 mg/m3)
for 4 to 12 weeks decreased the hepatic content of cytochrome P45o-
Daily exposures for shorter periods do not appear to influence hepatic
P450 content, but they do modify the activity of other enzyme systems. Kurppa
and Vainio (1981) exposed rats at 500 and 1000 ppm DCM (1735 and 3470 mg/m3)
6 hours per day for 5 and 10 days. They reported no change in hepatic P450
content but they observed a marked decrease of NADPH-cytochrome C (35 percent)
and a two-fold increase in UDP-glucuronsyl transferase. The hepatic GSH content
remained unchanged. Toftgard et al. (1982) exposed rats to 500, 1500, and 3000
ppm DCM (1735, 5211, and 10,422 mg/m3) 6 hours/day for 3 days and found no change
in hepatic P4s0 content, but a dose-related increase of microsomal metabolism of
biphenyl and of benzopyrene was noted. These changes were postulated to be
caused by a change in the proportions of different cytochrome P4s0 isozymes
resulting from an DCM-inducing effect on some specific forms of cytochrome P4s0-
In contrast to these observations, Pritchard et al. (1982) recently administered
DCM to male mice for 3 days by gavage (5, 50, 100, 250, 500, 1000 mg/kg doses in
corn oil) and found no significant changes in hepatic weight, microsomal protein,
P450> cytochrome bs contents, or activities of aminopyrene N-demethylase and
biphenyl 4-hydroxylase. Furthermore, 28 days of administration of DCM in
005DC5/C 4-40 11-14-83
-------
drinking water, to provide daily doses ranging from 5 to 1000 mg/kg, also did
not affect these parameters or hepatic glutathione content.
4.4 COVALENT BINDING TO CELLULAR MACROMOLECULES
The likelihood of significant covalent binding of reactive metabolites
from the metabolism of OCM to cellular macromolecules is predicated on the
postulated reactive intermediates: formyl chloride from microsomal oxidative
metabolism and S-chloromethyl glutathione and formaldehyde from cytosolic
metabolism (Figure 4-10). These compounds may be capable of acylating cellular
nucleophiles. S-chloromethyl glutathione is structurally similar to the
reactive bis-halomethyl ethers.
Anders et al. (1977) have studied the extent and pattern of binding to
microsomal lipid and protein after aerobic incubation of rat hepatic microsomes
with 14C-DCM. Table 4-13 shows that metabolites of DCM become covalently
bound to both microsomal protein and lipid under conditions optimal for meta-
bolism of DCM to CO. Furthermore, microsomes from rats pretreated with pheno-
barbital showed increased binding. Thus, the formyl chloride intermediate may
either acylate macromolecules or decompose to CO.
Cunningham et al. (1981) have investigated covalent binding of inter-
mediates from the metabolism of 14C-DCM by rat hepatocytes. Rat hepatocytes in
suspension have been shown to metabolize DCM and other dihalomethanes to CO
(Stevens et al., 1980). The results of irreversible binding of 14C-DCM to
cellular macromolecules in this system, in comparison to 14C-carbon trichlo-
roethylene, are given in Table 4-14.
The covalent binding of carbon tetrachloride to lipids and protein was
greatly enhanced in the absence of 0- and was almost eliminated in the presence
TABLE 4-13. I_N VITRO COVALENT BINDING OF 14C-DCM
TO MICROSOMAL PROTEIN AND LIPID
Conditions
nmoles 14C bound/mg/min ± SD
Protein Lipid
Normal rat microsomes
Microsomes from pheno-
barbital-treated rats
0.24 ± 0.02
0.57 ± 0.03
0.27 ± 0.02
1.97 ± 0.19
Source: Anders et al., 1977.
S.D. = standard deviation.
005DC5/C
4-41
11-14-83
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presence of 02 (Table 4-14). In contrast, binding of DCM and trichloroethylene
was enhanced in the presence of oxygen; this is consistent with the microsomal
oxidative metabolism of these compounds to an epoxide and formyl chloride, re-
spectively. Glutathione-depleted hepatocytes showed markedly decreased covalent
binding of 14ODCM to both lipids and protein, suggesting inhibition of cytosolic
DCM GSH conjugation with a decreased production of reactive S-chloromethyl glu-
tathione and of formaldehyde. However, at least part of the decrease in binding
may be caused by diethylmaleate inhibition of microsomal oxidation (Stevens et
al., 1980), as well as by diethyl maleate depletion of cellular GSH. Phenobar-
bital pretreatment also markedly decreased binding from 14C-DCM hepatocyte meta-
bolism. The explanation of this effect is not readily apparent because pheno-
barbital is not known to deplete cellular glutathione but does increase DCM
microsomal oxidative metabolism to CO in hepatocytes (Stevens et al., 1980).
Perhaps the most important observation made by Cunningham et al. was that
14C-DCM metabolism by isolated hepatocytes did not result in the alkylation of
the nucleic acids RNA or DNA, whereas metabolites of carbon tetrachloride and
trichloroethylene labeled nucleic acids under the conditions.
Reynolds and Yee (1967) studied labeling patterns of 14C-DCM and 14C-formal-
dehyde in rat liver after jm vivo injection. They found similar patterns of
labeling of DCM and its metabolite, formaldehyde. Binding occurred most often
at the amino acid locus corresponding to serine and on the acid-soluble cell
constituents, with smaller amounts in lipid and nucleic acids. However, for-
maldehyde or formic acid can directly combine with tetrahydrofolic
acid and consequently be incorporated into de novo nucleic acid synthesis;
therefore, the association of 14C-activity with nucleic acids does not necessarily
indicate covalent binding.
4.5 KINETICS OF CARBOXYHEMOGLOBIN FORMATION
Blood COHb accumulates when the amount of endogenous or exogenously derived
CO in the body exceeds that of pulmonary elimination. Pulmonary elimination
of CO is a first-order process that involves the exchange of hemoglobin-bound CO
with oxygen and the diffusion of CO across capillary and alveolar endothelium
into alveolar lung space. In humans, the half-time of elimination of CO is 4 to
5 hours (NIOSH, 1972; Lambertsen, 1974), and in the rat the time is 1.8 to 2.5
hours (McKenna et al., 1982; McKenna and Zempel, 1981). The half-time of pul-
monary elimination is independent of the blood COHb concentration but is depen-
dent on factors such as lung function and dysfunction, pulmonary rate and am-
plitude, regional blood flow, and cardiac function.
005DC5/C 4-42 11-14-83
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TABLE 4-14. COMPARATIVE COVALENT BINDING OF DCM, CARBON TETRACHLORIDE,
AND TRICHLOROETHYLENE TO LIPID AND PROTEIN IN RAT HEPATOCYTES
Atmosphere3
(0^/N^)
Substrate
ecu.
TCE
DCM
Protein
0.09
9.63
8.53
Lipid
0.06
5.08
11.96
GSH Depletion*3, %
Protein
100
74
44
Lipid
100
74
38
Phenobarbital
induction, %
Protein
145
288
44
Lipid
120
238
40
aRatio of covalent binding observed under oxygen and nitrogen atmosphere.
Values are the mean of three to six determinations for each condition.
Thirty minutes prior to hepatocyte isolation, rats were treated intraperi-
toneally with 0.6 ml/kg of diethylmaleate. Values are expressed as the mean
percent of binding observed in hepatocytes from untreated rats, with N=5.
GRats were pretreated with three consecutive daily doses of phenobarbital
(80 mg/kg, intraperitoneally) beginning 4 days before isolation of hepatocytes.
Values are expressed as the mean percent of binding observed in hepatocytes from
untreated rats, with N=5.
Source: Cunningham et al., 1981.
TCE = trichloroethylene
Ordinarily, the sole endogenous source of CO, and hence COHb, is 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 the normal human is about 20 umoles/hr, producing a blood
COHb concentration of approximately 0.4 percent. A close linear correlation
exists between the molar rate of CO production and the 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 catabolism 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 (e.g., ambient-air CO, tobacco smoking)
005DC5/C 4-43 11-14-83
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is decreased by increased alveolar ventilation or increased inspired partial
pressure of oxygen (Lambertsen, 1974).
Since Stewart and his associates (1972a,b) reported the remarkable increase
of blood COHb (up to 15 percent from 0.6 percent pre-exposure) in persons acutely
exposed by inhalation to DCM vapor, numerous investigations of the phenomenon
have been made in experimental animals and humans. Studies have been undertaken
to determine the dose-response relationship of blood COHb level with DCM air
concentration, with duration of exposure, with time-course of COHb blood con-
centration rise and decline, and with the magnitude of its occurrence in the
industrial setting. Because CO generated from DCM is additive to exogenous
environmental CO, DCM exposures at high levels could pose an additional health
burden. Of particular concern are smokers who maintain significant constant
levels of COHb, i.e., 4.6 to 5.2 percent (Stewart et al., 1974a,b; Kahn et al.,
1974), and others who may have increased sensitivity to CO toxicity, such as
pregnant women and persons with cardiovascular disease or pulmonary dysfunction.
Indeed, Stewart et al. (1972a,b) have noted that exposure to concentrations of
(R)
DCM that do not exceed the industrial TLV^ (100 ppm, 347 mg/m3) may yield COHb
levels exceeding those allowable from exposure to CO (35 ppm, 38.5 mg/m3; about
5 percent COHb blood concentration).
As previously discussed, the recent findings of DiVincenzo and Kaplan
(1981a,b) with sedentary individuals and those engaged in physical exercise
while being exposed to DCM indicate that blood levels of COHb are increased
but are within the biological' TLV® recommended by NIOSH (1976). NIOSH has
recommended that the TWA exposure to DCM should not produce COHb levels that
exceed 5 percent. DiVincenzo and Kaplan (1981a) found that an 8-hour exposure
to 150 ppm DCM (520 mg/m3) in sedentary individuals is equivalent to an 8-hour
exposure to 35 ppm CO; either exposure has been shown to result in blood levels
of COHb of about 5 percent. In their companion study in which individuals either
exercised or smoked cigarettes during exposure, DiVincenzo and Kaplan (1981b)
observed an additive increase in blood COHb concentrations. Individuals were
exposed for 7.5 hours to 100 ppm DCM (347 mg/m3). The effects of exercise on
physiological parameters are presented in Table 4-2. Moderate to heavy work-
loads produced about a two-fold increase in blood COHb levels compared to the
increase for sedentary individuals.
From review of the literature, an important observation that can be made of
DCM-induced COHb is that the blood concentration, although DCM-dose dependent and
005DC5/C 4-44 11-14-83
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exposure-time dependent, never exceeds 10 to 12 percent in either humans or ani-
mals in usual circumstances of free breathing and normal pulmonary function
(Figures 4-5, 4-6, 4-11). This limiting blood concentration is clearly deter-
mined by the resultant of first-order pulmonary elimination of CO and zero-order
kinetics of hepatic DCM metabolism to CO (Section 4.2.2). Because COHb is essen-
tially confined to the blood compartment, a one-compartment open kinetic model
with zero-order input can be used to describe the time course of blood COHb as
follows:
(4-3)
where V is the volume of the Hb compartment (90 percent CO is distributed in
this compartment) (Luomanmaki and Coburn, 1969), kQ is the zero-order rate of
COHb formation, k is the first-order rate constant for pulmonary elimination
of CO from COHb, and C is the the concentration at any time t of COHb formed.
Then,
(4-4)
This equation (Wagner, 1975) describes the time course of rising COHb concen-
tration with zero-order formation of COHb. With long periods of exposure to
DCM (6 to 8 hours), the relation becomes
C
COHbt
ko
Vke
1-e "kgt
COHb4
= k /Vk
o e
(4-5)
That is, a steady-state plateau concentration of blood COHb is reached (see
Figure 4-12). Therefore, maximal blood COHb is mandated when KQ) V, and kg are
constants.
4.5.1 Studies in Human
In a series of studies, Stewart and his associates (Stewart et a!., 1972a,b,
1973, 1974a,b; Forster et al., 1974; Hake et al., 1974) have shown that blood
COHb levels achieved in response to DCM exposure are related to the inhaled
concentration and to the duration of exposure. Male nonsmokers were exposed to
005DC5/C
4-45
11-14-83
-------
CH2CI2, ppm
O—0500
EXPOSURE, days.
Figure 4-11.
Blood COHb level in men during an 8-hour exposure for 5 consecu-
tive days to 500 ppm and 100 ppm DCM. COHb percent saturation is
equal to |jg CO per ml blood divided by 2.5.
Source: Fodor and Roscovanu, 1976.
005DC5/C
4-46
11-14-83
-------
DCM for 1, 2, and 7.5 hours daily, 5 days/week. Blood COHb levels were deter-
mined for daily pre-exposure and post-exposure times. Their data, which are
replotted in part in Figure 4-5, indicate that maximum COHb concentrations occur
with 400 to 500 ppm DCM (1390 to 1737 mg/m3) exposure and increase with duration
of exposure. Similar results were reported in women nonsmokers exposed 1, 3, and
7.5 hours to 250 ppm DCM (869 mg/m3) for 5 consecutive days (Hake et al., 1974),
and in male volunteers exposed to 100 and 500 ppm DCM (347 and 1737 mg/m3) daily
for 5 days (Fodor and Roscovanu, 1976) (Figure 4-11). 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 ob-
served (Figure 4-11); however, the half-time of COHb disappearance was signi-
ficantly longer than expected. The longer apparent half-time of pulmonary eli-
mination of CO produced by hepatic metabolism of DCM results from storage of
DCM in adipose tissue (Section 4.1.2), with conversion to CO continuing subse-
quent to termination of exposure. In these circumstances, the biological half-
life of COHb derived from DCM (10 to 15 hours) is proportional to the body burden
of DCM, in contrast to the constant half-life of 4 to 5 hours from CO inhalation
(Stewart et al., 1972a,b, 1976a,b; Fodor et al., 1973; Peterson, 1978; Peterson
and Stewart, 1970b). However, when workplace exposures were simulated, the bio-
logic half-life for COHb was similar to that for CO (DiVincenzo and Kaplan,
1981a).
DiVincenzo and Kaplan (1981a) found that blood COHb saturation in seden-
tary male volunteers is not attained during a 7.5-hour exposure to DCM at con-
centrations ranging from 50 ppm (173 mg/m3) to 200 ppm (694 mg/m3). Peak blood
COHb during the 200-ppm exposure was 6.8 percent. At the TLV for DCM, the
authors estimated that an 8-hour exposure would produce a blood COHb level
of about 3 percent. A linear relationship was found between blood COHb and
DCM exposure concentrations. Repeated exposure to DCM levels of 100, 150, or
200 ppm (347, 620, or 694 mg/m3) for 7.5 hours daily for 5 consecutive days
produced slightly higher blood COHb levels than those found for single expo-
sures. Following the weekend, blood COHb levels returned to pre-exposure
levels. Blood COHb declined at a slower rate than DCM in expired breath during
post-exposure. About 24 hours was required for blood COHb to return to pre-
exposure values.
The findings in a companion study by DiVincenzo and Kaplan (1981b), with
smokers and individuals engaged in physical activity indicated that exposure
005DC5/C 4-47 11-14-83
-------
100.0
E ' I I I ' 1 ' I I I ' I I I ' I I I 'E
10.0
0.1
EXPOSURE'
J 1 I I I I 1
A 50 ppm
• 500 ppm
• 1500 ppm
I I I 1 I I I I I , I , I
0123456/01234
TIME, hours
Figure 4-12. Blood HbCO concentrations in rats during and after a
6-hour inhalation exposure to DCM. Each data point is the mean
± standard error for two to four rats.
Source: McKenna et al. (1982).
4-48
-------
to 100 ppm DCM (347 mg/m3) may result in slightly higher blood COHb than in
sedentary nonsmokers. The investigators concluded that workers performing phy-
sical exercise while exposed to DCM at 100 ppm (347 mg/m3) are unlikely to exceed
the COHb biological TLV® recommended by NIOSH (1976).
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/m3) with no measurable CO. At
the beginning of the work day, blood COHb levels averaged 4.5 percent. After an
8-hour exposure, COHb levels rose to about 9 percent, then declined exponen-
tially to 4.5 percent by the next working day (16 hours later) with an apparent
half-time of CO pulmonary elimination of 13 hours. These prolonged half-times
for CO in alveolar air and for COHb in blood are difficult to interpret in
light of the contrasting results of Stewart et al. (1972a,b) and DiVincenzo
and Kaplan (1981a,b). Ratney et al. (1974) reported that pre-exposure levels
of CO in expired air and COHb in blood were elevated above the values expected
for nonsmokers. Three of the seven individuals that were monitored were not
previously exposed to DCM; therefore, it is surprising that the CO levels in
their alveolar air and blood COHb values were as high as reported in the
paper. Ratney et al. (1974) reported a mean pre-exposure CO concentration in
expired air of about 29 ppm. Normal levels of CO in alveolar air are expected
to range between 1 and 5 ppm.
The studies of Stewart and associates, Fodor and Roscovanu, and Ratney et
al. are consistent in their findings and lead to the following conclusions.
(1) COHb derived from CO of DCM metabolism is additive to COHb derived
from CO of exogenous sources.
(2) Blood COHb concentration attained from DCM metabolism is a function of
both DCM inhalation concentration and of exposure duration, with a maximal attain-
able concentration of 10 to 12 percent COHb.
(3) A steady-state blood COHb concentration in sedentary individuals is not
attained within 8 hours for single exposures of DCM.
(4) The half-time of first-order pulmonary elimination of COHb is 4 to 5
hours, although a longer pseudo half-time of 10 to 15 hours is observed because
of continued metabolism of DCM post-exposure, from solvent stored in fat compart-
ment during exposure.
005DC5/C 4-49 11-14-83
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(5) Blood COHb concentrations from multiple daily exposures reach a steady-
state level within 3 to 5 days, and the level is only slightly higher than that
after a single 8-hour exposure.
These observations on the kinetics of COHb formation during DCM inhalation
exposure are consistent with Michaelis-Menten kinetics for DCM metabolism to CO
(Section 4.2.2). The data plot of Stewart and associates of Figure 4-5 (7.5-hour
exposures) illustrates dose-dependent formation of COHb and is of the form of
the Michaelis-Menten equation:
V S
dC = max (4-6)
dt K + S
m
where dC/dt is the rate of COHb formation and S is the exposure concentration
in parts per million. Estimates of V and K were obt
K ^ max m
data in accordance with the linear form of the equation.
in parts per million. Estimates of V and K were obtained from plotting the
max m
d£ - -K dC s + y (4.?)
dt m dt max
This form provides values of V s 15% COHb/7.5 hr and K = 200 ppm (694 mg/m3)
Ml
DCM. These values indicate a' maximum obtainable COHb blood level in humans
caused by DCM exposure in air of about 25 percent, and a saturation- of hepatic
metabolism of DCM to CO in humans at 400 ppm DCM (1388 mg/m3) in inhaled air
(2 x K ).
The COHb blood levels resulting from DCM inhalation are dependent on pul-
monary function status as well as on metabolism of DCM to CO. Physical activity
or exercise during exposure to DCM markedly increases pulmonary absorption and
body retention (Section 4.1.3), but tends to diminish the maximum blood COHb
level achieved by the end of the exposure period, although higher 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; DiVincenzo
and Kaplan, 1981b). These findings may be explained by the decrease in half-
life of pulmonary CO elimination affected by the increased pulmonary ventila-
tion rate and increased cardiac output with physical activity (Lambertsen, 1974).
Conversely, compromised pulmonary function (e.g., adult respiratory distress
005DC5/C 4-50 11-14-83
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syndrome, emphysema, asthma) can be expected to increase COHb blood levels by
decreasing CO pulmonary elimination. A comprehensive kinetic model describing
in humans the parameters of absorption, distribution, and elimination of DCM, as
well as metabolism to CO and COHb, would be valuable in evaluating the effects of
physical activity, pulmonary and cardiovascular diseases, and concomitant envi-
ronment xenobiotic exposures including 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 COHb
formation 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.
4.5.2 Studies in Animals
The kinetics of COHb formation from DCM exposure elucidated in humans have
largely been confirmed in animals. Figure 4-6 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 1000 ppm DCM (3,474 mg/m3). Fodor and Roscovanu (1976) noted that a 3-hour
exposure with 200 ppm DCM (695 mg/m3) in humans produced COHb levels of about
4.3 percent, but found nearly twice this level in the rat (Figure 4-6); this
difference suggests that metabolic capacity for CO formation is greater in rats
than in humans. Hogan et al. (1976) found that rats exposed to 440 ppm DCM (1529
mg/m3) for 3 hours had maximal COHb levels of about 7 percent, and exposure to
2300 ppm (7,990 mg/m3) produced no further increase. Pretreatment of the animals
with phenobarbital increased the rate of rise of COHb levels and the time the
maximum level was maintained, but did not increase the highest level. These
investigators suggest that endogenous CO inhibition of the P450 metaboli-
zing system by CO binding to the cytochrome may occur at very high levels of
DCM exposure. However, their observations also are consistent with the Michaelis-
Menten kinetics of hepatic CO formation from DCM, with saturation of metabolic
conversion from exposure to 400 ppm (1,388 mg/m3). Kurppa and Vainio (1981)
exposed rats to 500 and 1000 ppm DCM (1737 and 3474 mg/m3), 6 hours/day, 5
days/week for 1 and 2 weeks. The test results are given in Table 4-15. Since
maximal blood COHb levels of 8 to 9 percent occurred with 500 ppm (1737 mg/m3)
exposure and no further increase was obtained with 1000 ppm (3474 mg/m3) or with
005DC5/C 4-51 11-14-83
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longer duration of exposure, these results also indicate that saturation of
metabolism of DCM to CO in rats occurs at a DCM exposure concentration of 500 ppm
(1737 mg/m3) or less.
McKenna et al. (1982) exposed rats to 50, 500, and 1500 ppm DCM (174, 1737,
and 5211 mg/m3) for 6 hours; during testing steady-state conditions were reached
between production of CO, maintenance of a given circulation blood COHb concen-
tration, and pulmonary CO excretion. These results are shown in Figure 4-12.
The blood percent COHb at steady-state was not linearly related to exposure con-
centration but reached a maximum of about 12.5 percent. Kinetic parameters for
the production of CO and formation of COHb can be estimated on the clear assump-
tion of dose-dependent Michaelis-Menten kinetics (Section 3.2.2). Estimates
calculated from the data in Figure 4-12 are V =13.5 percent COHb/6-hour expo-
sure, and K = 170 ppm DCM (590 mg/m3). These values indicate a saturating DCM
exposure concentration of about 350 ppm (1,215 mg/m3) (2 x K ) for the rat.
The determination of Michaelis-Menten kinetics of DCM metabolism to CO
and an upper limit to the attainable blood COHb concentration provides an ex-
planation of the observations of Roth et al. (1975) and of Haun et al. (1971,
1972). Roth et al. found that concentrations of COHb in the blood of rabbits
after very short exposures (20 minutes) to DCM inhalation concentrations ranging
from 2000 to 12,000 ppm (6948 to 41,688 mg/m3) were a linear function of DCM
exposure concentration. COHb levels were approximately 5.5 percent at 2000
ppm (6,948 mg/m3) and 13 percent at 12,000 ppm (41,688 mg/m3). However, with
TABLE 4-15. BLOOD COHb AND Hb CONCENTRATIONS IN RATS EXPOSED TO DCMa
Exposure
(ppm)
None
500
1000
Carboxyhemogl obi n, %
Five days
0.
8.
9.
4
1
2
± 0.1
± 0.6
± 0.6
Ten
0.4
8.1
8.5
days
± 0.
± 0.
± 0.
2
7
6
Hemoglobin, g/1
Five
155.2
144.0
151.5
days
± 5.5
± 21.2
± 6.0
Ten
156.
155.
158.
days
6 ±
4 ±
0 ±
6.2
3.5
4.5
a
The results are given as mean ± standard deviation of five rats per group.
Source: Kurppa and Vainio, 1981.
005DC5/C 4-52 11-14-83
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4 hours of exposure at about 7000 ppm (24,318 mg/m3), 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. Therefore, Roth
et al. (1975) suggested that increased CO production from DCM at the microsome
in response to a phenobarbital-induced increase of the cytochrome P45o system
inhibits further DCM metabolism. A more likely explanation of these results is
that the short exposures did not provide sufficient time for the establishment
of body equilibrium to inhaled DCM; therefore, even at high inhalation concentra-
tions, the DCM presented to the hepatic-metabolizing system was on the linear
portion of the Michaelis-Menten curve.
In a comparison of dogs and monkeys continuously exposed to 25 and 100 ppm
DCM (87 and 348 mg/m3) for 6 to 13 weeks, Haun et al. (1971) found that steady-
state levels of blood COHb were maintained throughout the exposure period, and
these concentrations were directly proportional to the exposure concentration.
However, these concentrations result from low-level exposures; therefore, they
are expected to be on the linear portion of the Michaelis-Menten function. Haun
et al. noted that dogs had higher steady-state blood concentrations of DCM than
monkeys at these inhalation concentrations, but monkeys had the higher COHb blood
levels, suggesting that monkeys have a greater hepatic capacity for CO formation.
4.5.3 Comparison of Kinetics of Humans and Rats
The kinetics of DCM metabolism to CO and of COHb formation are strikingly
similar for humans and rats. Both species demonstrate Michaelis-Menten dose-
dependent kinetics with similar estimates for V (15 and 13.5 percent COHb/time
unit, respectively) and K (200 and 170 ppm DCM, respectively). Hence, the satur-
ating DCM inhalation concentration for the metabolism of DCM to CO and forma-
tion of COHb are comparable for the two species (350 vs. 400 ppm; 1215 vs. 1388
mg/m3).
The metabolism of DCM involves two pathways: the microsomal oxidative
pathway leads to CO, and the cytosolic pathway using GSH converts DCM to for-
maldehyde, formic acid, and C02. Comparison of the kinetic parameters estimated
above for the CO pathway in the rat with those obtained by McKenna et al. (1982)
for the total overal1 (both pathways) metabolism of DCM in these rats (Section
4.2.2) reveals that the K = 400 ppm (1388 mg/m3) for overall metabolism is about
005DC5/C 4-53 11-14-83
-------
twofold greater than for the CO pathway alone. Furthermore, this figure is an
average overall value, suggesting that the cytosolic GSH-dependent pathway has
a much higher «m value than that of the CO pathway. Andersen (1981) has called
the CO metabolism a high-affinity, low-capacity pathway and the GSH-dependent
pathway low-affinity but high-capacity.
4.6 MEASURES OF EXPOSURE AND BODY BURDEN IN HUMANS
In the controlled laboratory setting, estimating the DCM absorbed into
the body by comparing inspired and alveolar air concentrations, or by measur-
ing blood levels of DCM and then extrapolating these parameters to body dose,
is an imprecise task. However, the goal of this research is to develop a suffi-
cient data base and knowledge of the kinetics of absorption, disposition, eli-
mination, and metabolism of DCM to enable assessment of the body burden of DCM
from acute or chronic exposure in the industrial or ambient setting where air
concentrations and exposure period 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 in human body burdens from recent
exposures. However, in addition to the lack of complete pharmacokinetic know-
ledge necessary to interpret these determinations into accurate and reliable
measures of body burden, the results also are subject to unknown interindividual
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 associates (1976a) advocate the use of breath analysis to moni-
tor DCM exposure because it is a noninvasive method and avoids the problems
associated with the multiple blood sampling required for determinations 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 spectroscopy or gas liquid
chromatography provides both an identification and a measurement 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 related directly to the average
inhalation exposure concentration. When the duration of exposure and work
005DC5/C 4-54 11-14-83
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intensity are known, the total body burden can be estimated by reference to
"standard" breath concentration curves. Because of large variations between
individuals exposed under identical conditions, only very approximate DCM body
doses can be estimated. The 2-hour period following exposure appears to be the
most reliable breath sampling time for estimating the TWA DCM exposure concen-
tration. Stewart et al. (1976a) provide little information on the statis-
tical reliability or the interindividual variation expected in the predictive
values for exposure burdens, although they point out the desirability of con-
structing "individualized" breath decay curves for every person expected to be
exposed to DCM. Peterson (1978), using the same experimental exposure data as
Stewart et al., has developed empirical equations relating post-exposure breath
concentrations with exposure time, duration, and blood COHb levels.
4.7 SUMMARY AND CONCLUSIONS
The metabolism and pharmacokinetics of DCM have been studied extensively.
At ambient temperature, DCM is a volatile liquid with high lipid solubility and
modest solubility in water; therefore, the principal routes of entry to the body
are by pulmonary and oral absorption. Comparatively fewer data are available on
the metabolism and pharmacokinetics of absorption and excretion of DCM in humans,
but these kinetics have been studied extensively in the rodent. Absorption from
the GI tract is rapid and complete, occurring by first-order passive processes in
the rat; the kinetics of peroral absorption and post-absorption disposition and
elimination are influenced by the dosing vehicle. Pulmonary absorption also
occurs by first-order diffusion processes in both humans and rats. There are
three distinct components whose rate constants correspond to tissue loading of
at least three major body compartments. At equilibrium with inspired air con-
centration, a blood/air partition coefficient of about 10.5 at 37°C has been
observed.
Distribution of DCM in the tissues is consistent with its lipophilic nature
and modest water solubility. The chemical readily crosses the blood-brain and
placenta! barriers and.distributes into breast milk. Concentrations occurring
in all major tissue organs are dose-related to inspired air concentration or
to oral dosage. During inhalation exposure, the quantity of DCM absorbed is
dependent also on body weight and fat content of the body; the adipose tissue/
blood coefficient at 37°C is about 7, and about 0.8 to 1.0 for brain and liver
tissues. The rate of tissue loading with a given inspired air concentration
is increased with physical activity, and with exposure duration, with steady-
state body equilibrium requiring more than 6 hours.
005DC5/C 4-55 11-14-83
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The kinetics of elimination of DCM from the body are complex and are domi-
nated by two major and parallel occurring processes: 1) pulmonary elimination of
unchanged DCM, and 2) hepatic metabolism of DCM. Pulmonary elimination follows
first-order kinetics, is independent of body dose, and exhibits at least three
distinct body compartments with half-times for humans of about 8 to 23 minutes,
40 to 80 minutes, and 360 to 390 minutes, and half-times for rats of 1 to 2 min-
utes, 12 to 16 minutes, and 47 minutes, respectively. The longest half-time is
associated with the lipid and adipose tissue body compartment. The differences
between humans and rats for the half-times of pulmonary elimination from body
compartments are presumed to be caused by differences in pulmonary and cardio-
vascular functions. Hepatic metabolism is body-dose dependent and saturable, and
follows nonlinear Michaelis-Menten kinetics. Body dose, as given by oral dosing,
also is subject to the kinetics of first-pass hepatic metabolism and first-pass
pulmonary excretion. The estimated K for overall metabolism of DCM in the rat,
as obtained from inhalation exposures, is about 400 ppm. This inhalation concen-
tration for 6 hours produces an estimated body dose of approximately 50 mg/kg.
For the body dose of 50 mg/kg, 70 percent of the DCM is metabolized, and for a
5.5 mg/kg body dose resulting from 50 ppm for a 6-hour exposure, 95 percent is
metabolized. The inhalation concentration saturating overall metabolism of
DCM is about 800 ppm (2 x K ).
The hepatic metabolism of DCM occurs by two enzymic pathways: 1) a cyto-
chrome P450-mediated microsomal oxidative dehalogenation to CO, and 2) a cyto-
solic glutathione transferase dehalogenation system yielding formaldehyde and
formic acid, which are further metabolized to CO^. The activities of these two
pathways are approximately equal to low body burdens of DCM, and both pathways
are saturable at high body burdens. Covalent binding to cellular macromolecules
(proteins and lipids) by putative reactive intermediates of metabolism, formyl
chloride from microsomal oxidative metabolism and S-chloromethyl glutathione and
formaldehyde from cytosolic metabolism, has been shown to occur in vitro with
rat hepatic microsome preparations and with intact isolated rat hepatocytes and
in vivo after 14C-DCM injections to rats. No evidence was found in these studies
for the occurrence of covalent binding to DNA.
The occurrence of increased blood COHb levels in humans and animals exposed
to DCM is a consequence of hepatic microsomal oxidative metabolism of DCM to CO.
The COHb produced is additive to COHb formed from exogenous CO. A functional
relationship exists between the DCM inhalation concentration, duration of exposure,
005DC5/C 4-56 11-14-83
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and the time course and peak blood COHb level. The blood COHb level achieved
is determined by the nonlinear kinetics (Michaelis-Menten kinetics) of hepatic
metabolism of DCM to CO and the parallel-occurring linear kinetics (first-order
kinetics) of pulmonary elimination of CO from circulating COHb. Because hepatic
metabolism of DCM to CO is saturable, the zero-order kinetics of CO production
constrains blood COHb accumulation to an upper, limited concentration fo 12 to
15 percent COHb, as observed experimentally. However, human studies have demon-
strated that exposures to DCM at levels up to about 150 ppm (620 mg/m3) are
unlikely to exceed the biological TLV© for blood COHb (5 percent) recommended
by NIOSH (1976). Kinetic parameters for the production of CO and formation of
COHb, calculated from experimental DCM exposure data of blood COHb concentra-
tions in humans and rats, have similar values. Estimations of V for humans
and rats are 15 and 13.5 percent COHb per 7-hour exposure, respectively, and
estimates of K are 200 and 170 ppm, respectively. These findings indicate
that the CO pathway is saturated with an inspired air concentration of appro-
ximately 400 ppm DCM (2 x K ) in both humans and rats.
005DC5/C 4-57 11-14-83
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5. HEALTH EFFECTS OF DICHLOROMETHANE
5.1 HUMAN HEALTH EFFECTS
5.1.1 Acute Exposures
5.1.1.1 Nervous System and Behavior—Virtually all experimental exposure
studies of DCM using humans have studied acute exposures and neural or behav-
ioral dependent variables. Using between three and eight subjects, Stewart et
al. (1972b) reported that subjects had symptoms of "lightheadedness" and had
difficulty with speech articulation at DCM levels higher than 868 ppm (3,012
mg/m3) for more than I hour. They also reported anecdotal data about increased
amplitude in certain visual evoked potentials at about the same exposure levels;
however, no statistical analyses were possible because of the small number of
subjects. In a later study, they reported that 50, 100, or 250 ppm (173, 347,
or 867 mg/m3) DCM had no effects on the Romberg equilibrium test, vigilance,
coordination, or arithmetic skill (Stewart et al., 1973).
Winneke (1974) and Winneke and Fodor (1976) exposed female human subjects
to 0, 300, 500, and 800 ppm (0, 1041, 1730, and 2,776 mg/m3) DCM for up to 3
hours. They measured performance on a series of hand-eye coordination tasks
including tapping pursuit rotor and hand steadiness. They also measured the
critical flicker fusion (CFF) frequency and performance on an auditory signal
detection task. Concentration-related decrements in signal detection, beginning
at 300 ppm (1041 mg/m3), were observed. CFF frequency also decreased in a con-
centration related fashion. Many motor decrements were observed at 800 ppm
(2,776 mg/m3), the only concentration at which these tests were conducted.
Fodor and Winneke (1971) reported a similar study in which 300 and 800 ppm (1,041
and 2,776 mg/m3) both decreased CFF frequency and impaired signal detection
behavior; however, the two DCM levels did not produce effects significantly
different from each other.
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—250, 500, 748 and 997 ppm (870, 1,740, 2,600, and 3,470
mg/m3)--through a face mask. Seven days later they were observed under con-
trol conditions. The second group was studied under identical conditions, but
in reverse order. The subjects were exposed to each concentration for 30 minutes
with no break in exposure; total exposure time for each individual was two hours.
005DC4/A 5-1 11/14/83
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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 DCM, no sta-
tistically significant impairment in any measured performance was observed,
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 to 40 years) to
201 ppm (700 mg/m3) of 99.5 percent pure DCM or to 7 ppm (80 mg/m3) CO for 4 hours
in an 8-m3 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 4 hours of OCM exposure was about 17 ppm
(60 mg/m3), and COHb was 5.1 percent. Alveolar CO after CO exposure for 4 hours
was 4 ppm (50 mg/m3), and COHb was 4.85 percent. Thus, there was COHb and alveo-
lar 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 de-
creases in performance after exposure to both compounds.
These preceding studies can only be considered a preliminary survey of
possible CMS and behavioral consequences of DCM exposure. Only one study
(Winneke, 1974) reported concentration effects data on CFF and signal detec-
tion, both possibly involving the alertness of the subjects. CFF is a primitive
measure of CMS activation. The lowest concentration of DCM reported to have
affected behavior was 200 ppm (694 mg/m3) (Putz et al., 1976). They also repor-
ted vigilance decrements in addition to compensatory tracking decrements. In
spite of these few preliminary findings, the results are consistent with the hypo-
thesis that DCM effects are depressant and also agree well with the available
laboratory animal data, reported elsewhere in this chapter.
Data are required on the effects of DCM upon other behaviors. No data
were found regarding cognitive skills, and no concentration-effects data were
available for motor skills or hand-eye coordination. No quantitative tests
of electrophysiological effects of DCM in humans were found. Finally, no
data regarding the effects of DCM in combination with other pollutants or
medicinals were found.
Putz et al. (1976) concluded that DCM produced its effects via its prin-
cipal metabolite, COHb. Stewart et al. (1972a) had suggested this possibility,
005DC4/A 5-2 11/14/83
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Winneke (1974) presented evidence suggesting that DCM is more toxic than CO. He
compared subjects exposed to 50 or 100 ppm CO for 5 hours to subjects exposed to
300, 500, and 800 ppm (1041, 1730, and 2776 mg/m3) DCM for 4 hours. OCM-exposed
subjects showed more effects on the same tasks. Comparison to data presented by
Putz et al. (1976) reveals that equivalent COHb levels were produced by 70 ppm
CO and 200 ppm (694 mg/m3) DCM. Examination of the behavioral results of the
study by Putz et al. reveals that DCM always produced greater decrements than
did CO. Logically DCM should be more toxic than CO, since it not only produces
COHb, but, because of its lipophilic qualities, it probably has some primary
neural effect. Additional data are required to explore this issue.
5.1.1.2 Other Experimental DCM Effects—Stewart et al. (1972a,b) reported no
effects of exposure to 500 or 1,000 ppm (1,730 or 3,460 mg/m3) DCM for 2 hours
on various hematological, hepatic, or renal measures. Continuous electrocardio-
graphic monitoring during one to two exposures to 1000 ppm (5,460 mg/m3) was not
reported to cause arrhythmias. Gamberale et al. (1975) reported that DCM expo-
sure up to 1,000 ppm (3,460 mg/m3) produced no alterations in heart rate. Based
on laboratory animal exposures, it is not reasonable to expect other internal
organ system involvement in healthy humans with low-level acute exposures.
Stewart and Dodd (1964) studied groups of three to five men and women
(25 to 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 1 hour, both the erythema and paresthesia subsided.
Studies of human blood jji vitro from individuals homozygous for sickle
cell anemia (Matthews et al., 1977, 1978) have shown that DCM caused 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., per-
sons 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 5-3 11/14/83
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5.1.1.3 Accidental Exposure—A number of case reports have implicated expo-
sure to DCM as one factor in human fatalities. Repeated paint stripping acti-
vities were associated with the death of a 66-year-old man in a report by
Stewart and Hake (1976). Although mixed exposure occurred, DCM is strongly
implicated as a causative agent because of the striking association between
exposure and chest pain, the high correlation between cardiovascular effects
of pure DCM and a commercially-formulated paint stripper (Aviado et al., 1977),
and the well-established metabolism of DCM to CO. Neither air levels of DCM
nor COHb levels were discussed in the report.
DCM was a probable factor in the death of a workman who became unconscious
and fell into a vat containing the solvent. Death occurred after repeated
myocardial infarctions (Kuzelova et al., 1975). Overexposure to DCM may have
been a factor in the death of one of four men rendered unconscious in a fac-
tory where DCM was used to extract oleoresin from dried plant materials.
Death was attributed "presumably" to CNS depression (Moskowitz and Shapiro,
1952).
Excessive prolonged exposure to DCM was judged by Bonventre et al. (1977)
to be the principal factor in the death of a 13-year-old boy who had used
paint remover containing DCM. Neither COHb nor ambient concentrations were
reported. Levels of DCM found in the liver, blood, and brain were 14.4 mg/
100 g, 51 mg/100 ml, and 24.8 mg/100 g, respectively.
High COHb levels in blood were found in a 40-year-old male who was found
unconscious after exposure to an unknown concentration of DCM leaking from
a degreasing factory (Benzon et al., 1978). When examined 1.5 hours after the
incident, this individual had an initial arterial blood COHb of 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. Another exposed
male, 50 years old and with a history suggestive of ischemic heart disease, did
not lose consciousness. When he was 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 follow-
ing 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 creative phos-
phokinase was not elevated, suggesting no myocardial injury. Subsequent ECGs
showed no further changes. Thirty-four hours after his admission to a hospital,
blood levels of COHb had decreased to 3 percent. He was subsequently discharged.
005DC4/A 5-4 11/14/83
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Exposure to DCM vapors as well as skin contact with DCM resulted in typical
symptoms of solvent intoxication for a worker who was exposed for about 4 hours
to DCM vapors emanating from an open drum and to DCM liquid from dipping his bare
hands into the liquid to clean copper gaskets (Hughes, 1954). There was no pro-
vision for air ventilation in the workrooms. Symptoms included excessive fatigue,
weakness, sleepiness, 1ightheadedness, chilly sensations, and nausea. About
2 hours after the exposure, cough and substantial pain developed and pulmonary
edema was diagnosed upon his admission to a hospital. DCM was considered to be
the pronounced respiratory irritant. In contrast to the observation of Stewart
and Dodd (1964), no adverse dermal effects were noted.
Asphyxia was judged to be the cause of death of a 20-year-old male found
slumped over a tank containing DCM (Winneke et al., 1981). Autopsy findings
included chemical burns on the forehead, moderate edema of the brain, and
pulmonary congestion with focal hemorrhage. The level of DCM in postmortem
blood was 29.8 mg percent.
Systematic conclusions from accidental exposure data are difficult. Exact
exposure levels and durations are unknown. Simultaneous exposure to other sub-
stances is almost always the case. Nonetheless, some observations can be sum-
marized.
Human case studies involving myocardial infarction have been reported and
have included fatalities resulting from, or closely associated with, exposure to
DCM. Nonfatal exposures have caused ECG changes that were similar to those
induced by CO. (It is as yet unclear what the relative contributions of DCM and
its metabolite, CO, are to those effects.) The case histories of certain exposed
individuals suggest the existence of underlying cardiovascular disease. This
effect may, therefore, be significant to this human subpopulation. Hepatoto-
xicity has not been reported in any human case report, even following fatal
exposures. The only evidence of human nephrotoxicity resulting from DCM exposure
was a finding of congested kidneys following a fatal exposure. Ocular toxicity
other than eye irritation and congested conjunctivae has not been reported in
humans exposed to DCM.
5.1.2 Chronic Effects
5.1.2.1 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 5 days/week
for 5 weeks to DCM concentrations of 50 ppm (173 mg/m3) in week 1, 250 ppm (867
005DC4/A 5-5 11/14/83
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mg/m3) in week 2, 250 ppm (867 mg/m3) in week 3, 100 ppm (346 mg/m3) in week 4,
and 500 ppm (1730 mg/m3) in week 5. Three subjects were exposed for 1 hour/day,
three subjects were exposed for 3 hour/day, and four subjects were exposed for
7.5 hour/day. This complex experimental design was further confounded by attempts
to separate smoking and 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 nine
female subjects to 250 ppm (867 mg/m3) DCM for 1, 3, or 6.5 hour/day for 5 days.
TABLE 5-1. COHb CONCENTRATIONS IN NONSMOKERS EXPOSED
TO DCM AT 250 ppm (869 mg/m3) FOR 5 DAYS
Exposure Time
h/day
Pre-exposure
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 DCM 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/day for not more than
5 successive days.
5.1.2.2 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 DCM estimated to range from 660 to 900 ppm (2,293 to 3,127
mg/m3) (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 5-6 11/14/83
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Collier (1936) reported four cases of occupational exposure to paint re-
mover containing approximately 96 percent DCM. The men, all of whom were pro-
fessional painters, had been exposed to lead for 5 to 14 years. During one
autumn, while they were removing paint, the workers complained of loss of appe-
tite, dullness, faintness, and giddiness while using the remover and during 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 DCM-induced
toxemia, since the acute symptoms subsided upon cessation of working with the
paint remover. The second painter, aged 45, experienced 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/m3) 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.
5.2 EFFECTS ON LABORATORY ANIMALS
Much of the experimental research on the effects of DCM has been done on
laboratory animals. Such work can be used to elucidate the general principles
of DCM action and to discover which target organs or systems are involved.
Generalization of laboratory animal findings to humans, however, is not straight-
forward. Human health effects must, in the final analysis, be assessed in man.
5.2.1 Acute Effects
5.2.1.1 Lethality--Qn1y preliminary work has been done on the lethality of
DCM. No systematic data exist which would make it possible to specify a time-
by-dose-by-lethality function.
Table 5-2 is a summary of lethality data. The inhalation data are remark-
ably consistent between rats and mice. The one report on guinea pigs suggests
that they may be more sensitive to DCM but procedural variation cannot be
ruled out. Data on shorter exposures are required since accidental exposures
and substance abuse frequently involve high concentration, short-term profiles.
A curve of LCbu for a range of exposure times would be valuable.
Oral and intraperitoneal injection LDbu data appear to be remarkably con-
sistent across investigations and species. Exceptionally low values (Zakhari,
1965) and high values (Ugazio et al., 1973) are possibly due to procedural
005DC4/A 5-7 11/14/83
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o
o
TABLE 5-2. ACUTE LETHAL TOXICITY OF DCM
i
CO
Route of
Administration
Coral
Intr aperi tonea 1
Inhalation
Species
mice
rats
rats
mice
mice
mice
mice
mice
dogs
mice
mice
rats
rats
guinea
pigs
Duration of
Dose Exposure
1987 mg/kg
4368 mg/kg
2388 mg/kg
448 mg/kg
1330 mg/kg
1995 mg/kg
1990 mgAg
1990 mgAg
1260 mgAg
14,100 ppm 6 hours
16,100 ppm 7 hours
ppm
25,600-28,000 1.5 hours
ppm
16,100-18,150 6 hours
ppm
11,550 ppn 6 hours
Effect
LD50
LD50
LD50
LD50
100%
survival
20%
survival
LD50
LD50
LD50
LD50
LJD50
(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 rng/m3.
CO
OJ
-------
differences. The mean LD50 from Table 5-2, excluding the low and high values
mentioned, for injection and oral doses in rodents is near 2000 mg/kg. No data
for other sites of injection were found. Oral and intraperitoneal data cannot
be compared with inhalation data because of 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.
The course of events during lethal exposures to DCM via inhalation was
described by Berger and Fodor (1968) who exposed rats to DCM concentrations
ranging from 2,800 to 28,000 ppm (9.7 to 97.3 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 brain electrical activity. Rats
exposed to concentrations to 25,000 to 28,000 ppm (87 to 97.3 g/m3) ceased to
exhibit electroencephalographic (EEC) activity after only 1.5 hours; those
exposed to concentrations between 16,000 and 18,000 ppm (55.6 and 62.5 g/m3)
ceased to exhibit EEC activity after 6 hours.
5.2.1.2 Nervous System and Behavior—The nervous system is a likely target
organ because DCM is lipophilic and thus is concentrated in myelin. Brain
tissue has been shown to have high concentrations of DCM following exposure
(Savolainen et al., 1977; Bergman, 1978; see also Section 4.1.3). Pankow
et al. (1979) showed that nervous tissue function is affected by DCM dosage.
They demonstrated a linear decrease in sciatic nerve conduction velocity, using
intraperitoneal injections of DCM at dose levels of 1 to 6 mmoles/kg.
EEC and rapid eye movement (REM) have been used to characterize sleep
during DCM inhalation. These data form a concentration-related continuum from
500 to 3000 ppm (1,735 to 10,410 mg/m3) in which REM sleep was reduced in
duration beginning at about 500 ppm (1,735 mg/m3) (Fodor and Winneke, 1971).
At 3,000 to 9,000 ppm (10,410 to 31,230 mg/m3), sleeping time was increased
with a further decrease in REM sleep (Berger and Fodor, 1968). Higher con-
centrations eventually produced a flat EEC after coma (Berger and Fodor, 1968).
The above may be viewed as a continuum of effects beginning with REM sleep
reduction at 500 ppm (1,735 mg/m3) and ending in brain death after 1.5 hours
at 27,000 ppm (93.7 mg/m3) or 6 hours at 17,000 ppm (59 mg/m3).
Measures of general activity level are commonly used variables to char-
acterize behavioral effects. However, general activity is not a simple or
005DC4/A 5-9 11/14/83
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unidimensional measure. Frequently, some measures of activity level are affected
though others are not. A concentration of 5,000 ppm (17,370 mg/m3) DCM admini-
stered for 1 hour to male rats was sufficient to decrease 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-hour exposure to 1,000 ppm (3,474 mg/m3) of DCM. Weinstein
et al. (1972) exposed female mice in groups to about 5,000 ppm (17,370 mg/m3)
DCM for 24 hours and observed progressive decreased spontaneous activity.
Little can be said about the effects of acute DCM exposure upon the
nervous system and behavior of laboratory animals. The effect appears to be
consistently depressive above 500 ppm (1,737 mg/m3). However, to say that an
effect is "depressive" is to oversimplify matters. Also, only activity level or
sleep has been measured with DCM acute exposure in laboratory animals. No work
has been found on the effects of acute DCM exposure upon motor tasks, sensory
discrimination, schedule-controlled behavior, or response acquisition in labora-
tory animals. Similarly, no research has been published concerning the effects
of acute DCM exposure on any electrophysiological variable other than nerve con-
duction velocity and in a secondary manner, brain electrical activity. There
have also been no follow-up studies to show whether acute exposure effects were
(or were not) reversible.
5.2.1.3 Cardiovascular Effects—Aviado et al. (1977) used pentobarbital-anes-
thetized, artificially ventilated, open-chested dogs to examine the cardio-
vascular 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/m3) and to the same concen-
trations 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 5 minutes. At the higher
doses (2.5 percent and 5 percent), all hemodynamic effects were consistent with
primary depression of myocardial contractility (Table 5-3): left ventricular
dp/dt (the time-dependent rate of rise of ventricular pressure, which is a mea-
sure of myocardial contractility), fell, as did LV pressure. Left ventricular
end-diastolic pressure (left ventricular filling pressure) rose. However, car-
diac output fell. Mean arterial pressure fell, but calculated peripheral vas-
cular resistance (MAP-central venous pressure/cardiac output) rose. Heart rate
fell nonsignificantly when 5 percent DCM (50,000 ppm) (174 g/m3) was employed.
005DC4/A 5-10 11/14/83
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TABLE 5-3. SUMMARY OF CARDIOTOXIC ACTION OF 5% DICHLOROMETHANE
Function
HR
MAP
LVP
CVP
LVEDP
LVdP/dt
CO
SV
VR
Taylor et
1%
0%
0%
0%
9%
17%
14%
14%
25%
al.
t
t
4-
4-
4-
t
(1976)
(NS)
(NS)
Aviado et
11%
4%
4%
—
125%
22%
36%
33%
44%
al.
4,
4-
4-
t
4-
4-
4-
t
. (1977)b
(NS)
(NS)
(NS)
(NS)
Comments:
Rabbits
Pentobarbi tal-anesthetized
Spontaneously breathing
Close-chested
1-min. exposure (except
1.5 min. for CO)
Static dose (5%)
Dogs
Pentobarbital-anesthetized
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-Diastolic 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
Results presented are for the highest dose (5%) of dichloromethane.
The authors concluded that the effects were compound-related and that DCM
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/m3)
DCM in air (except for cardiac output, which was measured 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
005DC4/A
5-11
11/14/83
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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 the
heart rate of 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 ventricular end-diastolic pressure did not change in rabbits, but signifi-
cantly increased in dogs.
Trained male beagles (size unspecified) were repeatedly 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 DCM, 0.008 mg/kg epinephrine was injected in-
travenously. 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.
In two reports, Aviado and coworkers (Aviado, 1977; Zakhari, 1977) men-
tioned arrhythmias occurring in dogs exposed to 5 percent (50,000 ppm; 174 g/m^)
DCM. The simultaneous administration of alcohols prevented these arrhythmias.
In a subsequent review Aviado (1975) stated 0.5 percent of (5,000 ppm; 17 g/m3)
DCM sensitized the heart to epinephrine. Levels of 2.5 and 5.0 percent (25,000
and 50,000 ppm) were reported to induce arrhythmias in anesthetized monkeys
(Aviado, 1975; Belej et al. , 1974) but produced only tachycardia in unanesthe-
tized monkeys (Aviado, 1975). However, Aviado and coworkers have not described
their experimental protocols or the nature of the arrhythmias in their dog and
monkey experiments.
005DC4/A 5-12 11/14/83
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Aviado and Belej (1974) ^xposed Swiss mice (25 to 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-Dawley 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
3 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 (sex not specified). Six were injected sub-
cutaneously with 90 mg/kg unspecified DCM. Six others were used as controls.
DCM reduced the blood pressure of the hypertensive rats by about 10 percent
but was ineffective in reducing blood pressure of the normotensive rats.
Adams and Erickson (1976) exposed eight trained mongrel 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) DCM for 2 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 2 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, coronary flow, and 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 DCM 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
A second exposure to DCM was given 1 hour after the first. After both
005DC4/A 5-13 11/14/83
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exposures, 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 concerning 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.
DCM used in the study of cardiopulmonary effects generally was at a very
high level (> 30,000 ppm; 104 mg/m3) in a short-term exposure (£ 5 minutes).
Knowledge of effects of such levels would be important for accidental or sub-
stance abuse exposures but are otherwise not particularly useful for health
assessment. For such short exposures, thresholds for effects are quite high.
It is hard to interpret such short-term exposures considering that asymptotic
blood level is not approached until 1 to 2 hours. Data on longer duration
acute exposures are not available.
5.2.1.4 Hepatic, Pancreatic, and Renal Effects—Idaassen and Plaa (1966)
injected Swiss-Webster mice (25 to 35 g) intraperitoneally with analytical
grade DCM. Doses of 13,300 mg/kg had no effect on Bromsylphalein (BSP)®
retention or on serum glutamic pyruvic transaminase (SGPT) activity 24 hours
after injection. No histopathological change was seen upon examination of the
1iver.
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
LDj-n was 1,260 mg/kg). Histopathology showed moderate neutrophilic infiltration
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 14C-DCM up to 2,210 mg/kg dissolved in mineral oil. The rats
were sacrified 24 hours later. No liver necrosis was seen and no change in glu-
cose-6-phosphatase activity was observed. Labelled DCM (14C) 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.
Weinstein et al. (1972) exposed female ICR mice (13 to 20 per group, 23
to 27 g) continuously to approximately 4,893 ppm (17,000 mg/m3) 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. Body
weight decreased, liver weight increased absolutely and as a ratio to body
005DC4/A 5-14 11/14/83
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weight, liver triglycerides increased (indicating liver toxicity), glycogen
decreased, and protein synthesis was reduced as shown by reduction 3H-leucine
incorporation.
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
vesicles, pernuclear cisternae dilated, and lipid droplets increased. Mitochon-
dria, 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 6 hours to a 5,181 ppm (18,000 mg/m3) vapor concentration of reagent
grade DCM. Animals were sacrified immediately after exposure and liver samples
were taken. Liver and serum were analyzed for triglycerides, which increased
markedly in the former and decreased in the latter. Liver phospholipids showed
no change, neither did serum-free fatty acid. Liver slices were incubated with
14C-palmitic acid. The uptake of 14C was similar to controls. Uptake after
14C-leucine did not differ between controls and exposed samples.
Differences in protein syntheis in the above two studies in protein syn-
thesis may be due to different periods of exposure and the use of different
animal species.
Harms et al. (1976) cannulated the bile duct of male Sprague-Dawley rats
(350 to 450 g) after pretreatment with intraperitoneal injections of 670 mg/kg
DCM dissolved in corn oil. Tritiated 3H-insulin was instilled into the duct
24 hours later. DCM 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, intraperitoneally injected 860 mg/kg DCM (dissolved in corn
oil) in male Sprague-Dawley rats (280 to 320 g). The bile duct was cannulated,
and bile duct pancreatic flow and its contents were measured and compared with
controls. DCM induced increased pancreatic bile duct flow, decreased protein
concentration, and increased 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 experiment with secretin indicated that these changes were not related
to that substance or, after an ancillary experiment with atropine, to any
cholinergic effect.
005DC4/A 5-15 11/14/83
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Plaa and Larson (1965) injected 10 male Swiss mice (18 to 30 g) intraperi-
toneally with 1330 mg/kg of DCM (source and quality unidentified) dissolved in
corn oil. No glycosuria or proteinuria was detected 24 hours later. Two sur-
viving mice of the 10 that had been injected with 1,995 mg/kg DCM 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.
Klaassen and Plaa (1966) also injected male Swiss-Webster mice (25 to 35
g) intraperitoneal ly with 13.3 g/kg analytical grade DCM. These mice showed
no glycosuria, proteinuria, or changes in BSP excretion 24 hours after injection.
Two groups of 10 mice each were gavaged for 3 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."
Kluwe et al. (1982) have investigated the effects of DCM on renal tubular
cells of adult male Fischer 344 rats by injecting DCM (1300 mg/kg), i.p., in
corn oil. Renal proximal tubular swelling was observed in the cortex and in
the outer medulla. Tubular cell functions measured included organic ion transport,
ability to maintain a potassium gradient, and the rate of oxygen utilization.
None of these functions were altered by DCM. Rats were sacrificed 2, 12, 24,
48, or 96 h after injection and kidneys removed and thin slices prepared. DCM
was not observed to alter glomerular, distal tubular, inner medullary or papil-
lary cell morphology. Although DCM did cause an increase in the fraction of
water in renal tissue, the lack of effect on other parameters suggested to the
authors that little or no functional cell disturbance had occurred.
5.2.1.5 Other effects-- 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 conjunc-
tivae for 2 weeks, conjunctiva! edema for a week, sloughing for 3 days, and
increased corneal thickness for 9 days. Iritis and keratitis appeared within
6 hours and lasted for 7 and 14 days, respectively. Intraocular pressures in-
creased also. Increased corneal thickness developed in rabbits exposed to DCM
vapor at concentrations of 1,750 and 17,500 mg/m3 (504 and 5,040 ppm).
Sahu and Lowther (1981) observed that inhalation of DCM by 2-month-old
Sprague-Dawley rats led to pulmonary injury, presumably rupture of type II
005DC4/A 5-16 11/14/83
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alveolar cell membranes and release of cell contents into the airways. Rats
were exposed to about 4000 ppm (13,896 mg/m3) for 5 h/day, 5 days/wk, for 4 weeks.
Pulmonary secretions were obtained by lung lavage. The soluble supernatant of
the lung homogenate and the cell-free lung lavage were used for determination
of lung lipid peroxidation. Lipid peroxidation was significantly elevated
(P < 0.05).
5.2.1.6 Summary of Effects of Acute Exposure—Of all the organ systems stu-
died, the central nervous system appears to be affected by DCM at levels rang-
ing from 500 to 1,000 ppm (1,730 to 3,470 mg/m3). While only short-term
exposures were used, levels of over 20,000 ppm (69 mg/m3) were required before
cardiac function changes were produced. Hepatic effects were reported at expo-
sure levels as low as 5,000 ppm (17,300 mg/m3). While it is difficult to compare
results of injection studies to those of inhalation exposures, it appears that
half the LDbU is required to produce hepatic or renal changes. The above com-
parisons are based upon the lowest level reported. The behavioral data were,
however, in two cases, for levels of exposure at or below 1,000 ppm. The
weight-of-evidence for short-term exposures indicates that the CNS is the pri-
mary target organ for DCM.
The concentrations of DCM necessary to depress cardiac function (25,000 to
50,000 ppm) in acute experiments are so high that chronic long-term exposures
of humans to levels considerably in excess of 250 ppm (865 mg/m3) would be
unlikely to have any effect. However, comparatively little research has been
done on acute DCM effects on laboratory animals.
5.2.2 Chronic Effects
5.2.2.1 CNS Effects—In early studies of DCM chronic exposure (Heppel et al.,
1944; Heppel and Neal, 1944), dogs, monkeys, rabbits, guinea pigs, and rats were
exposed 4 hours/day, 5 days/week for 8 weeks to 5,000 or 10,000 ppm (17,300 or
34,600 mg/m3) DCM. Unfortunately, only qualitative observations of behavioral
"symptoms" were reported. No symptoms were noted in groups exposed to 5,000 ppm
(17,300 mg/m3). At 10,000 ppm (34,600 mg/m3) all species were affected but in
different ways. Dogs became excitable and hyperactive. Monkeys became pro-
gressively more inactive and, by the end of each daily exposure, lay prostrate
with barely perceptible respiration. Rabbits first were excitable and then
became inactive toward the end of each daily session. Guinea pigs and rats
simply became more inactive. All species appeared well within 1 hour after
cessation of exposure. Only monkeys (n = 2) appeared to develop behavioral
005DC4/A 5-17 11/14/83
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tolerance. No other cumulative effects were reported over the course of the
8 weeks.
Weinstein et al. (1972) exposed female mice to 5,000 ppm DCM continuously
for 7 days. Only qualitative behavioral observations were reported. For the
first few hours of exposure, the animals exhibited an increased activity level
and increased food and water intake. This was followed by decreased activity,
"hunched" posture, dehydration, and the appearance of roughened yellow
coat. By the fourth day of exposure, many of the mice had adapted so that by
the end of the study they were virtually normal.
Thomas et al. (1972) studied activity level in mice continuously exposed
to 25, 100, or 1,000 ppm (87, 347, or 3,470 mg/m3) for 14 weeks. The lowest expo-
sure level elevated activity level. There was no effect at 100 ppm (347 mg/m3),
and at 1,000 ppm (3470 mg/m3), there was reduced activity level. The increased
activity level in the 25 ppm (87 mg/m3) exposed group was, if replicable, pro-
bably due to the increased sensory stimulation due to the odor of DCM.
Savolainen et al. (1977) exposed male rats to 500 ppm (1,735 mg/m3) DCM, 6
hours/day for 4 days. Exposed rats engaged in more grooming behavior than con-
trols during the first exposure hour but by the 17th exposure hour (the 3rd day)
exposed rats no longer differed from controls. Other behaviors were observed,
but by their absence in the results summary, it may be assumed that only grooming
was affected. 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. Relatively small increases in acid proteinase and
nonspecific cholinesterase activities were reported, but the determinations for
treated animals were made by only two assays and, therefore, may be of ques-
tionable significance.
Since only one study of activity level is available, it is difficult to
conclude much about neurobehavioral effects of chronic exposures. As in acute
exposure studies, no other behaviors, such as sensory, motor response acqui-
sition, or schedule-controlled behaviors, were studied. Where effects were
seen, no follow-up studies were conducted to determine the irreversibility of
effects.
Suggestions of species differences in DCM sensitivity occurred in the
studies by Heppel et al. (1944) and Heppel and Neal (1944). Based on these
qualitative observations, one could conjecture that DCM behaves in a manner
similar to other anesthetics: there is an early excitatory phase followed
005DC4/A 5-18 11/14/83
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by progressive debilitation. If some species had different sensitivities to
DCM, they might remain in the excitatory phase or progress more rapidly into
debi1itation.
Several reports mentioned an increased tolerance to DCM over the duration
of chronic exposures (Weinstein et al. , 1972; Savolainen et al. , 1977). Heppel
et al. (1944) also reported monkeys to have adapted somewhat. Probably adap-
tation, like sensitivity, is a function of species, exposure level, and duration.
Because of the paucity of data and the qualitative approaches used, these
conclusions must be regarded as tentative. Effects have been reported at expo-
sure levels as low as 1,000 ppm (3,470 mg/m3) but not lower. The one case of
increased activity level at 25 ppm (87 mg/m3) must be discounted.
5.2.2.2 Hepatic and Renal Effects—Many of the "high-dose" chronic animal stu-
dies with DCM have revealed a certain degree of liver and kidney involvement as
target organs. The magnitude of this involvement increases 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) observed moderate
centrilobular congestion and fatty degeneration of the liver in dogs and guinea
pigs exposed to 10,000 ppm (34,740 mg/m3), 4 hours/day, 5 days/wk for 8 weeks.
Weinstein et al. (1972) reported identical findings after exposing mice to
5,000 ppm (17,370 mg/m3) continuously for 7 days. High mortality was observed
by MacEwen et al. (1972) where 14 weeks of continuous exposures at 1,000 or
5,000 ppm (3,480 or 17,400 mg/m3) resulted in severe toxic effects and a high
degree of mortality in mice,'rats, dogs, and monkeys.
More importantly, chronic mouse studies by Weinstein and Diamond (1972)
and Haun et al. (1972) have revealed that continuous exposure to even such low
levels of DCM as 100 ppm (347 mg/m3) can effect changes in both liver function
and cell architecture.
Weinstein and Diamond (1972) exposed ICR mice (17 to 25 g) continuously for
3 days to 10 weeks to 100 ppm (347 mg/m3) 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. Tri-
glycerides increased approximately threefold at week 2, nearly fourfold by week 3,
then decreased to about double at week 4. Four mice withdrawn at 3 days showed
no abnormalities, but at 7 days, centrilobular fat accumulation was seen, accom-
panied by a decrease in liver glycogen. These abnormalities persisted to the
005DC4/A 5-19 11/14/83
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termination of the experiment at 10 weeks. During this 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.
Haun et al. (1972) exposed mice, rats, dogs, and monkeys to 25 and 100 ppm
(87 and 347 mg/m3) 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.
Norpoth et al. (1974), when studying the possibility of enzyme induction
in inhalation of hydrocarbon solvents, tested DCM. Male SPF Wistar rats (80
to 100 g) were exposed 5 hours/day to vapors containing 0, 500, or 5,000 ppm
(0, 1,737, or 17,370 mg/m3 DCM) of the hydrocarbon solvents for 10 days or
250 ppm (869 mg/m3 DCM) of the solvents for 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 P4S(j and microsomal aminopyrine demethylase activity were
determined.
Following the 10-day exposure, a significant increase in liver cytochrome
P45u in animals exposed to 500 ppm (1,737 mg/m3) but not in animals exposed to
5,000 ppm (17,370 mg/m3) of the compound was reported. In contrast, aminopyrine
demethylase activity was not elevated in animals exposed to 500 ppm (1,737 mg/m3)
DCM but was substantially elevated in those exposed to 5,000 ppm (17,370 mg/m3)
DCM. 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 P45u 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.
Loyke (1973) induced chronic renal hypertension in 13 Sprague-Dawley 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 3
005DC4/A 5-20 11/14/83
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months, 11 experimental rats ^nd 3 controls were injected subcutaneously 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.
In the Dow Chemical (1980) chronic inhalation study in rats, exposure to
500, 1500, or 3500 ppm (1,735, 5,205, or 12,145 mg/m3), for 6 hours/day, 5 days/
week, and 2 years resulted in exposure-related non-neoplastic hepatic lesions
in both males and females. Grossly, the effect was most prominent in females
exposed to 3500 ppm and consisted of increased numbers of dark or pale foci.
The percentages of total rats with any degree of vacuolization were 17 percent,
38 percent, 45 percent, and 54 percent in the males of the 0, 500, 1,500, and
3,500 ppm exposure groups, respectively, and 34 percent, 52 percent, 59 percent,
and 65 percent in the females, respectively. The degree of severity tended to
increase with the dose. Further information relating to protocol and additional
observations are discussed in Section 5.3.3.1.1.
In the Dow Chemical (1980) 2-year inhalation study in hamsters at the same
levels described above for rats, a variety of gross and histopathologic observa-
tions were recorded for hamsters sacrificed at 6-, 12-, or 18-month interim
kills. Histopathologically, exposure-related differences consisted of hamsters
with amyloidosis of the liver, kidney, adrenals, thyroid, and spleen. Further
observations are discussed in Section 5.3.3.1.3.
In the Dow Chemical (1982) 2-year inhalation study in Sprague-Dawley rats
(Spartan substrain) exposed to 50, 200, and 500 ppm (174, 696, and 1,735 mg/m3),
no definite exposure-related histopathologic findings were noted. An exception
was the interim sacrifice of females at 15 months. Some had a focus or foci of
altered liver cells. There were significant increases in non-neoplastic liver
lesions (i.e., hepatocellular vacuolization and multinucleated hepatocytes) in
female rats at 500 ppm (1,935 mg/m3). Further observations are discussed in Sec-
tion 5.3.3.1.4.
Histopathologic changes in liver cells of Fischer 344 rats exposed to vari-
ous levels of DCM in drinking water over a 2-year exposure period have been
reported by the National Coffee Association (1982). Details are described in
Section 5. 3. 3.1.5.
005DC4/A 5-21 11/14/83
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5.2.2.3 Morbidity and Mortality--Heppel et al. (1944) conducted two inhalation
experiments. One used 1,700 mg/m3 (489 ppm) commercial DCM for 7 hours daily,
5 days/week for 6 months. The other involved exposure for 4 hours/day, 5 days/
week, for 7 to 8 weeks at a concentration of 34,000 mg/m3 (9,789 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
depression, usually becoming prostrate at the end of each daily 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.
5.2.2.4 Summary of Effects of Chronic Exposures—Chronic exposure to high levels
of DCM has been reported to produce alterations in behavior and in hepatic and
renal function. When deaths occurred, they were frequently due to pulmonary
congestion. Apparently these effects begin at about 500 to 1,000 ppm with be-
havioral effects in evidence early, followed by changes in internal organs.
No data are available to determine if the effects seen were reversible after
cessation of chronic exposures. This issue could best be addressed through
additional research.
5.3 TERATOGENICITY, MUTAGENICITY, AND CARCINOGENICITY
5.3.1 Teratogenicity, Embryotoxicity, and Reproductive Effects
It is not possible, on the basis of limited available data, to define the
full potential of DCM to produce adverse teratogenic or reproductive effects.
005DC4/A 5-22 11/14/83
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Human epidemiology studies tMt evaluate the effects of DCM on the exposed
population are difficult to conduct. Each of the available mammalian studies
had methodological drawbacks that do not allow for conclusive evaluation of the
ability of DCM to produce a teratogenic response over a wide range of doses,
which should include doses high enough to produce signs of maternal toxicity
and lower doses that do not produce this effect. Other studies in chicken
embryos have indicated that DCM disrupts embryogenesis in a dose-related manner
(Elovaara et al., 1979). However, since administration of DCM directly into
the air space of chicken embryos is not comparable to administration of dose
to animals with a placenta, interpretation of this result related to the poten-
tial of DCM to cause adverse human reproductive effects is not possible. 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.
5.3.1.1 Animal Studies—The following discussion subscribes to basic viewpoints
and definitions of the terms "teratogenic" and "fetotoxic" as summarized by
the Office of Pesticides and Toxic Substances (U.S. EPA, 1980):
Generally, the term "teratogenic" is defined as the tendency to produce
physical and/or functional defects in offspring J_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, reduc-
tions in fetal weight, enlarged renal pelvis edema, and increased incidence of
supernumerary 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 contin-
uous, 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 is 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 5-23 11/14/83
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be embryo or fetal toxicity as comprised of those effects which are potentially
reversible. This subcategory would therefore include such effects as weight
reductions, reduction in the degree of skeletal ossification, and delays in
organ 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 is performed on the neonate, however, no conclusion may be drawn
concerning permanent organ functional 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 irreversible manner.
5.3.1.2 Mice--Swiss-Webster mice were exposed via inhalation to 1,250 ppm
(4,350 mg/m3) of DCM for 7 hours daily during days 6 through 15 of gestation
(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 a vaginal plug was
observed. Caesarean sectioning of dams was performed on day 18 of gestation.
Dams were evaluated for body weight gain and various organ weights. Mater-
nal COHb 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 malformations (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 COHb values with return to
control levels after 24 hours. On the basis of the maternal liver weight obser-
vations, 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 increase in liver weight per animal, the observed increased maternal
weight gain might not have been a result of gains in liver weight alone.
Of the 12 litters examined, a statistically significant number of litters
contained fetuses that had a single extra center of ossification in the sternum.
005DC4/A 5-24 11/14/83
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This common variation in mice is thought to reflect the degree of embryonic
development. It is not known if this observation resulted from an accelera-
tion 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 this may be caused
by the average slightly smaller litter size compared to controls (10 vs. 12).
The litters in this treatment group also had a lower incidence of delayed ossi-
fication of the sternebrae (17 vs. 23 percent), split sternebrae (8 vs. 18 per-
cent), and ossification of the skull bones (25 vs. 36 percent). Cleft palate
and "rotated kidney" were observed in two (17 percent) of the DCM exposed fetuses,
and not observed in any of the control litters. Because of the low incidence
of these effects, these effects may reflect spontaneous malformation rates.
5.3.1.3 Rats--A study using the same design as that used for the mice (Section
5.3.1.2) was performed in Sprague-Dawley rats (Schwetz et al., 1975). Rats
inhaled DCM at 1,250 ppm (4,350 mg/m3) for 7 hours daily on days 6 through 15
of gestation, with day 0 being the day spermatozoa were observed in vaginal
smears. Dams were Caesarean sectioned on day 21 of gestation. All other pro-
cedures 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 not effect on the relative weight of the
liver. Carboxyhemoglobin values in the dams increased significantly 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 might indicate a slight but reversible
delay in development similar to delays in sternal ossification. However, since
this study evaluated only one dosage level, it is not possible to firmly estab-
lish the significance of this effect or its reversibility.
Hardin and Manson (1980) used Long-Evans rats to evaluate the effect of
exposure to DCM via inhalation at 4,500 ppm (15,660 mg/m3) for 6 hours daily,
7 days/week to determine whether exposures before and during gestation were
more detrimental to the developing conceptus than exposures before gestation
only. Minimal maternal toxicity, consisting of increased absolute and relative
liver weight and elevated COHb levels, was observed. The litters of rats exposed
005DC4/A 5-25 11/14/83
-------
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 DCM from the Hardin and Manson (1980) study.
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 that 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 demon-
strated 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
confirm these effects. Also, the entire field of behavioral teratology is in
its early stages of development (Buelke-Sam and Kimmel, 1979), and the signi-
ficance of alterations in behavioral effects to human risk assessment is not
clearly defined.
005DC4/A 5-26 11/14/83
-------
5.3.2 Mutagenlcity
Dichloromethane has been tested for mutagenic activity in bacteria, yeast,
insects, nematodes, mammalian cells in vitro, and rodents. These studies are
discussed below and are summarized in Tables 5-4 to 5-9.
5.3.2.1 Gene Mutations—
5.3.2.1.1 Bacteria. There are fourteen reports in the literature concerning
the mutagenic potential of DCM in bacteria; the Salmonella histidine reversion
assay was used in all of these studies (Simmon et al., 1977; Simmon and Kauhanen,
1978; Kanada and Uyeta, 1978; Jongen et al., 1978; McGregor, 1979; Snow et al.,
1979; Green, 1980; Rapson et al., 1980; Barber et al., 1980; Nestmann et al.,
1980; Green, 1981; Gocke et al., 1981; Nestmann et al., 1981; and Jongen et al.,
1982). Kanada and Uyeta (1978) also tested DCM in the B. subtil is rec assay.
DCM tested positive in all studies using Salmonella without or with metabolic
activation in strains TA100, TA1535, or TA98 when assays were performed in
sealed gas tight exposure chambers. Negative responses were reported by Rapson
et al. (1980) and Nestman et al. (1980) in standard assays, but these tests
are judged to be inadequate because DCM was added directly to the agar medium,
and no precautions were taken to prevent excessive evaporation and escape of
the test material. The tests were carried out at 37°C, which is very close to
the boiling point of DCM (39°C). It is very likely excessive evaporation
occurred. Data were presented in many of the reports, and a clear dose-related
response is apparent for each, with 10-fold or greater increases in revertants
observed at the highest doses compared to negative controls. The doses employed
and the responses observed are summarized in Table 5-4.
The purity of the test material was not given in any report. Because of
this, most positive responses must be viewed with caution; they may be caused by
substances other than DCM. For instance, formaldehyde, a metabolite of DCM,
could conceivably also form nonenzymatically in the aqueous solutions used for
biological testing by hydrolysis of DCM (March, 1977). However, because of the
consistency of positive responses with several different samples, it is more
likely the DCM itself is mutagenic. For instance, in their tests of DCM, Barber
et al. (1980) used a redistilled sample estimated to be >99.9 percent pure,
5-27
-------
TABLE 5-4. MUTAGENICITY TESTING OF OCM IN BACTERIA
i
INi
CO
Reference Test System Strain
Simmon et al . Salmonella/S9 TA1535
1977 vapor exposure TA1537
TA1538
TA98
TA100
Simmon and Sa1mone11a/S9 TA100
Kauhanen 1978 vapor exposure
Activation
System
None
Aroclor-1254
Induced rat
liver
mlcrosome
S9 mix
Concentration/Results
(Extrapolated from
Fig. 17) 0, 50,
100, 200, 400, and
800 ul/9 liter
desiccator
0 and 1 ml/9 liter
desiccator for 6.5
and 8 hours
(Extrapolated
from Fig. 17)
TA100
Dose (ul) jtevertants/plate
0
50
100
200
400
800
Met.
»h Act.
6.5
+
8
+
170
210
300
400
650
1350
TA100
revertants
Treated Control
688 133
1344 130
830 174
912 158
Comments
1. Toxicity not reported.
2. Number of revertants
observed for TA100 not
specified numerically.
3. Data not presented for
strains other than TA100.
4. Purity and source of
compound not provided.
5. Positive response.
1. Toxicity not reported.
2. Purity and source of
compound not provided.
3. Used as a positive
control In the testing of
2-chl oroethy 1 -chl oro-
formate.
4. Positive response.
-------
TABLE 5-4. (continued)
i
INi
Reference
Kanada and
Uyeta 1978
Jongen et al .
1978
Test System Strain
Salmonella/S9 TA98
and TA100
B. subtlUs
rec assay
testing
Salmonella/S9 TA98
vapor exposure TA100
Activation
System
PCB-lnduced rat
liver mlcrosome
S9 mix
Phenobarbltal-
Induced rat
liver mlcrosome
S9 mix
Concentration
Not reported
(ppm x 103)
0
5.7
11.4
14.1
22.8
57.0
Result
DCM reported negative
for both strains 1n
B. subtlUs and positive
for both in S. typhimurlum
TA100* TA98*
+S9 -S9 +S9 -S9
15Z+19 1Z9+1Z Zl+4 19+5
329~37 248+32 54+5 44+8
515+76 407+47 74+4 56+10
757~82 582+56 93+9 66+12
865+82 653+89 123+10 96+11
1201+191 740+94 149+42 110+42
Comments
1. Results summarized
1n abstract form.
2. Positive results
of "Ames" testing
supports reports by
other authors using
same system.
1. Testing conducted
1n gas tight perspex
boxes.
2. Only highest dose
exhibited less than
83% survival.
3. Purity of DCM not
reported.
4. Positive response.
(continued on the following page)
-------
TABLE 5-4. (continued)
tri
i
CO
O
Reference Test System Strain
Snow et al. Salmonella/S9 TA98
1979 vapor exposure TA100
Activation
System
DCM-1nduced Syrian
Golden hamster liver
S9 mlcrosome mix
Dose
(ul /Chamber)
0
100
300
500
1000
+"59
"55
177
463
642
972
Result
TA100*
-S9
63
142
274
468
632
TA98
+S9
38
47
69
92
39
-S9
19
31
46
61
72
Comments
1. Purity of DCM not
reported.
2. No Information about
variability of results.
3. Positive response.
*Mean calculated from three plates/dose.
(continued on the following page)
-------
TABLE 5-4. (continued)
en
i
CO
Reference Test System Strain
McGregor Salmonel1a/S9 TA1535
1979 vapor exposure
Nestmann et Salmonella/S9 TA1535
al. 1980 vapor exposure TA1537
TA1538
TA98
TA100
Activation
System
None . Atmospheric
Theoretical
0
0.5
1.0
2.0
4.0
10
Aroclor-lnduced
rat liver S9
Concentration/Results
Concentration
Actual
nd
0.14
0.33
0.67
1.60
nd
X Plate Concentration
ug
nd
245
600, 595, 530
1400
2425
nd
Revertants
15
20
25
50
75
80
Comments
1. Purity of DCM
not reported.
2. Positive response.
1. Data not presented.
2. Negative response
1n standard test.
3. Positive response
1n gas tight chamber.
Doubling 1n revertant
counts for TA1535;
sixfold Increase for
TA100.
(continued on the following page)
-------
TABLE 5-4. (continued)
Reference
Test System
Strain
Activation
System
Dose
Result
Comments
Green 1980 Salmonel1a/S9 TA1535
vapor exposure TA100
Rat liver fractions
en
i
CO
ro
Dose
(% in air)
0
1.4
2.8
5.5
8.3
TA 100
+S9 -S9
"69+3 :
283+10 267+20
506T27
825~34
1050+88
462+28
872727
997+88
1. Preliminary results
presented in abstract form.
2. Metabolic studies
conducted in rat tissue and
TA100. Similar metabolism
1n both systems. Radiolabel
reported to bind to bacterial
DNA but not to rat liver DNA.
3. Purity of DCM not
reported.
4. Positive response.
Author thinks this is due to
close proximity of cyto-
plasmic enzymes and inter-
mediates to DNA in bacteria,
and that negative responses
would be obtained in higher
organisms. Positive
responses in other tests
argue against this. See
discussion in text.
(continued on the following page)
-------
TABLE 5-4. (continued)
Revertants/Plate
TA1535
Reference Test System ppm Vapor
en
i
oo
OJ
Barber et al . Salmonella/S9
1980 vapor exposure
3
7
9
10
0
.600
,200
.100
,900
umoles/plate*
0
38
76
96
115
-S9
23
40
59
78
64
+S9
28
36
51
78
50
TA98
-S9
23
259
441
459
741
+S9
39
288
297
322
479
TA100
-S9
254
752
1440
2640
3060
+59
264
1152
960
1096
3240
Comments
1. Tested redistilled sample of DCM
> 99. 91 pure.
2. Revertants/nmole at highest dose
for TA1535, TA98, and TA100 were
0.0006, 0.006, and 0.03, respectively.
3. Data shown for testing In gas
tight chamber.
4. Negative response 1n standard
test; positive response in gas tight
chamber.
(continued on the following page)
-------
TABLE 5-4. (continued)
CO
-pi
Activation
Reference Test System Strain System
Concentration/Result
Nestmann et Salmonella/S9 TA1535 Aroclor 1254-
al. 1981 vapor exposure TA1537 Induced rat
TA1538 liver S9
TA98
TA100
Material
Type Weight (mg)
Added
Paint 0
remover 203
370
790
1435
Mix 0
(90:5:5 0.1
v/v/v DCM/ 0.2
methanol/ 0.4
ethanol) 0.8
4NQO 0.001
(Average
values from
triplicate
plates In 4
experiments)
Vaporized
„
144
241
469
903
(ml)
0.1
0.2
0.4
0.8
his* Revertants/Platea
TA1535
16
22
14
23
31
13
15
24
25
34
28
TA100
144
310
433
563
785
154
268
401
789
1084
878
TA98
25
31
42
76
60
32
43
73
138
164
162
Comments
1. Levels
of DCM in
chambers related din
mutatlonal dose-effei
three paint removers
2. Data shown for 01
remover only. Other
similar response.
3. Purity of DCM noi
4. Positive responsi
removers likely due 1
Exposure Level (mg/1 )
Time
DCM
Max 5h
Averaged0 Calculated0
...
12.7
21.9
40.1
80.2
12.2
26.9
50.6
94.1
15.5
25.5
49.9
95.4
12.8
25.3
50.8
101.0
Measured
13.0
23.0
45.0
86.0
11.5
27.5
50.0
94.5
Methanol
<0.5
<0.5
0.7
1.9
<0.5
<0.5
0.9
2.6
exposure
sctly to the
ct curves of
•
rte paint
two gave
t reported.
; for paint
to DCM.
Ethanol d
...
...
...
—
<0.9
0.7
1.1
2.7
concentration against time. (c). Calculated
in 91 chamber, (d). Maximum measured.
,. Determined from an area under curve for
rom amount vaporized assuming only DCM vaporized,
(continued on the following page)
-------
TABLE 5-4. (continued)
CO
CJ-,
Reference Test System Strain
Green Salmonella/S9 TA100
1981 vapor exposure
Gocke et Salmonella/39 TA1535
al. 1981 vapor exposure TA1537
TA1538
TA98
TA100
Jongen et Salmonella/S9 TA100
al . 1982 vapor exposure
Activation
System
Aroclor-1254
Induced rat liver
S9, microsomes,
and cytosol
Aroclor-1254
rat liver S9
Aroclor-1254
rat liver S9,
microsomes,
and cytosol
Dose
Result
Revertants
% Vapor
0
2.8
5.0
8.4
+S9
TOlT
458
700
950
-S9
TTO
386
720
900
Revertants
ul /desiccator
B
125
250
500
750
Activation
S9
Cytosol
Microsomes
+S9
30 + 0
54 + 7
68 + 32
105 ~ 7
203 _+ 32
0
150
150
150
150
-S9
40~T"0
85 + 7
110 + 14
195 + 21
295 _+ 7
% DCM
0.35 0.7
210 350
240 410
220 420
215 380
Comments
1. Bacterial and mammalian meta-
bolism similar.
2. Cytosol and glutathione
catalyze DCM to formaldehyde and
C02- S-chloromethylglutathione
1s a putative Intermediate.
3. DCM converted to carbon monoxide
1n the presence of microsotnes.
Formyl chloride is a putative
Intermediate.
4. Purity of DCM not given.
5. Positive response. See entry
for Green 1980.
1. Spontaneous rervertants for
TA100 too low.
2. No information presented for
toxicity.
3. Purity of DCM not given.
4. Equivocal positive response.
1.4 1. Purity of DCM not given.
550 2. Positive response.
730
810
610
-------
containing only traces of 1,1- and 1,2-dichloroethane, chloroform, chloromethane,
and an unidentified C^^Q aliphatic material (Dr. E. Barber, Eastman Kodak,
personal communication). Nestmann et al. (1981) reported that their sample was
"gas chromatographically pure," and Gocke et al. (1981) checked their sample
for the "correct melting point (sic) and elementary analysis." From this
information we conclude that the consistent positive responses in Salmonella
can be attributed to DCM. Barber et al. (1980) conducted their tests in a
chemically inert, closed incubation system and analyzed the concentrations of
DCM in the vapor-phase head space and in the aqueous phase of a test plate by
gas-liquid chromatography (Barber et al., 1981). Based on this information, the
mutagenic responses at the highest dose (i.e., 115 ^moles/plate) for TA1535,
TA98, and TA100 were 0.0006, 0.006, and 0.03 revertants per umole, respectively,
indicating DCM is a weak mutagen for Salmonella under the conditions of the
test.
The results discussed above clearly show that DCM is mutagenic in Salmonella.
However, questions have been raised about the applicability of these results
to predicting mutagenicity in other species, especially mammals. DCM is metabol-
ized, apparently via mutagenic intermediates, to CO and C02 in both rodents and
humans (see Chapter 4). CO is produced by an oxidative dechlorination of DCM by
the microsomal 9450 mixed function oxidase system. Formyl chloride is believed
to be an intermediate in this pathway. A second cytosolic glutathione transferase
system dehalogenates DCM to produce formaldehyde, which is further oxidized to
C02- This pathway is thought to proceed via an S-chloromethyl glutathione
intermediate (Ahmed and Anders, 1978; Kubic and Anders, 1975). Formyl chloride
and S-chloromethyl glutathione are highly reactive alkylating agents. They are
highly unstable compounds, but like formaldehyde, they are likely mutagens if
they reach DNA. Salmonella also metabolizes DCM to C02 and CO apparently by
reaction pathways similar to those occurring in mammals (Green, 1980, 1981).
Because of the reactivity of formaldehyde, formyl chloride, and S-chloromethyl
glutathione, and the proximity of bacterial DNA to bacterial cytoplasmic
enzymes, it has been hypothesized that these chemical substances are more
effective as mutagens when they formed by bacterial metabolism than when they
are formed outside the bacterial cell by rat liver fractions (Green, 1980,
1981). The basis for this hypothesis is that rat liver fractions used for
5-36
-------
metabolic activation have little effect on increasing the mutagenicity of
methylene chloride in the Ames test. The implication is made that as organismic
complexity is increased there is less likelihood of DCM causing mutations. It
is argued that compartmentalization of DMA into the nucleus protects the genetic
material from exposure to the mutagenic metabolites of DCM (i.e., they would
react with other cellular constituents first) and thus there is little or no
mutagenic risk. The positive results using eukaryotes, discussed in the follow-
ing sections, argue against this hypothesis.
5.3.2.1.2 Yeast. Callen et al. (1980) studied the ability of DCM obtained
from Fisher Scientific Company (purity not reported) and six other halogenated
hydrocarbons to cause gene conversion, mitotic recombination, and reverse
mutations in Saccharomyces cerevisiae (Table 5-5). Strain D7 log phase cells
were incubated for 1 hour in culture medium containing 0, 104, 157, and 209 mM
DCM. The precent survival for these doses were 100, 77, 42, and <0.1,
respectively. Due to the toxicity of the compound, the genetic endpoints
were not measured at the highest dose. The response for the other doses
(0, 104, and 157 mM) expressed per 10^ survivors were: gene conversion at
the trp-5 locus (18, 28, and 107); mitotic recombination for ade-2 (310, 190,
and 4,490); total genetic alterations for ade-2 (3,300, 3,900, 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. No exogenously applied metabolic activation was used in these
experiments, which indicates that yeast metabolize DCM intracellularly to
a mutagenic intermediate(s) that reaches nuclear DMA. In another genetic
study employing yeast, Simmon et al. (1977) reported that DCM (source and
purity not given, but stated to be the highest available purity) did not
increase mitotic recombination in strain D3 of Saccharomyces cerevisiae
when cells (1 x 10^) in suspension culture were exposed for 4 hours at 30°C
(Table 5-5). The doses used and the actual experimental values obtained for
mitotic recombination were not reported. The discrepancies between the work
by Callen et al. (1980) and Simmon et al. (1977) may be due to a number of
factors including the different strains used (D3 vs. D7), exposure time differ-
ences (4 hours vs. 1 hour), or differences in the incubation temperature (30°C
vs. 37°C). Call en et al. (1980) reported that an increase in the treatment
time of D7 cells with DCM from 1 hour to 4 hours significantly reduced the level
5-37
-------
TABLE 5-5. GENE MUTATIONS AND MITOTIC RECOMBINATION IN YEAST
Response/10^ Survivors
LTl
I
CO
co
Reference
Callen
et al. 1980
Simmon
et al. 1977
Test System
Saccharomyces
cerevlslae
Saccharomyces
cerevlslae
suspension test
Dose
Strain (mM) % Survival
07 0 100
104 77
157 42
209
D3
ade-2
trp-5 Total Genetic 1lv-l
Conversion Recombination Alterations Revertants Comments
18 • 310 3300 2.7 1. Positive response.
28 190 3900 4.4 2. Active metabolites
107 4490 14000 5.8 produced by this system
are made intracellularly
rather than by an
exogenously employed
activation system.
1. Data not provided,
but reported negatlv
for mitotlc recomblna-
tion.
2. Strain differences
and differences in
treatment conditions
(i.e., time and temp-
erature) may be the
cause of differences
between this study and
that of Callen et al.
1980.
3. Cytochrome P450
concentration not known.
Callen et al. (1980)
report different yeast
strains have different
levels.
-------
of induced mutation. Other variables, such as a lower level of P45Q enzymes in
strain D3, could conceivably account for the discrepancy in the results. At this
time, DCM is considered to be a positive mutagen in yeast.
5.3.2.1.3 Drosophila. Two reports are available concerning the ability of DCM
to induce sex-linked recessive lethal mutations in Drosophila melanogaster
(Table 5-6). Abrahamson and Valencia (1980) reported negative results, while a
positive response was reported by Gocke et al. (1981). Abrahamson and Valencia
(1980) conducted their sex-linked recessive lethal tests using two routes of
administration: feeding and injection. Due to the low solubility of DCM in
aqueous solutions, high concentrations of the test substance were not used in
these experiments, and the negative response observed may be due, in part, to
the fact that sufficiently high doses were not tested. In the feeding study,
male Canton S flies were placed in culture vials containing glass microfiber
paper soaked with a saturated solution of 1.9 percent DCM in a sugar solution
(224 mM DCM) for 3 days. (The feeding solution was added twice daily to compen-
sate 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. DCM gave a level of 0.204 percent lethal mutations compared to 0.215
percent for controls. However, because of the volatility and insolubility of
DCM, 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
percent DCM was administered to male flies. This exposure level resulted in 30
percent post-injection mortality. However, the post-injection mortality observed
for the controls was not reported. Because 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, concurrent negative controls upon which to base
conclusions concerning toxicity of the test chemical are necessary. After
mating, 8,262 chromosomes from the offspring of treated parents and 8,723
chromosomes from the offspring of control parents were assessed for recessive
lethal mutations. No evidence of mutagenicity was observed by this route of
5-39
-------
TABLE 5-6. GENE MUTATIONS IN MULTICELLULAR EUKARYOTES IN VIVO
CJ-i
i
-p.
o
Reference Test System
Abrahamson Orospphila
and Valencia sex-linked
1980 recessive
lethal test
Gocke et Drosophila
al. 1981 sex-linked
recessive
lethal test
Strain Chemical Route
FM6 EMS Fed
females, Tr1s Fed
Canton Neg. Controls Fed or Inj
S males DCM Fed
Inj
iFChromosomes
Tested I Lethal s
773 44
2442 35
94491 230
14682 34
8262 18
Base Lethal s/Brood
females, DCM (mM) 1 ^ 3
Berlin 0 19/7130
K males (0.27)
125 16/3632
(0.44)
620 8/1213
(0.66)
Total 24/4845
treated (0.50)
P < 0.05
8/5525 13/3416
(0.14) (0.38)
2/2579 6/1310
(0.08) (0.46)
3/735 5/1005
(0.41) (0.50)
5/3114 11/2315
(0.16) (0.47)
Corrected
Lethal s (X) Comments
5.69 1. No precaution taken
1.43 to design exposure
0.233 chambers to prevent
0.204 evaporation of the
0.157 test compound for
feeding experiment.
2. No concurrent
negative controls
reported for the
Injection experiment.
3. Negative response at
dose tested (224 mM).
1. Positive response for
Brood 1 indicating DCM
is mutagenlc to sperm in
Drospphila.
T. Higher dose used than
in test by Abrahamson and
Valencia (1980).
(continued on the following page)
-------
TABLE 5-6. (continued)
tr,
i
Reference
Samolloff
et al.
1980
Test System
Panagrelus
redivlvus
sex-linked
recessive
lethal test
Concentration
(mol/L)
DCM
W*
io-6
io-4
Proflavlne
10-8
io-6
io-4
Mutation Frequency
(Lethal mutations/105 loci)
6.
10.
9.
12.
10.
28.
0
1
8
5
0
6
(Survival
L2
Juveniles
l.OZ
1.02
1.00
Toxlcity
rel. to controls)
L2-L3
Molt
0.99
1.00
1.00
L3-L4
Molt
0.97
0.86
0.88
L4
Adult
Molt
0.4b
0.15
0.17
Comments
1. Equivocal positive re-
sponse.
2. Not dose-related.
3. Some positive controls
gave negative (e.g., EMS)
or only marginally positive
response (e.g., 3-methyl
cholanthrene).
4. Test system not
validated.
-------
administration. Flies injected with 0.2 percent DCM had 0.157 percent lethals
compared to 0.206 percent for controls.
Gocke et al. (1981) also tested.DCM (Merck, Darmstadt, FRG, purity not
given) for its ability to induce sex-linked recessive lethal mutations in
Drosophila. Two solutions, 125 mM and 625 mM in 2 percent DMSO and 5 percent
saccharose, were fed to wild-type Berlin K male flies for an unreported period
of time. The highest dose (625 mM) is reportedly close to the 1050. These
males were then mated to Base females. Three broods were scored (i.e., off-
spring from virgin females mated to treated males on days 1 through 3, 4 through
6, and 7 through 10 after exposure). There were significantly more lethals (24
out of 4,845 chromosomes scored) for brood 1 (0.50 percent lethal mutations)
compared to the negative controls (19 lethals out of 7,130 chromosomes, 0.27
percent lethal mutations), P £0.05. Elevated, but not statistically signifi-
cant increases in lethal mutations were noted in broods 2 (0.16 percent compared
to 0.14 percent lethals) and 3 (0.47 percent compared to 0.39 percent lethals)
of the treated flies compared to the controls. As noted in Table 5-6 the
incidence of lethals is dose-related. The incidence of lethals for the high
dose in brood 2 is elevated nearly three-fold over the corresponding negative
control value (0.41 vs. 0.14 lethals, respectively), but this may not be a
significant increase because of the small sample size (735 vials). This test
indicates DCM is mutagenic to sperm in Drosophila (a multicellular eukaryote).
The discrepancies in results between Abrahamson and Valencia (1980) and Gocke
et al. (1981) may be due to stock specific differences (Canton S. vs. Berlin K)
in the metabolic activation of DCM or more likely to the larger doses of DCM
employed by Gocke et al. (1981).
5.3.2.1.4 Nematodes. In another sex-linked recessive lethal test, Samoiloff
et al. (1980) tested DCM for its ability to mutate the nematode Panagrellus
redivivus (Table 5-6). Individual females homozygous for the X-linked mutation
b7 (coiled phenotype in liquid medium) were grown for 120 hours in the presence
of several concentrations of DCM ranging from 10~3M to 10~8M. They were then
washed and mated to S-15 males who carry an X-chromosome crossover suppressor
extending at least 15 recombination units to either side of b7. One hundred
5-42
-------
female progeny were collected and mated to wild-type (C-15) males and their
progeny scored for the presence of the b7 phenotype. The absence of b7 male
progeny indicates lethality of the X-chromosome marked with b7 derived from a
female grown on DCM. Three replicate experiments were performed. A non-dose-
related increase in the level of lethals was observed in the progeny of DCM-
treated worms compared to the negative controls. For worms treated with 10-8,
1CT6, and 10~4M DCM, the corresponding lethal mutations/105 loci were 6.0, 10.1,
and 9.8, respectively, compared to an estimated spontaneous mutation frequency of
2.2 x 10~6 mutations/locus. Some of the positive controls tested concurrently,
such as proflavine, yielded a positive response (12.5, 10.0, and 28.6 lethals/105
loci at 10-8, ID'6, and 10~4M, respectively). But others, such as aflatoxin B
and ethyl methanesulfonate (EMS), did not cause an increase in lethal mutations.
This study suggests that DCM is mutagenic in nematodes, but firm conclusions
cannot be made because the assay is not validated and more importantly because
of the negative responses obtained with some of the positive controls.
5.3.2.1.5 Mammalian cells in culture. Jongen et al. (1981) tested DCM for its
mutagenic potential in several mammalian short-term tests. Testing for the
induction of forward mutations at the HGPRT locus will be described here (Table
5-7). Testing for the ability of DCM to cause sister chromatid exchanges (SCE),
unscheduled DMA synthesis (UDS), and inhibition of DNA synthesis (IDS) will be
discussed later in the section on other indicators of DNA damage.
In their testing of the ability of DCM to cause forward mutations, Jongen
et al. (1981) incubated log phase CHO and V79 cells with 1, 2, 3, 4, and 5
percent DCM or 1, 2, 3, and 4 percent DCM, respectively, at 37°C for 1 hour in a
closed glass container without exogenous S9 mix. DCM was obtained from Merck
(analytical grade). The cells were exposed to gaseous DCM and then DCM in
solution for 15-minute intervals each by alternately tilting the plates then
placing them horizontally. After growth to allow for an 8-day (CHO cells) or
6-day (V79 cells) expression period, mutant cells were selected in thioguanine-
containing medium. DCM failed to increase the mutation frequency of either
cell line at any dose compared to controls. However, DCM was not very cytotoxic
to either cell line. At the highest dose, survival decreased only 20 percent.
5-43
-------
TABLE 5-7. GENE MUTATIONS IN MAMMALIAN CELLS IN CULTURE
V79
Concentration Mutants/105
Reference
Jongen et
al. 1981
Test System
6-Thioguanine
resistance In
V79 and CHO
cells
(X)
DCM 0
1
2
3
4
5
EMS 0
2
4
Survivors
2
1.8
2
1.7
1.6
2
13
33
r.Hfl
Survival Mutants/105
(%)
100
98
95
85
80
Survivors
1.9
1.8
1.2
0.9
2.1
2.5
Survival
(%)
100
90
85
80
73
76
Comments
1. Equivocal negative response.
2. Highest dose only resulted 1n 20%
decrease 1n survival. Higher doses up
to about 80% toxicity should be tested.
-------
It would be appropriate to repeat the experiment using higher doses of DCM.
EMS yielded a positive, dose-dependent increase in mutation induction in V79
cells, but it was not tested in CHO cells.
Based on the positive responses in bacteria, fungi, nematodes, and insects,
DCM is judged to be capable of causing gene mutations. Metabolic activation to
highly reactive mutagem'c metabolites apparently accounts for this response,
and although these are thought to be unstable, they seem to be capable of
interacting with genetic material of higher eukaryotes.
5.3.2.2 Chromosomal Aberrations—Three studies on the ability of DCM to cause
chromosomal aberrations were evaluated. Burek et al. (1980) subjected four
groups of 10 Sprague-Dawley albino rats (Spartan substrain, SPF-derived, 5 males
and 5 females) to 0, 500, 1,500, or 3,500 ppm DCM by inhalation 6 hours/day,
5 days/week for 6 months. The animals were then sacrificed, bone marrow cells
were collected, chromosome preparations were made, and slides were coded and
subse quently analyzed. Two hundred metaphases per animal were scored and
aberrations were tabulated (See Table 5-8). No increase in the total frequency
of abnormal cells or in the frequency of any specific type of aberration was
noted in the treated animals compared to the controls. There were 1.1 +_ 1.3,
0.6 +_ 0.7, 0.8 +_ 1.2, and 1.1 +_ 0.9 percent cells with chromosome aberrations
in animals treated with 0, 500, 1,500, and 3,500 ppm DCM, respectively.
Thilagar and Kumaroo (1983) treated CHO cells grown in either plastic or
glass culture flasks with 0, 2, 5, 10 and in one experiment 15 ul/ml (i.e., 0,
31, 78, 156, and 234 mM) DCM for 2 hours with or 12 hours without S9 mix derived
from Aroclor-induced rat livers. DCM was obtained from Fisher Scientific
(certified A.C.S., lot no. 713580). After the exposure period, the cells were
washed, refed, and allowed to grow before being arrested at metaphase with
colcemid and harvested for chromosome preparation. Slides were coded and read
"blind;" 100 cells were scored for each dose level (50 cells/duplicate flask).
DCM induced a dose-related increase in chromosome aberrations (see Table 5-8)
ranging from 0.02 aberrations/cell in the negative controls to 1.44 aberrations/
cell at 15 ul/ml (234 mM). The response was greater in cells treated in glass
culture flasks and was not dependent on the presence of the exogenous metabolic
activation system.
5-45
-------
TABLE 5-8. TESTS FOR CHROMOSOMAL ABERRATIONS
en
i
Reference
Burek et
al. 1980
Reference
Thi lagan
and
Kumaroo
1983
Route of
Strain/Tissue Exposure
Male and female Inhalation
Sprague-Dawley
rat/bone marrow
Test System Dose RCG*
Cultured CHO DCM (ul/ml)
cells 0 100
2 98.4
5 75.3
10 66.7
TEM
TH? N.D.
Dose
ppm
0
500
1500
3500
Chromatld
2
4
8
12
22
Breaks
Chromatld
0.9 + 0.99
0.5 T 0.71
0.5 T 0.97
0.7 jf 0.48
Breaks
Isochromatld
0
0
14
34
30
Chromosome
0.2 + 0.42
0.2 7 0.42
0.1 ~ 0.32
0.2 jf 0.42
Exchange
0
2
8
10
42
D1 centrlcs
0
0
0
0 0.
Number of
Aberrations/Cell
0.02
0.06
0.34
0.56
0.96
Rings Exchanges Comments
0 0
0 0
0 0.1 + 0.
2 _* 0.42 0
Cells w/
Aberration (%)
2
6
26
38
66
1. 5 animals/sex/
dose.
32 2. 200 cells/
animals.
3. Dose to bone
marrow cells may
have been low.
4. Negative res-
ponse.
Comments
1. Positive response.
2. Four experiments
yielded similar
response.
*RCG
Relative cell growth.
Reference
Gocke et
al. 1981
Strain/Tissue
Hale and female
NMRI mice /bone
marrow
Route of
Exposure
l.p.
Injection
Dose
ppm
No. injection x
0
2 x 425
2 x 850
2 x 1700
Breaks
Chromatld Chromosome Di Gentries Rings Exchanges
mg/kg Mlcronuleated Polychromatic Erythrocytes (%)
1.9
1.9
3.5
2.8
Comments
1. Suggestive pos-
itive response;
authors Indicate
negative response.
2. Dose to bone
marrow cells may
have been low.
-------
Gocke et al. (1981) assessed the ability of DCM (Merck, Darmstadt; purity
not given) to cause micronuclei in polychromatic erythrocytes (PCE). Two male
and two female NMRI mice were used for each of three dose levels (425, 850, and
1,700 mg/kg/intraperitoneal injection). The highest dose approximated the 1059
for mice. Intraperitoneal injections of each dose were given at 0 and 24 hours,
the animals were sacrificed at 30 hours, bone marrow smears were made, and
1,000 PCEs per animal were scored for the presence of micronuclei. An increase
in PCEs with micronuclei was observed at the two highest doses, but the response
was not dose-related and was not double the control value. Thus, the results
are considered suggestive of a positive response but are not conclusive.
(There were 1.9 percent micronuclei in the untreated controls compared to 3.5
percent micronuclei in the animals receiving two injections of 850 mg/kg, and 2.8
percent micronuclei at the highest dose).
Based on the positive response reported by Thilagar and Kumaroo (1983), DCM
is tentatively judged to be capable of causing chromosomal aberrations. The
negative responses reported by Burek et al. (1980) and Gocke et al. (1981) are
not inconsistent with these results. Thilagar and Kumaroo (1983) exposed
mammalian cells in culture to DCM. In the studies by Burek et al. (1980) and
Gocke et al. (1981), exposure occurred in vivo. The dosage received in the bone
marrow cells may have been insufficient to yield a detectable positive response.
5.3.2.3 Other Indicators of DNA Damage—
5.3.2.3.1 Sister chromatid exchange (SCE). Two papers have been published on
the ability of DCM to induce SCEs (Table 5-9). Jongen et al. (1981) tested the
ability of 0.5, 1.0, 2.0, 3.0, and 4 percent DCM (i.e., 58, 118, 235, 353, and
471 mM) to induce SCEs in Y79 cells. Log phase cells were incubated at 37°C
for 1 hour in a closed glass container. The cells were exposed to DCM in the
gaseous phase and in the medium by tilting the plates for 15 minutes, then
placing them horizontally. The experiment was conducted seven times and each
yielded a dose-related increase in SCEs/cell, which approached but did not
exceed a twofold increase above the control level. Taken together, these
results are statistically significant (P < 0.001). Increasing the exposure
time to 2 hours or 4 hours or using S9 from rat liver did not alter the shape
5-47
-------
of the dose-response curve, which plateaued at 1 percent DCM. The authors
suggest that this phenomenon is due to a saturation of the metabolic activation
system of V79 cells.
Thilagar and Kumaroo (1983) exposed CHO cells to 0, 2, 5, 10, and in one
experiment 15 ul DCM/ml of medium (0, 31, 78, 156, and 234 mM DCM) for 2 hours
with and 24 hours without metabolic activation. The cells were grown for 24
hours in BrdUrd followed by a mitotic shake off, fixing, and staining by a
fluorescence-plus-Giemsa technique, and then the coded slides were scored
"blind." Slight dose-related elevations in SCE values were noted (see Table 5-9),
but they never exceeded a 50 percent increase at the highest dose. The authors
judged their test to be negative, but the results are consistent with those
reported by Jongen et al. (1981) discussed above. Thus, DCM is judged to be
capable of causing DMA damage resulting in SCE.
5.3.2.3.2 DNA repair assays. In their study of the genotoxic potential of
DCM, Jongen et al. (1981) also measured UDS and IDS in V79 cells and primary
human fibroblasts (AH cells). These experiments were conducted by exposing 105
cells attached to glass coverslips (UDS assay) or to glass petri plates (IDS
assay) to 0.5, 1.0, 2.0, 3.0, and 5.0 percent DCM (58, 118, 235, 353, and 471 mM,
respectively) without metabolic activation. UDS experiments were done in
duplicate, and at least 25 nuclei of non-S phase cells were scored for the
number of silver grains/nucleus at each dose level. DCM had no detectable
effect on UDS in either cell line. In the IDS assays, the relative rate of DNA
synthesis was determined radioisotopically immediately after DCM exposure and
0.5, 1.5, and 3.5 hours later. The average of duplicate samples revealed that
DCM inhibited DNA synthesis in V79 and AH cells at all dose levels compared to
controls but that synthesis recovered with time after exposure in all cases.
This is unlike the persistent inhibition of DNA synthesis by the positive
control 4-nitroquinoline-l-oxide. The authors conclude that DCM was not induc-
ing genetic damage in cells but was inhibiting DNA synthesis by an effect on
cell metabolism.
Perocco and Prodi (1981) also performed a UDS assay using DCM. They
collected blood samples from healthy individuals for their studies, separated
5-48
-------
TABLE 5-9. TESTS FOR SISTER-CHROMATIO EXCHANGE
Reference Test System Dose
Results
Comments
SCE/Cell
Jongen et SCE/V79 cells X
al. 1981 In culture DCM
0
Experiment #
0
1
.26 +
0
.02
0
.30
7
+ 0
.03
1.
2.
SCEs
3.
Exposure time 1 hour.
Positive response. Significant Increases 1n
(P < 0.001)
Same type of
experiments 2-6
0.5
1.0
2.0
3.0
4.0
0
0
0
0
.40^
.46 _f
.45^
...
.51 _+
0
0
0
0
.02
.02
.03
.03
0
0
0
0
.47
.51
.58
.61
—
± °
± °
± °
±°
.02
.03
.03
.03
4.
DMSO did not
•
dose-response observed 1n
(data not shown).
Increase Incidence of SCE.
(continued on the following page)
-------
TABLE 5-9. (continued)
Reference Test System Dose
SCE/Cel 1
(X _+ SO) Range of SCE's MI MI + M2 Comments
Thllagar Cultured CHO DCM (ul/ml)
en and cells 0
1 Kumaroo
o 1983 2
5
10
Trlethylene
10.28 i 3.17
11.36 +. 3.09
12.56 +_ 2.95
12.36 +_ 3.35
melamlne
5-17 0 3 97 1. Marginal, but not significant Increases
1n SCEs.
3-19 0 16 84 2. Three other experiments yielded similar
responses.
7-18 6 54 40 3. Results not Inconsistent with test by
Jongen et al. (1981) where highest dose
7-21 4 56 40 was three times greater (I.e., 471 mM vs.
156 mM).
0.025 ug/ral 47.74 i 4.76
39-61
49 47
-------
the lymphocytes, and cultured 5 x 105 cells in 0.2-ml medium for 4 hours at
37°C in the presence or absence of DCM (Carlo Erban, Milan, Italy or Merck-
Schuchardt, Darmstadt, FRG, 97 to 99 percent pure). The tests were conducted
both in the presence and in the absence of PCB-induced rat liver S9 mix. A
comparison was made between treated and untreated cells for scheduled DMA
synthesis (i.e., DMA replication) and UDS. No difference was noted between the
groups with respect to scheduled DMA synthesis measured as dpm of [3H] deoxythy-
midylic acid (TdR) after 4 hours of culture (2,661 _+ 57 dpm in untreated cells
compared to 2,356 +_ 111 dpm in cells treated with 5 ul/ml [78 mM] DCM). Subse-
quently, 2.5, 5, and 10 ul/ml (39, 78, and 156 mM) EDC was added to cells
cultured in 10 mM hydroxyurea to suppress scheduled DMA synthesis. The amount
of unscheduled DMA synthesis was estimated by measuring dpm from incorporated
[3H]TdR 4 hours later. At 10 ul/ml DCM, 532 +_ 31 and 537 +_ 39 dpm were counted
without and with exogenous metabolic activation, respectively. Both values
were lower than corresponding negative controls of 715 +_ 24 and 612 +_ 26 dpm,
respectively. No positive controls were run to ensure that the system was
working properly, although testing of chloromethyl methyl ether (CMME) with
activation resulted in a doubling of dpms over the corresponding negative
control values (1,320 +_ 57 at 5 ul/ml CMME vs. 612 _+ 26 untreated). The authors
calculated an effective DNA repair value (r) for each chemical based on the
control and experimental values with and without metabolic activation. DCM was
evaluated by the authors as negative in the test, but they did not state their
criteria for classifying a chemical as positive. None of the experimental
values from cells treated with DCM had higher dpm values than the controls.
Based on these experiments there is no evidence that DCM specifically in-
hibits DNA synthesis or causes UDS. However, UDS assays are designed to detect
the occurrence of a specific type of DNA repair (i.e., "long-patch" excision
repair). Xenobiotics that cause damage that is repaired by another process may
not be readily detected.
5.3.2.4 Summary and Conclusions. Dichloromethane has been tested for its
ability to cause gene mutations (in Salmonella, yeast, Drosophila, Panagrellus,
5-51
-------
and cultured mammalian cells), chromosomal aberrations (in rats, mice, and
cultured mammalian cells), and other indicators of DNA damage (sister chromatid
exchange formation [SCE] in cultured cells, unscheduled DNA synthesis, and
inhibition of DNA synthesis).
Commercially available samples of DCM have been shown to be mutagem'c in a
wide range of organisms, including bacteria (Salmonella), fungi (Saccharomyces),
nematodes (Panagrellus), and insects (Drosophila). The responses were weak under
treatment conditions used and were obtained without the addition of metabolic
activation systems (e.g., S9 mix). The data suggest that DCM is metabolized to a
mutagem'c metabolite(s). Several metabolism studies (including one with bacteria)
indicate such activation occurs "in vivo." Negative results have been reported
for gene mutation tests in fungi (Saccharomyces) and mammalian cells in culture,
but these may represent false negative results because of the treatment condi-
tions used. DCM has also been reported to induce chromosomal aberrations in
cultured mammalian cells but not in bone marrow cells from animals exposed j£
vivo, perhaps because a sufficient dose of DCM did not reach the bone marrow
cells to cause observable effects there. DCM causes a weak increase in SCEs
but has not been shown to cause UDS or inhibit DNA synthesis.
Based on the weight of available evidence, showing positive responses in
four different organisms, DCM is judged to be capable of causing gene mutations
with the potential to cause such effects in exposed human cells. A positive
response in cultured mammalian cells indicates it causes chromosomal aberra-
tions, but additional testing (e.g., another in vivo or in vitro chromosomal
aberration assay) is needed to resolve this point. If such tests are conducted,
care should be taken to insure that the test cells are sufficiently exposed to
DCM. If such precautions are not taken, false negative responses may be ob-
tained.
5-52
-------
5.3.3 Evaluation of the carcinogenicity of PCM
The purpose of this section is to provide an evaluation of the likelihood
that DCM is a human carcinogen and, on the assumption that it is a human carcin-
ogen, to provide a basis for estimating its public health impact, including a
potency evaluation in relation to other carcinogens. The evaluation of carcino-
genicity depends heavily on animal bioassays and epidemiologic evidence. How-
ever, other factors, including mutagenicity, metabolism (particularly in rela-
tion to interaction with DNA), and pharmacokinetic behavior, are important to
the qualitative and quantitative assessment of carcinogenicity. The available
information on these subjects is reviewed in other sections of this document.
This section presents an evaluation of the animal bioassays, the human epidemi-
ologic evidence, the quantitative aspects of assessment, and finally, a summary
and conclusions dealing with all of the relevant aspects of the carcinogenicity
of DCM. Further, the National Toxicology Program (NTP) rat and mouse gavage
bioassay draft technical report (1982) on DCM was cancelled because of data
discrepancies at their contract laboratory (memo from John A. Moore dated July
25, 1983).
5.3.3.1 Animal Studies--
5.3.3.1.1 Dow Chemical Company (1980) inhalation study in rats. A total of
1,032 male and female Sprague-Dawley rats (129/sex/exposure concentration) were
exposed by inhalation to DCM at 0, 500, 1,500, or 3,500 ppm for 6 hours/day, 5
days/week (excluding holidays), in a 2-year toxicity and oncogenicity study.
Approximately 95 rats/sex/exposure concentration were part of the chronic
toxicity and oncogenicity portion of the study. This number also included those
animals that died spontaneously, were killed moribund during the study, or were
killed at the end of the 2-year exposure. The remaining animals were sacrificed
as part of the cytogenetic 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 marked for
identification with metal ear tags. All rats were maintained on a 12-hour
light/dark cycle. They were observed daily, including weekends and holidays,
for general health status and signs of possible toxicity.
Dichloromethane representative of technical grade material was obtained
from Dow Chemical Company, Plaquemine, Louisiana, and was used throughout the
5-53
-------
exposure. Fourteen different samples of DCM were analyzed during the 2 years
of animal exposure; each sample showed 99 percent pure DCM, with a few trace
chemical contaminants that varied slightly from sample to sample, as shown in
Table 5-10. The concentration of DCM vapor in chambers was considered well
within the range of expected variability. Hematologic determinations, serum
clinical chemistry, urinalysis, bone marrow collection, and blood carboxyhemo-
globin (COHb) determination were done in animals sacrificed at 6, 12, 15, and
18 months (interim kills). Plasma estradiol determination was done at the 12-
and 18-month interim kills. This included samples from six controls/sex and
four high exposure animals (3,500 ppm)/sex from the 12-month kill, which were
pooled together (two animals/sample) to give three control samples and two
high exposure (3,500 ppm) samples/sex. Ten individual samples/sex (not pooled)
from the high exposure and control groups were also sent from the 18-month kill.
All animals that either died spontaneously, were killed in moribund con-
dition, or were killed at the interim or terminal kills were subjected to
complete gross and microscopic pathological examinations by a veterinary path-
ologist. Liver samples for possible electron microscopic evaluation were
collected.
In females exposed to 3,500 ppm, there was a statistically significant
increase of mortality from the. 18th through the 24th months that may be exposure-
related. The remaining treated groups in males or females did not differ
significantly from the controls (Table 5-11). There was no exposure-related
difference in body weights of either male or female rats exposed to 500, 1,500,
and 3,500 ppm DCM.
Although some hematologic values were increased and others were decreased,
the mean values were within the normal range of biological variability. Serum
glutamic pyruvic transaminase (SGPT), blood urea nitrogen (BUN), and serum
alkaline phosphatase (AP) values were in the normal range. It is noted that the
females had significantly increased (P < 0.025) plasma estradiol levels at 18
months, which may be related to the higher incidence of mammary tumors in the
exposed (3,500 ppm) group. Urinalysis findings were in the normal range, with
the exception of a few statistically significant values in specific gravity in
males exposed to 1,500 ppm at 6 months and males and females exposed to 3,500 ppm
at 12 months. Rats exposed to 500, 1,500, or 3,500 ppm had elevated COHb values
but with no evidence of either dose-response or increased values with prolonged
exposure. 5_54
-------
TABLE 5-10. ANALYTICAL ANALYSIS OF DICHLOROMETHANE
(Dow Chemical Company 1980)
1
Specific gravity 1.320
HC1, ppm 14.2
H20, ppm 207
Nonvolatile
material, ppm ND
Methyl chloride, ppm <1
en
^ Chloroform, ppm 60
en
Vinylidene
chloride, ppm 60
Trans 1,2-dichloro-
ethylene, ppm 560
Cyclohexane, ppm 365
Ethyl chloride, ppm 6
Vinyl chloride, ppm <1
Methyl bromide, ppm 23
Carbon tetrachloride, <2
ppm
2
ND
1.8
560
<7
<4
<27
52
561
385
9
--
--
__
3
ND
2.3
37
<7
<4
399
53
266
247
5
--
--
__
4
ND
2.9
55
<7
4.5
48
90
706
467
11
--
--
__
Sample Number
5 6 7
ND
1.8
52
<7
4.5
48
75
550
365
8
--
--
ND
1.1
27
<7
4.5
48
65
550
374
11
--
--
ND
2.5
112
<7
4.5
32
60
487
335
5
--
--
__
8
ND
--
--
ND
1
52
66
--
399
2
1
1
1
9
ND
--
--
ND
1
576
72
321
266
2
1
1
15
10
ND
1
324*
ND
1
64
62
653
426
2
1
1
. 1
11
ND
--
--
ND
1
562
78
323
262
2
1
1
13
12
ND
1
264*
ND
1
515
70
318
268
6
1
1
16
13
ND
1
340*
ND
1
547
77
337
288
1.5
1
1
20
14
ND
1
3
NL)
1
460
72
298
242
6
1
1
12
*0riginal analysis was lost; sample was subsequently reanalyzed.
ND = not determined.
-- = not detected.
-------
TABLE 5-11. CUMULATIVE PERCENT MORTALITY OF RATS
2-YEAR DICHLOROMETHANE INHALATION STUDY
(Dow Chemical Company 1980)
Males
Month of
study 0 ppm 500 ppm 1,500 ppm 3,500 ppm
Females
0 ppm 500 ppm 1,500 ppm 3,500 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
0.8
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
6.8
10.9
10.9
11.8
16.2
20.0
36.2
45.3
52.6
56.8
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*
*Significantly different from control by Fisher's Exact Test, P < 0.05.
5.3.3.1.1.1 Gross and histopathologic observations of rats from the 6-, 12-,
15-, and 18-month interim kills. Numerous gross and histopathologic observations
were recorded for control and DCM-exposed rats at each time period, and most
were typical of spontaneous or naturally-occurring lesions normally seen in
rats of this strain. There were many palpable masses in males and females.
Some palpable masses appeared to be abscesses of the preputial or clitoral
glands, while others were cyst-like lesions of the skin. The total number of
masses in the 3,500 ppm group of males was significantly increased over the
5-56
-------
controls at 15, 18, and 21 months, but not at 23 months. Female rats exposed
to 500, 1,500, and 3,500 ppm showed an exposure-related increase in total
number of masses. There was also a trend of increased benign mammary tumors in
females exposed to 1,500 and 3,500 ppm. The total numbers of animals with
benign mammary gland tumors were 9/28 (0 ppm), 10/29 (500 ppm), 11/29 (1,500
ppm), and 14/27 (3,500 ppm), whereas the total numbers of benign mammary gland
tumors were 17/28 (0 ppm), 17/29 (500 ppm), 28/29 (1,500 ppm, P = 9.23 x 1Q-4),
and 37/27 (3,500 ppm, P = 2.33 x lO"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-neoplastic effects in both males and females at all exposure concentrations.
Grossly, the effect was most prominent in females exposed to 3,500 ppm and con-
sisted of increased numbers of dark or pale foci. The control group had an
incidence of 0/28, while the 3,500 ppm DCM-exposed female rats had a significantly
greater number of foci (11/27). Some rats from the 3,500 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 3,500 ppm). Because of the
limited number of rats at each interim kill, this latter change may be related
to exposure but may also be due to biological variability.
Histologically observed, exposure-related lesions were present in the
livers of both males and females exposed to 500, 1,500, or 3,500 ppm. In males,
the total number of animals with any degree of vacuolization consistent with
fatty changes were 5/29 (0 ppm), 19/29 (500 ppm, P = 2.05 x 10'4), 21/29 (1,500
ppm, P = 2.47 x ID"5), and 23/27 (3,500 ppm, P = 2.81 x 10'7). The livers of
female rats also had alterations considered to be related to DCM exposure. The
total numbers of females with any degree of vacuolization consistent with fatty
changes were 13/28 (0 ppm), 20/29 (500 ppm, P = 7.16 x 10'2), 20/29 (1,500 ppm,
P = 7.16 x 10-2), and 22/27 (3,500 ppm, P = 7.16 x 1Q-3). Because of these ef-
fects, it may be considered that this experiment was performed at the maximum
tolerated dose (MTD).
5.3.3.1.1.2 Gross and histopathologic observations of rats killed moribund or
dying spontaneously during the study and those from terminal sacrifice (24 months).
Non-neoplastic observations--The liver was affected in both males and females
exposed to 500, 1,500, or 3,500 ppm. The percentage of total rats with any de-
gree of vacuolization was 17 percent, 38 percent, 45 percent, and 54 percent in
5-57
-------
the males of the 0, 500, 1,500 and 3,500 ppm exposure groups, respectively, and
34 percent, 52 percent, 59 percent, and 65 percent 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 histologically
observed decrease in the number of cases of adrenal cortical necrosis, but the
nodular hyperplasia incidence (unilateral or bilateral) was increased: 18/95 (0
ppm), 30/95 (500 ppm, P = 3.07 x 10-2), 31/95 (1,500 ppm, P = 2.2 x 10-2), and
24/97 (3,500 ppm).
Tumor or tumor-like lesions—The Sprague-Dawley strain of rats used in
this study historically has had a high spontaneous incidence of benign mammary
tumors. The incidence varies slightly from study to study, but normally exceeds
80 percent in females and about 10 percent in males by the end of a 2-year study.
The mammary gland tumors have been classified, based on their predominant morpho-
logical 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-statistically significant increase in the
number of male rats with a benign mammary tumor exposed to 3,500 ppm (14/95
as compared to 7/95, 3/95, and 7/95 in the 0, 500, or 1,500 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), 1,500 ppm (11/95), or 3,500 ppm
(17/97, P = 4.6 x 10-2).
The total number of female rats with a benign mammary tumor did not
increase in any exposure group (0, 500, 1,500, or 3,500 ppm groups had totals
of 79/96, 81/95, 80/95, and 83/97 benign mammary tumors, respectively). How-
ever, 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, 1,500, and 3,500 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 1,500 ppm, and to 3.0 in those exposed to 3,500 ppm. This effect is
exposure-related, and a dose-response relationship was apparent. There was no
indication of an increased number or incidence of malignant mammary tumors in
either males or females.
5-58
-------
The number of malignant tumors (Table 5-12) increased in male rats exposed
to 3,500 ppm. This increase did not appear to correlate clearly with an in-
creased number of any one tumor type or location. However, this observation
led Dow Chemical Company to re-evaluate the gross and histopathologic data on
all tumors arising in or around the salivary glands. Table 5-13 lists the
specific individual animal data for these salivary gland area tumors, showing
the palpable mass data, specific histopathologic diagnoses, and the number of
sarcomas with metastases. Table 5-14 summarizes the incidence of salivary
gland region sarcomas in male rats.
Grossly, these tumors were large (several centimeters in diameter), cystic,
necrotic, or hemorrhagic. They appeared to invade all adjacent tissues in the
neck region, and often completely replaced the normal salivary gland tissue.
Histologically, all were sarcomas. They were composed of cells that varied from
round to spindle-shaped, but that appeared to be of mesenchymal cell origin.
Mitotic 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 in 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 in the controls (1/93) compared to 0/94
in the 500 ppm exposure group, 5/91 in males from the 1,500 ppm exposure group,
and 11/88 in males from the 3,500 ppm exposure group (P = 0.002). Historically,
a spontaneous incidence (0 to 2 percent) of this tumor type has been observed in
Dow's laboratory. Therefore, the 12.5 percent incidence (11 of the 88 rats,
Table 5-14) found in the males from the 3,500 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 1,500 ppm had five of these tumors, which was also slightly higher than
expected, but was not statistically signficant. Therefore, this effort appeared
to be exposure-related in the males exposed to 3,500 ppm.
The total number of male rats with malignant tumors was similar in the
control, 500 ppm, and 1,500 ppm exposure groups. Males exposed to 3,500 ppm
had an increase in this category, since 69 of the 124 rats had malignant tumors
compared to 57, 59, and 57 in the 0, 500, and 1,500 ppm exposure groups, respec-
tively.
5-59
-------
TABLE 5-12. SUMMARY OF TOTAL TUMOR DATA FOR RATS ADMINISTERED DICHLOROMETHANE FOR 2 YEARS BY INHALATION
(Dow Chemical Company 1980)
I
O^
o
Males
Spontaneous
Total number of rats
examined
Total number of rats
with a tumor
Total number of rats
with a benign tumor
Total number of rats
with a malignant tumor
Concentration
In ppm
0
500
1500
3500
0
500
1500
3500
0
500
1500
3500
0
500
1500
3500
Interim
kill
29
29
29
27
9
7
9
11
6
3
8
9
6
4
5
7
deaths and
killed
moribund
81
81
89
90
53.
52
67
68
33
29
42
37
39
45
48
55
Terminal
kill
14
14
6
7
13
13
6
7
11
11
6
5
12
10
4
7
Cumulative
total s
124
124
124
124
75
72
82
86
50
43
56
51
57
59
57
69
Interim
kill
28
29
29
27
14
15
14
14
14
15
14
14
2
3
1
2
Females
Spontaneous
deaths and
killed
moribund
75
71
82
93
67
68
79
91
65
60
73
84
23
28
31
32
Terminal
kill
21
24
13
4
21
24
13
4
21
24
12
4
13
14
7
2
Cumulative
total s
124
124
124
124
102
107
106
109
100
99
99
102
38
45
39
36
-------
TABLE 5-13. TIME-TO-TUMOR, PALPABLE MASS, AND HISTOPATHOLOGY DATA FOR SALIVARY GLAND REGION SARCOMAS IN INDIVIDUAL MALE RATS
EXPOSED TO DICHLOROMETHANE BY INHALATION FOR 2 YEARS
(Dow Chemical Company 1980)
cr>
Animal
number
76-3301
76-3733
76-3809
76-3722
76-3743
76-3749
76-3583
76-3698
76-3580
Month of study Size of tumor
Exposure tumor first when first
group (ppm) observed observed (cm)
0
1500
1500
1500
1500
1500
3500
3500
3500
15 5
16 3
Not observed
24 1.5
14 2
18 3
Not observed
14 3
21 2
Month of study Size of tumor
for necropsy at necropsy (cm)
15
17
19
24
14
20
24
15
24
7x6x4
5x4x2
Not detected
(slightly enlarged
salivary gland)
4x3x2
No size given
(6 grams)
5x4x4
Not detected
7x6x3
9 x 6 x 4.5
Evidence of
Histological distant
diagnosis metastases
Subcutaneous - undif-
ferentiated sarcoma
Salivary gland -
malignant schwannoma
Salivary gland -
malignant schwannoma
Salivary gland - undif-
ferentiated schwannoma
Subcutaneous - round
cell sarcoma
Subcutaneous - round
cell sarcoma
Salivary gland -
fibrosarcoma
Salivary gland -
carcinosarcoma
Subcutaneous -
None
None
None
None
None
None
None
Yes
Yes
neurofibrosarcoma
-------
TABLE 5-13. (continued)
Animal
number
76-3663
76-3682
c^ 76-3578
r-o
76-3608
76-3621
76-3666
76-3671
76-3597
Month of study Size of tumor
Exposure tumor first when first
group (ppm) observed observed (cm)
3500 20 2
3500 21 6
3500 16 8
3500 18 2.5
3500 17 4
3500 15 3
3500 Not Observed
3500 19 6
Month of study
for necropsy
21
21
16
18
18
16
14
19
Evidence of
Size of tumor Histological distant
at necropsy (cm) diagnosis metastases
No size given Subcutaneous -
fibrosarcoma
7x4x4 Subcutaneous -
' undifferentiated
round cell sarcoma
7 x 7 x 3.5 Subcutaneous - undif-
ferentiated sarcoma
6x4x3 Subcutaneous -
pleomorphic sarcoma
5x5x3 Subcutaneous -
pleomorphic sarcoma
8x6x4 Subcutaneous -
pleomorphic sarcoma
3.5 Subcutaneous -
neurofibrosarcoma
7 x 7 x 3.5 Subcutaneous -
Yes
None
None
None
None
Yes
None
None
fibrosarcoma
-------
TABLE 5-14. SUMMARY OF TALIVARY GLAND REGION SARCOMA INCIDENCE IN MALE
RATS IN A 2-YEAR INHALATION STUDY WITH DICHLOROMETHANE
(Dow Chemical Company 1980)
Dose
0 ppm
500 ppm
1500 ppm
3500 ppm
Incidence*
1/93 (IX)
0/94 (0%)
5/91 (5.5%)
11/88 (12.5%)
Fisher's exact test
—
—
(P = 0.10, N.S.)
(P = 0.002)
*Cochran-Armitage test for linear trend, P<0.0001.
N.S. = Not significant.
5.3.3.1.2 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 ex-
posed by inhalation to 0, 500, 1,500, and 3,500 ppm of DCM. 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 the hamsters were 61 to 70 grams
when they were received. The hamsters were marked by unique toe clips for
group identification and by ear punch for individual identification within the
cages.
The mortality data for males and females are presented in Table 5-15.
Female hamsters exposed to DCM at 3,500 ppm had a statistically significant
decreased mortality from the 13th through the 24th month of the study. Females
exposed to 1,500 ppm also had statistically significant decreased mortality
from the 20th through the 24th month. This decreased mortality in females
exposed to 3,500 and 1,500 ppm was considered to be exposure-related. The
remaining exposure groups of male (500, 1,500, and 3,500 ppm) and female (500
ppm) hamsters had no exposure-related differences in mortality. Some hamsters
in each group had alopecia at 5.5 months into the study, but this alopecia was
secondary to a mange mite (Demodex species) infection. This parasite did not
result in increased mortality or morbidity. No treatment-related differences
were observed in the body weights of either males or females exposed to 500,
1,500, or 3,500 ppm of DCM.
5-63
-------
TABLE 5-15. CUMULATIVE PERCENT MORTALITY OF HAMSTERS,
2-YEAR DICHLOROMETHANE INHALATION STUDY
(Dow Chemical Company 1980)
Males
Month of
study 0 ppm 500 ppm 1500 ppm 3500 ppm
Females
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
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
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
68.8
75.3
88.2
0
0
0
0.9
3.7
3.7
5.8
5.8
6.7
9.6
11.5
12.5
17. 2t
24.2
26.3
33.3
34.3
37.4
42.6
55. 3t
60.6
69.1
74.5
85.1
0.9
0.9
1.9
1.9
2.8
3.7
3.9
4.9
4.9
5.8
9.7
13.3*
22.6
26.9
32.3
36.6
41.9
52.7
63.6
71.6
80.7
88.6
94.3
100.0
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. 8t
61.lt
68. 4t
75. 8t
89. 5t
0
0
0.9
0.9
0.9
0.9
2.9
3.9
3.9
4.9
4.9
4.9
7. It
11. 2t
15. 3t
23. 5t
27. 6t
30. 6t
40. 9t
45. 2t
53. 8t
72. Ot
80. 6t
90. 3t
*Five males and five females died due to food deprivation. These animals were
subsequently deleted from mortality calculations.
tSignificantly different from controls by Fisher's Exact Test, P < 0.05.
Based on the information available to the Carcinogen Assessment Group (CAG),
it is very difficult to conclude whether the MTD was used. Dow Chemical Company
has not submitted a 90-day dose-finding study, but a 30-day inhalation study
has been reported in a letter from Dr. J. Burek to Dr. D. Singh, dated May 1,
1981.
5-64
-------
"The study was conducted prior to the two-year study, but has not been
reported. CD-I mice, Golden Syrian hamsters, Sprague-Dawley and CDF (F-344)
rats were exposed to 0, 2,500, 5,000, or 8,000 ppm DCM 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, 2,500, and 5,000 ppm groups, and organ/body
weight ratios were calculated. Animals exposed to 8,000 ppm DCM exhibited
anesthetic effects, increased blood urea nitrogen levels in Sprague-Dawley
rats, and decreased body weights in rats. The animals exposed to 5,000 ppm
showed slight anesthesia, decreased body weight in male rats, increased SGPT
values in female mice and Sprague-Dawley rats and increased liver weights in
female mice, hamsters and rats. Animals exposed to 2,500 ppm DCM 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, 8,000 and 5,000 ppm DCM were considered
to be too high a dose level for the two-year study, and 2,500 ppm did not
appear to have produced a severe enough response over the 30-day period.
Therefore, concentrations of 3,500 ppm of DCM was [sic] chosen as the top dose
for the two-year study."
Dow conducted a subchronic dose-finding study for a period of only 30 days
rather than for the 90-day period that is usual in most animal bioassays.
Further, body weight or mortality rate in the experimental group of hamsters
does not decrease as compared to the controls. Hematologic determinations,
serum clinical chemistry, urinalysis, bone marrow collection, and blood COHb
determination were done in animals at the 6-, 12-, 18-, and 24-month interim
kills. No treatment-related effects were observed in any of the parameters
evaluated in male or female hamsters after 6, 12, 18, or 24 months of exposure
to 500, 1,500, or 3,500 ppm of DCM, respectively.
Carboxyhemoglobin determinations were performed on the blood of male and
female hamsters following 22 months on test. Males and females exposed to DCM
at 500, 1,500, or 3,500 ppm all had significantly elevated COHb values. There
was a slight trend in a dose-response relationship in females, since the mean
5-65
-------
COHb value for those exposed to 500 ppm was 23.6 percent, while the values
for females exposed to 1,500 or 3,500 ppm were 30.2 percent and 34.6 percent,
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 meta-
bolism of DCM 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 COHb 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 rela-
tionship 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.
5.3.3.1.2.1 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,
and consisted of decreased numbers of hamsters with amyloidosis of the liver,
kidney, adrenals, thyroid, and spleen. A few animals in each group may or may
not represent the trend of amyloidosis in males.
5.3.3.1.2.2 Gross and histopathologic observations of hamsters killed moribund
or dying spontaneously during the study and those from terminal sacrifice
(24-months). Neoplastic and non-neoplastic observations. Gross and histopath-
ologic examinations were 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 histopathologic observations for males and females are presented in the Dow
Chemical Company report (1980), tables 124 and 127. The observations shown
include all the neoplastic 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 hamsters had in-
creased incidences of lymphosarcoma in the experimental group. The incidences
were 1/96, 6/95, 3/95, and 7/97 (P = 0.033) in the 0, 500, 1,500, and 3,500 ppm
groups, respectively (letter from Hugh Farber, Dow Chemical Company, to EPA,
5-66
-------
dated April 14, 1981). A re-evaluation of lymphosarcoma data of female
hamsters by the CAG resulted in the following incidences: 1/91, 6/92, 3/91,
and 7/91 (P = 0.032) in the 0, 500, 1,500, and 3,500 ppm groups, respectively.
The differences between the denominators above reflect 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 denominators
included the total number of animals. Also, only a small number of mammary
gland tissues were examined, and no lesions were found. Table 5-16 summarizes
the total tumor data. The total number of hamsters with benign tumors increased
significantly in females at 3,500 ppm; the total number of hamsters with malignant
tumors increased significantly in males at 1,500 ppm.
5.3.3.1.3 Summary of the Dow Chemical Company (1980) rat and hamster inhalation
studies. 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 1,500 or 3,500 ppm appeared to have an increased
number of sarcomas in the ventral midcervical 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, 1,500, or 3,500 ppm, respectively. Based on routine sections, special
stains, and ultrastructural evaluations, these tumors appeared to be of mesen-
chymal 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, fibrosarcoma), and still other tumors
had cell types that were undifferentiated 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 local-
ized small tumors described above.
Therefore, there was an apparent association between the increased incidence
of sarcomas in the salivary gland region of male rats and prolonged exposure
via inhalation to 1,500 or 3,500 ppm DCM. There were no salivary gland sarcomas
5-67
-------
TABLE 5-16. SUMMARY OF TOTAL TUMOR DATA FOR HAMSTERS ADMINISTERED DICHLOROMETHAHE BY INHALATION FOR 2 YEARS
(Dow Chemical Company 1980)
en
i
01
CO
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 a
malignant tumor
Concentration
(ppm)
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
moribund
76
74
82
78
18
17
17
18
14
10
6
10
6
8
12
8
Terminal
kill
16
20
11
14
3
9
8
7
2
7
5
6
1
4
3
1
Cumulative
total s
107
104
103
107
25
29
27
29
20
19
13
19
7
13
15t
10
Interim
kills
15
10
10
14
2
0
2
2
2
0
1
1
0
0
1
1
Females
Spontaneous
deaths and
killed
moribund
91
88
81
82
17
20
13
27
11
8
9
22
8
13
4
9
Terminal
kill
0
4
10
9
_
1
4
3
_
1
3
3
_
0
2
0
Cumulative
totals
106
102
101
105
19
21
19
32t
13
9
13
26t
8
13
7
10
*Does not Include hamsters that escaped from their cages, or hamsters that were severely autolyzed, or
severely cannibalized. Also, this total does not Include 500 ppm or 1500 ppm male and female hamsters
from the 6-month Interim kill because no hlstopathology was done on these animals except for a liver
special stain (GomoM's Prussian Blue Iron Reaction).
tSlgnlflcantly different from controls when analyzed by Fisher's Exact Test, P<0.05.
-none examined or not applicable.
-------
in 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 ongoing National
Toxicology Program inhalation study.
2. Male and female rats exposed to DCM had increased numbers of benign
mammary tumors as compared to control values. Female rats exposed to 500, 1,500,
or 3,500 ppm of DCM had increased numbers of benign mammary tumors per tumor-
bearing rat in comparison with 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 benign
mammary tumors was not statistically increased in any exposure group (0, 500,
1,500, or 3,500 ppm groups had a total of 79, 81, 80, and 83 animals with benign
mammary tumors, respectively). Sprague-Dawley rats have very high incidences
of spontaneous mammary tumors. However, the total number of benign mammary
tumors has 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, 1,500, or 3,500
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 1,500^ ppm, and to 3.0 in those
exposed to 3,500 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. The number of rats with benign mammary tumors in males
exposed to 3,500 ppm increased but this increase was not statistically signifi-
cant. The total number of benign mamary tumors in males exposed to 1,500 or
3,500 ppm increased slightly. As was the case in females, these effects in
males exposed to 1,500 or 3,500 ppm were considered to be exposure-related.
There were no mammary gland tumors in male or female hamsters. Also only
28/92, 44/93, 30/94, and 27/93 mammary gland tissues were examined in the 0,
500, 1,500, and 3,500 ppm groups, respectively. Not a single lesion was recog-
nized 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, 1,500,
5-69
-------
and 3,500 ppm groups, respectively (letter from Hugh Farber, Dow Chemical Com-
pany, to EPA, dated April 14, 1981). A re-evaluation of lymphosarcoma data of
female hamsters by the CAG resulted in the following incidences: 1/91, 6/92,
3/91, and 7/91 (P = 0.032) in the 0, 500, 1,500, and 3,500 ppm groups, respectively.
The differences between the denominators above reflect 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 denominators included the
total number of animals. Dow Chemical Company believed that the females exposed
to 3,500 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. These data are not statistically signifi-
cant (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
increased particularly toward the latter part of the experiment, whereas the
body weights of female rats were unaffected in any experimental group. Exposure
to 3,500 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
1,500 and 3,500 ppm, while mortality in male hamsters was unaffected at 500, 1,500,
and 3,500 ppm based on only a 30-day rat and hamster inhalation (dose-finding)
study. Based on this information, it is difficult to judge whether the animals
were given a dose equal to the MTD.
5.3.3.1.4 Dow Chemical Company (1982) inhalation toxicity and oncongenicity
study in rats. A 2-year inhalation study of DCM with Sprague Dawley rats and
Golden Syrian hamsters by Dow Chemical Company (1980) has been described earlier
in this document. In that study, animals were exposed 6 hours/day, 5 days/week
for 2 years to DCM at 0, 500, 1,500, and 3,500 ppm. The carcinogenic response
was positive in rats but negative in hamsters. In rats, the liver appeared to
be the target organ affected by exposure. Hepatocellular vacuolization, consis-
tent with fatty change, was observed in male and female rats inhaling 500,
1,500, or 3,500 ppm DCM. There was an increased incidence of multinucleated
hepatocytes upon exposure to 500, 1,500, or 3,500 ppm, and an increased number
of foci and areas of altered hepatocytes at 3,500 ppm in female rats. Benign
5-70
-------
mammary tumors were increased in male rats inhaling 1,500 or 3,500 ppm, and in
female rats inhaling 500, 1,500, or 3,500 ppm DCM. Male rats exposed to 1,500
or 3,500 ppm DCM had an increased number of sarcomas in the region of the
salivary gland. Female rats inhaling 3,500 ppm DCM had an increased mortality
rate. Carboxyhemogiobin levels in the blood of rats exposed to DCM were higher
than control levels; however, no differences were observed in COHb levels of
animals inhaling DCM at 500 ppm versus animals inhaling it at 3,500 ppm. The
objective of this second study was to further investigate the toxicity of DCM
at concentrations far below those that may cause saturation of the metabolic
processes in rats.
A total of 360 male and 492 female Sprague-Dawley rats (Spartan substrain,
6 to 8 weeks old) were used in this study. Groups of 90 rats/sex were exposed
by inhalation to 0 (control), 50, 200, and 500 ppm DCM (technical grade, lot
#TA 05038, with purity of at least 99.5 percent) 6 hours/day, 5 days/week, for
20 months (males) or 24 months (females). Occasionally an exposure was shorter
than 6 hours, due to vapor generation or mechanical problem. In addition, 30
extra female rats, identified as 500/0, were exposed to 500 ppm DCM for the
first 12 months of the study and were housed as control rats for the duration
of the study (last 12 months). Another 30 female rats, identified as 0/500,
were housed in the same manner as control rats for the first 12 months of the
study and were exposed to 500 ppm DCM for the remaining 12 months of the study.
To determine the rate of DMA synthesis in the liver, 18 female rats were included
in each group. After 6, 12, 15, and 18 months of exposure, five rats/sex/expo-
sure level were sacrificed. In addition, five female rats from each of the
500/0 and 0/500 groups were sacrificed at the 18-month interim necropsy.
Clinical laboratory tests for chemistry, plasma hormone levels, and DNA
were made on interim sacrificed rats. Gross and microscopic examinations were
made of animals at interim and terminal sacrifice, as well as of animals dying
spontaneously and those that were killed moribund during the study.
As reported by the authors, the nominal and analytical concentrations of
DCM in the chambers were in close agreement, indicating no detectable loss or
decomposition of test material during vaporization. Approximately 2 months
after the initial exposure to DCM, symptoms consistent with sialodacryoadenitis
(SDA virus) were observed in male and female rats in each experimental group,
5-71
-------
including control groups. The rats from all exposure groups appeared to be
equally affected, and the symptoms were not apparent 3 weeks after the initial
observation.
No significant difference in body weight gain was noticed in male rats,
but the mean body weights of female rats at 50, 200, or 500 ppm were signifi-
cantly higher throughout the study period in comparison with controls. Although
the authors of this study consider this to be a reflection of biological varia-
bility, the CAG considered that the highest dose was not the MTD because the
same strain of rat tolerated a dose of 3,500 ppm by inhalation in the previous
study at the same laboratory (Dow Chemical Company, 1980).
No increase in mortality rate from that of the control group was observed
in male or female rats. According to the authors, "...due to the high mortality
rate in all groups of male rats, the terminal necropsy for male rats occurred
during the 21st month of exposure to methylene chloride to ensure adequate numbers
of surviving animals for pathologic evaluation." This is not consistent with the
findings in Tables 5-17 and 5-18.
No significant effect on absolute or relative organ weight was seen in
male or female rats. Blood COHb levels were significantly elevated (P < 0.05)
above controls in all experimental groups of rats. Incorporation of ^H-thymidine
into hepatic DNA was unaffected in female rats at 6 and 12 months. DMA synthesis
in rats was not determined at 15 months, as originally scheduled in the protocol,
due to the number of mammary tumors observed in female rats at 200 and 500 ppm.
Results from the 6-, 12-, 15-, and 18-month interim necropsies showed no
definite exposure-related gross or histopathologic findings in male and female
rats from any of the interim sacrifices. An exception was the interim sacrifice
of female rats at 15 months, where 1, 3, 4, and 5 females inhaling DCM at 0,
50, 200, or 500 ppm, respectively, had a focus or foci of altered cells in the
liver. This effect was not apparent in female rats from 6-, 12-, or 18-month
i
interim sacrifices, nor was it apparent from rats dying spontaneously, killed
moribund during the study, or terminally sacrificed.
There were no significant histological lesions observed in other organs,
with the exception of the liver. Data on the liver lesions are given in Tables
5-19 and 5-20. In males, the incidence of hepatocellular vacuolization increased
slightly (Table 5-19). The liver lesions in female rats were significantly in-
creased for foci of altered cell, hepatocellular vacuolization, and multinucleated
5-72
-------
TABLE 5-17. MONTHLY MORTALITY DATA FOR MALE RATS IN
A TWO-YEAR DICHLOROMETHANE INHALATION TOXICITY AND ONCOGENICITY STUDY
(Dow Chemical Company 1982)
Month
of
study
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Terminal sacrifice
0 ppm
0*
0
0
0
0
0
0
0
1(1/85)
1(1/85)
4(3/85)
4(3/85)
5(4/80)
15(12/80)
20(16/80)
33(25/75)
37(28/75)
47(35/75)
59(41/70)
70(49/70)
74(52/70)
50 ppm
0
0
1(1/90)
1(1/90)
1(1/90)
1(1/90)
2(2/85)
2(2/85)
4(3/85)
4(3/85)
4(3/85)
5(4/85)
9(7/80)
13(10/80)
18(14/80)
20(15/75)t
37(28/75)
45(34/75)
64(45/70)
70(49/70)
73(51/70)
200 ppm
0
0
0
1(1/90)
1(1/90)
1(1/90)
2(2/85)
4(3/85)
4(3/85)
4(3/85)
5(4/85)
7(6/85)
11(9/80)
14(11/80)
23(18/80)
31(23/75)
37(28/75)
53(40/75)
69(48/70)
79(55/70)
81(57/70)
500 ppm
0
0
0
0
0
0
0
0
1(1/85)
1(1/85)
1(1/85)
1(1/85)
4(3/80)
8(6/80)
13(10/80)
23(17/75)
25(19/75)
41(31/75)
50(35/70)
66(46/70)
73(51/70)
*Percent mortality (number dead/original number of animals minus animals
sacrificed for an interim necropsy).
tSignificantly different from control value by Fisher's Exact Test.
5-73
-------
TABLE 5-18. MONTHLY MORTALITY DATA FOR FEMALE RATS IN
A TWO-YEAR DICHLOROMETHANE INHALATION TOXICITY AND ONCOGENICITY STUDY
(Dow Chemical Company 1982)
Month
of
study
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Terminal
sacrifice
(after 24
months)
0 ppm
0*
0
0
0
0
0
0
0
1(1/85)
1(1/85)
2(2/85)
4(3/85)
4(3/80)
5(4/80)
9(7/80)
12(9/75)
15(11/75)
19(14/75)
30(21/70)
36(25/70)
43(30/70)
53(37/70)
60(42/70)
64(45/70)
50 ppm
0
0
0
0
0
0
0
0
0
0
0
2(2/85)
5(4/80)
5(4/80)
9(7/80)
15(11/75)
15(11/75)
17(13/75)
27(19/70)
36(25/70)
46(32/70)
53(37/70)
63(44/70)
76(35/70)
200 ppm
1(1/90)
1(1/90)
1(1/90)
1(1/90)
1(1/90)
1(1/85)
1(1/85)
2(2/85)
2(2/85)
2(2/85)
2(2/85)
2(2/85)
5(4/80)
5(4/80)
10(8/80)
13(10/75)
15(11/75)
21(16/75)
30(21/70)
34(24/70)
49(34/70)
54(38/70)
64(45/70)
67(47/70)
500 ppm
0
0
0
0
0
1(1/90)
1(1/85)
1(1/85)
1(1/85)
1(1/85)
1(1/85)
4(3/85)
6(5/80)
9(7/80)
15(12/80)
17(13/75)
19(14/75)
28(21/75)
37(26/70)
39(27/70)
47(33/70)
50(35/70)
54(38/70)
61(43/70)
0/500
0
0
0
0
0
0
0
0
0
0
0
0
3(1/30)
3(1/30)
3(1/30)
7(2/30)
7(2/30)
17(5/30)
24(6/25)
32(8/25)
36(9/25)
48(12/25)
60(15/25)
72(18/25)
500/0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Ot
Ot
3(l/30)t
8(l/25)t
8(2/25)t
16(4/25)t
36(9/25)t
48(12/25)t
52(13/25)
*Percent mortality (number dead/original number of animals minus animals
sacrificed for an interim necropsy).
tSignificantly different from control value by Fisher's Exact Test.
5-74
-------
TABLE 5-19. NON-NEOPLASTIC LIVER LESIONS IN MALE RATS
(Dow Chemical Company 1982)
Exposure level
(ppm)
Foci of
altered cells
Hepatocellular
vacuolization
Terminal
kill
0
50
200
500
1/18
3/19
5/13*
7/19*
3/18
9/19*
7/13*
8/19
Moribund and
spontaneous
death
Combined
0
50
200
500
0
50
200
500
6/52
6/51
6/57
4/51
7/70
9/70
11/70
11/70
19/52
9/51
14/57
20/51
22/70
18/70
21/70
28/70
*Fisher's Exact Test, P < 0.05.
5-75
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TABLE 5-20. NON-NEOPLASTIC LIVER LESIONS IN FEMALE RATS
(Dow Chemical Company 1982)
Exposure Level
(ppm)
Foci of
altered cells
Hepatocellular
vacuolization
Multinucleated
hepatocytes
Termi nal
kill
Moribund and
spontaneous
death
Combined
0
50
200
500
0/500
500/0
0
50
200
500
0/500
500/0
0
50
200
500
0/500
500/0
9/25
5/17
10/22
17/27*
5/7
8/12
12/45
12/53
17/48
14/43
7/18
5/13
21/70
17/70
27/70
.31/70
12/25
13/25
23/25
15/17
21/22
23/27
6/7
12/12
18/45
27/53
23/48
30/43*
9/18
4/13
41/70
42/70
44/70
53/70*
15/25
16/25
4/25
4/17
5/22
16/27*
1/7
6/12*
4/45
2/53
7/48
11/42*
2/18
3/13
8/70
6/70
12/70
27/70*
3/25
9/25*
*Fisher's Exact Test, P < 0.05.
0/500 = rats exposed to 500 ppm DCM for first 12 months.
500/0 = rats exposed to 500 ppm DCM for last 12 months.
hepatocytes at 500 ppm (Table 5-20) as compared to controls. The significance
of these alterations in the liver is not known. Further, the number of liver
lesions appeared to be increased in the 500/0 group at terminal sacrifice,
combined sacrifice, and death as compared to controls, but this was not the
case in the 0/500 ppm group. There were no significant differences in any tumor
type for liver, kidney, spleen, brain, salivary gland, lung, skin, pancreas,
5-76
-------
and mammary gland in male and female rats, with the exception of mammary gland
tumors, which were significantly higher in females (Table 5-21).
In summary, there were significant increases in non-neoplastic liver lesions
(i.e., hepatocellular vacuolization and multinucleated hepatocytes) in female
rats at 500 ppm. There was an increase in benign mammary tumors (adenoma,
fibroma, and fibroadenoma) in female rats. The number of benign mammary
tumors/tumor-bearing rats observed in female rats was 2.0, 2.3, 2.2, and 2.7
in rats inhaling DCM at 0, 50, 200, and 500 ppm, respectively. Female rats of
group 500/0 showed effects that were similar to rats exposed to 500 ppm for 24
months, but the 0/500 group did not differ from the control group.
TABLE 5-21. SUMMARY OF MAMMARY GLAND TUMORS IN FEMALE RATS
(Dow Chemical Company 1982)
Exposure
level (ppm)
Rats with a
benign mammary
tumor
(adenoma,
fibroadenoma,
or fibroma)
Total number
of benign
mammary tumors
(adenomas,
fibroadenomas,
and fibromas)
0
50
200
500
0/500
500/0
0
50
200
500
0/500
500/0
Number
of rats
70
70
70
70
25
25
70
70
70
70
25
25
Moribund and Terminal
spontaneous kill
35
43
41
32
17
11
69
97
91
68
35
27
17
15
20
23
6
12*
36
36
44
79
15
33
Cumulative
52
58
61*
55
23
23
105t
133
135
147
50
60
*Significantly different from control when analyzed by Fisher's Exact
Test, P < 0.05.
tData could not be analyzed by Fisher's Exact Test.
0/500 = rats exposed to 500 ppm DCM for first 12 months.
500/0 = rats exposed to 500 ppm DCM for last 12 months.
5-77
-------
In conclusion, the results of this study offer very limited evidence
of the carcinogenicity of DCM. However, the highest dose in this study is half
of the lowest doses in both the NTP gavage study (1982 draft) and the ongoing
NTP inhalation study.
5.3.3.1.5 National Coffee Association (1982) study in rats. On August 11,
1982, Hazelton Laboratories America, Inc., reported on a chronic study in
Fischer 344 rats administered DCM in deionized drinking water for 24 months.
This study was sponsored by the National Coffee Association (NCA), and utilized
1,000 animals in seven different dose groups or regimens (Table 5-22). The
actual mean daily consumption levels of DCM in the drinking water were similar
to the expected target levels (Table 5-23).
Interim sacrifices were performed at 26, 52, and 78 weeks of treatment with
5, 10, or 20 rats/sex/dose group, respectively, with the exception of the animals
in Group 7 (the recovery group, which were only sacrificed with the remaining
terminal animals at 104 weeks). The effects of compound administration were
evaluated using the following criteria: survival, body weight gains, total food
consumption, water consumption, clinical observations, ophthalmoscopic findings,
clinical pathology, organ and tissue weights, and gross and microscopic pathology.
TABLE 5-22. GROUP ASSIGNMENT OF FISCHER 344 RATS ADMINISTERED
DICHLOROMETHANE IN DEIONIZED DRINKING WATER FOR 24 MONTHS
(NCA 1982a)
Group Number
1.
2.
3.
4.
5.
6.
7.
Control
Control
Low-dose
Mid- dose 1
Mid-dose 2
High-dose
Hi gh-dose/recovery
Number of Animals
Males
85
50
85
85
85
85
25
Females
85
50
85
85
85
85
25
Target dose
(mg/kg/day)
0
0
5
50
125
250
250
(78 weeks/26 weeks)
-------
TABLE 5-23. MEAN DAILV CONSUMPTION OF DICHLOROMETHANE IN 24-MONTH
CHRONIC TOXICITY AND ONCOGENICITY STUDY IN FISCHER 344 RATS
(NCA 1982a)
Target level
Group (mg/kg/day) Males Females
3 5
4 50
5 125
6 250
7 250*
5.85
52.28
125.04
235.00
232.13
6.47
58.32
135.59
262.81
268.72
*The designated recovery group (Group 7) mean Is for the first 78
weeks only.
No compound-related findings were reported for either survival, clinical
observations, opthalmoscopic findings, gross necropsy findings, or organ
weight data. Throughout the study, small but significantly decreased body
weight gains and water consumption were reported for both male and female rats
in Groups 5, 6, and 7. Food consumption also decreased, but this criteria was
only monitored for the first 13 weeks. These decreased effects were attributed
to DCM administration. Low-magnitude statistical increases in mean hemotocrit,
hemoglobin, and red blood cell counts were noted in both male and female animals
of Groups 4, 5, and 6. In most cases, these were within the range of historical
control values of Fischer 344 rats.
Histopathological alterations were described in the livers of rats of
both sexes in Groups 4, 5, 6, and 7. These changes consisted of an increased
incidence of foci/areas of cellular alteration in Groups 4, 5, 6, and 7 and of
fatty changes in Groups 5 and 6 after 78 and 104 weeks of treatment. The
incidence of neoplastic nodules and/or hepatocellular carcinomas in female
Fischer 344 rats (Table 5-24) was derived from the data presented in Volumes
I-IV of the August 11, 1982, NCA report (NCA 1982a) and in the Addition to the
5-79
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TABLE 5-24. INCIDENCE OF HEPATOCELLULAR TUMORS IN MALE AND FEMALE FISCHER 344 RATS
ADMINISTERED DICHLOROMETHANE IN DEIONIZED DRINKING WATER FOR 104 WEEKS
(NCA 1982a, b)
Cn
00
o
Diagnosis
Neoplastic nodule (NN)
Hepatocellular carcinoma
(HC)
Combined NN and HC
Total livers examined
Neoplastic nodule (NN)
Hepatocellular carcinoma
(HC)
Combined NN and HC
Total livers examined
Treatment group (mg/kg/day)
0
4(4.7.)
2(2.4)
6(7.1)
85
0(0)
0(0)
0(0)
85
0
5(10)
2(4)
7(14)
50
0(0)
0(0)
0(0)
49
5
2(2.4)
0(0)
2(2.4)
85
1(1.2)
0(0)
1(1.2)
85
50
Male*
3(3.6)
0(0)
3(3.6)
84
Female*
2(2.4)
2(2.4)
4(4. 8)t
83
125
3(3.5)
0(0)
3(3.5)
85
1(1.2)
0(0)
1(1.2).
85
250
1(1.2)
1(1.2)
2(2.4)
85
4(4. 7)t
2(2.4)
6(7. l)t
85
250 (Recovery)
4(16)
0(0)
4(16)
25
2(8.0)
0(0)
2(8. 0)t
25
*Numbers in parentheses represent percent incidence for the particular lesion.
tStatistically significant (P < 0.05) by the Fisher's Exact Test using a combined control incidence of 0/134.
-------
Final NCA Report of November 5, 1982 (NCA 1982b). Male rats did not show
an increased incidence of liver tumors in treated animals versus controls
(Table 5-23). These statistically significant increases in the incidences
of liver tumors in female rats were within the range of historical control
values at this laboratory (Table 5-25), as presented in a letter from
Dr. John Kirschman of General Foods to Dr. Dharm Singh of the CAG, dated
February 17, 1983. Therefore, based on a review of the NCA study, DCM
administered in deionized water at doses up to 250 mg/kg/day was borderline
for carcinogenicity to Fischer 344 rats.
5.3.3.1.6 National Toxicology Program (1982 draft) gavage study in rats and
mice. The National Toxicology Program (NTP) conducted a 2-year carcinogenesis
bioassay of food-grade DCM and reported on their results in a draft technical
report dated September 22, 1982 (NTP, 1982). Dichloromethane was administered
in corn oil by gavage to male and female Fischer 344/N rats and B6C3F1 mice.
The DCM was more than 99.5 percent pure, with vapor phase chromatographic
analysis detecting the presence of vinylidene chloride and trans-dichloroethylene
up to 0.4 percent. A 13-week dose finding study was conducted to evaluate the
toxicity of the compound. Based on survival, body weight gain, and histopathol-
ogical examination, doses of 500 and 1,000 mg/kg by gavage were selected for male
and female rats and mice for the 2-year study.
The NTP announced on July 25, 1983 that the draft NTP report would not be
issued as a final report due to discrepancies in experimental data that compro-
mise a clear interpretation. NTP further allowed that pending the results of
an in-depth audit, select and relevant information from these gavage studies
might be incorporated into the future draft technical report for DCM inhalation
studies.
5.3.3.1.7 Theiss et a!. (1977). A pulmonary tumor bioassay in mice was reported
by Theiss et al. (1977). Groups of 20 male strain A mice were injected intra-
peritoneally three times a week with 0, 160, 400, or 800 mg/kg DCM for a total
of 16 or 17 injections. Mice were sacrificed 24 weeks after the first injection,
and the lungs were examined under a dissecting microscope for surface adenomas.
Some adenomas were confirmed by histology.
5-81
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TABLE 5-25. HISTORICAL CONTROL DATA OF LIVER NEOPLASIA IN FEMALE FISCHER 344 RATS
AT HAZELTON LABORATORIES AMERICA, INC.
(Kirschman, 1983)
en
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IN3
LIVER (No.
Neoplastic
% Incidence
Study: A ' B C
Group: 12 11
examined) 57 70 37 40
nodules 54----
8.8 5.7 —
Hepatocellular carcinoma
% Incidence
_.
D E
T T
38 43
1
2.3
1 1
2.6 2.3
F
1
11
1
9.1
1
9.1
G
~~7 T
14 39
4
28.6
1
7.1
H I
r T
40 17
2
11.8
--
__
J
T
13
2
15.4
1
7.8
Study
Total
419
19
4
5
1
s
.5
.2
— = Not reported.
-------
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 two highest dose levels (Table 5-26). At the lowest dose,
a highly significant increase in the number of tumors was observed (P = 0.013).
Therefore, this study was marginally positive for carcinogen!city.
TABLE 5-26. PULMONARY TUMOR BIOASSAY IN STRAIN A MICE
(adapted from Theiss et al. 1977)
Dose
(mg/kg)
0
160
400
800
Total dose
given
0
2,720
6,800
12,800
No. of mice
at beginning
20
20
20
20
No. of mice
exami ned
for tumors
15
18
5
12
Tumors/mouse
0.27
0.94
0.80
0.50
Significance*
—
<0.013
>0.1
>0.1
*The test of significance used is the exact test of ratio of two Poisson parameters.
5.3.3.1.8 Heppel et al. (1944). Heppel et al. (1944) exposed dogs, rabbits,
guinea pigs, and rats to DCM by inhalation at levels of 5,000 ppm for 7 hours/day
and 10,000 ppm for 4 hours/day, 5 days/week, for 6 months. No tumors developed
in any animals.
5.3.3.1.9 McEwen et al. (1972). McEwen et al. (1972) exposed dogs to DCM by
inhalation at 500 ppm for 14 weeks; no tumors were reported, but edema of the
meninges of the brain occurred. Neither this study nor the Heppel et al.
(1944) study could have detected a carcinogenic response because of the shortness
of the observation times and the fact that these studies were not originally
designed to test for carcinogenicity.
5-83
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5.3.3.1.10 Other animal studies in progress. The NTP sponsored a 2-year inhala-
tion study in male and female Fischer 344/N rats and B6C3F1 mice at exposure con-
centrations of 0, 1,000, 2,000, and 4,000 ppm for rats and 0, 2,000, and 4,000
ppm for mice. The animals have been sacrificed (April 1983), but the pathology
report is not yet available.
A 2-year study was sponsored by the National Coffee Association in which
male and female B6C3F1 mice were administered daily doses of 0, 60, 125, 185,
or 250 mg/kg DCM in drinking water. The animals have been sacrificed, and the
results of this study should be available by the end of 1983.
5.3.3.1.11 Cell transformation studies. Price et al. (1978) exposed Fischer
rat embryo cell cultures (F1706, subculture 108) to DCM liquid at concentrations
of 1.6 x 102 and 1.6 x 103 yM for 48 hours. Dichloromethane was diluted with
growth medium to yield the appropriate doses. The DCM sample, obtained from the
Fisher Scientific Company, was >_ 99.9 percent pure. The cells were grown in
Eagles minimum essential medium in Earle's salts supplemented with 10 percent
fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential ami no acids, 100 y g
penicillin, and 100 yg streptomycin/ml. Quadruplicate cultures were treated at
50 percent 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 DCM 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 cells grown in medium alone or in
the presence of a 1:1,000 acetone concentration, even after a subculture.
Twenty and 27 microscopic foci per three dishes with the low and high DCM doses
respectively, were found in 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 before staining.
Subcutaneous injection of cells treated with 1.6 x 102 yM DCM five subcul-
tures 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 in the presence of a 1:1,000 concentration of
acetone. Exposure of cells to 3.7 x 10'1 yM 3-methylcholanthrene produced 124
microscopic foci per three dishes in the inoculation test described above by 37
5-84
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days of incubation, and local fibrosarcomas in 12/12 rats by 27 days following
subcutaneous injection of cells. The exposure of 3-methylcholanthrene was
attained by initial dilution in acetone to 1 mg/ml followed by further dilution
in growth medium to 0.1 ug/ml (personal communication from Dr. P. J. 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 percent. The original
study was done in quadruplicate and in each case the Fischer rat cells were
transformed. Since the publication, the same batch of methylene chloride was
sent to Andy Sivak at Arthur D. Little to be run against the Kakunago clone A31
of BALB/c 3T3. It did not transform his cells. We then repeated the study in
Fischer rat cells and at the same time tested methylene chloride sent to us by
the National Coffee Producers Association. The test was run in triplicate.
The Fisher methylene chloride again transformed the cells, while the National
Coffee Producers' (supplied by Diamond Shamrock) was negative.' The differing
responses in the two experiments may have been due to differences in the levels
of impurities present in the samples used. The chemical compositions of DCM
samples from different suppliers are given in Table 5-27.
The Fischer rat embryo cell line contains the genome of the Rauscher
leukemia virus, but there is no basis for minimizing the positive results.
Since the mode of action of DCM is not known, this transformation may be due to
activation of the virus.
5.3.3.2 Epidemiologic Studies—
5.3.3.2.1 Friedlander et al. (1978), Hearne and Friedlander (1981). Friedlander
et al. (1978) performed mortality analyses of male Eastman-Kodak employees
exposed to low levels of DCM. This study was updated by Hearne and Friedlander
(1981). Measurements from 1959 to 1975 ranged from 30 to 120 ppm. Both the
original 1978 analysis and the 1981 update found no increase in neoplasms,
heart disease, or any other cause of death in comparison with the two control
groups, which were composed of other Kodak employees and of New York State
males. The population was relatively stable and the workers were rotated
throughout the work area, and thus exposure was averaged among all the workers.
Dichloromethane had been used for 30 years as the primary solvent in this
Eastman-Kodak operation.
5-85
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TABLE 5-27. DICHLOROMETHANE ANALYSES*
Methyl chloride
Vinyl chloride
Ethyl chloride
Vinylidine chloride
Carbon tetrachloride
Fisher D-123t
Lot 761542 Diamond ShamrockS
(ppm) (ppm)
0.5 <1
0.8 <1
329 <1
3.6 <1
Dow#
(ppm)
3
33
Chloroform <0.1 26 <5
Trichloroethylene <0.1 <1 <1
1,2-Dichlorethylene 369 20 86
Methyl bromide — — 11
Cyclohexane — 300 305
*Presented by Drs. Sivak and Kirschman at the Science Advisory Board
Meeting, Sept. 4-5, 1980.
tFisher sample used by Dr. P. Price in two series of cell transformation studies.
§This material is under test in NCA's chronic rat study on DCM in
drinking water. It was also used in Dr. Price's follow-up study.
#This material, also analyzed by NCI, with the same results as given by
Drs. Sivak and Kirschman, was used in the following tests:
NCA's 90-day studies in rats and mice
Dr. Sivak's neoplastic transformation assay
Dr. P. Price's follow-up series of cell transformation tests
NCI's chronic bioassay studies in rats and mice presently under way
5-86
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Two separate mortality analyses were done. In the earlier paper, one
approach used the proportionate mortality ratio to examine 334 deaths of
DCM-exposed workers during 1956 to 1976. Seventy-one neoplasms were found; 73
were expected based on other Kodak employee mortality ratios. Furthermore, no
single site was over-represented.
A second approach, included in the first paper and used exclusively in the
update, was a cohort mortality study of all 751 employees who were in the DCM
work area in 1964. The results of the update are shown in Tables 5-28 and
5-29, taken from that paper. There were 110 deaths during the 16-year follow-
up (retrospective). Two control groups were used: other Kodak employees, and
New York State males. The expected numbers of deaths in the exposed groups
based upon the control group experiences were 105 and 168, respectively. The
differences between the observed and the expected deaths based on the controls
are either not statistically significant (other Kodak employees) or the expected
deaths based on New York State males are significantly increased over the
observed numbers.
The results show that malignant neoplasms accounted for 24 of the 110 deaths
in the study cohort, which was less than the 28.6 or 38.5 expected malignancies
based on the control data. Nine of these 24 deaths were from respiratory cancer
(7.6 and 13.6 were expected based on the control groups) and seven were from can-
cers of the digestive organs (less than expected). Only the two deaths associated
with brain or nervous tissue represented a higher than expected total (SMR =
169 and SMR = 227 vs. two control groups), but these SMRs (standard mortality
ratios) 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 59 deaths occurred:
13 were due to malignant neoplasms (17.8 and 24.7 expected based on the two
control groups) and 32 were due to circulatory diseases (37.9 and 59.7 expected).
A further analysis of the 252 males shows that this cohort had a median
age of approximately 54 years in 1964. With this group the more common cancers
would have to be markedly increased for there to be a reasonable probability of
detecting tKe increase. For example, following the cohort for 16 years, cancer
mortality at this age would require 10 deaths from respiratory cancer to detect
a significant result at the P = 0.05 level. This represents an increase of at
5-87
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TABLE 5-28. OBSERVED AND EXPECTED DEATHS, 1964-1980,
1964 HOURLY MALE DICHLOROMETHANE COHORTS FROM KODAK
{Hearne and Friedlander, 1981)
en
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oo
Cohort exposed minimum
Diagnostic group
Malignant neoplasms
Circulatory diseases
Ischemic heart disease
Other circulatory
Respiratory diseases
Accidents
All other causes
All causes
No.
observed
13
32
27
5
5
2
7
59
KP* no.
expected
17 .77
37.91
27.34
10.57
3.64
2.35
4.27
65.94
20 years
NYSt no.
expected
24.72t
59.735
45.505
14.23
7.11
3.90
10.60
106. 06§
Total 1964 CH2C12 cohort
No.
observed
24
62
48
14
8
6
10
110
KP* no.
expected
28.64
59.11
43.40
15.71
5.17
5.53
6.63
105.08
NYSt no.
expected
38.54§
90.905
69.545
21.36
10.32
9.80
18.44
168.005
*Based on age-sex specific mortality of hourly men, rest of Kodak Park, 1964-1976.
tBased on age-sex specific mortality New York State (excluding New York City) for the years 1965, 1970, 1975,
SObserved-expected differences significant (P £0.05).
-------
TABLE 5-29. MALIGNANT NEOPLASMS, OBSERVED AND EXPECTED DEATHS, 1964-1980,
1964 HOURLY MALE DICHLOROMETHANE COHORTS FROM KODAK
(Hearne and Friedlander, 1981)
i
oo
Cohort exposed minimum 20 years
Total 1964 CH2Cl2 cohort
Diagnostic Group
Digestive organs
Pancreas
Other
Respiratory
Genitourinary
Brain and other nervous
Lymphatic and hematopoietic
Other malignant neoplasms
Total malignant neoplasms
No.
observed
5
2
3
4
0
1
0
3
13
KP* no.
expected
7.08
1.34
5.74
4.60
2.19
0.62
1.44
1.84
17.77
NYSt no.
expected
7.73
1.48
6.25
8.675
3.48
0.44
2.00
2.40
24.72S
No.
observed
7
3
4
9
1
2
1
4
24
KP* no.
expected
10.93
2.36
8.57
7.56
<=, 3.23
1.18
2.68
3.06
28.64
NYSt no.
expected
11.68
2.24
9.44
13.62
4.94
0.88
3.45
3.97
38.54§
*Based on age-sex specific mortality of hourly men, rest of Kodak Park, 1964-1976.
tBased on age-sex specific mortality New York State (excluding New York City) for the years 1965, 1970, 1975.
§0bserved-expected differences significant (P < 0.05).
-------
least 100 percent over that expected, the expected probability of lung cancer
death for this cohort being 0.018 over the 16 years, based on other Kodak
employees' rates. The statistical power to detect 100 percent increases (at a
= 0.05, one-sided) is about 95 percent for all malignancies and 45 percent for
respiratory cancer deaths. 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 16 years mortality might fail to detect even a moderate
effect, since expected cancer mortality in this age group is so low.
5.3.3.2.2 Ott et a!. (1983a, b, c, d. e). Ott et al. (1983a, b, c, d, e)
recently reported the results of a health evaluation of employees of one fiber
production plant who were exposed to DCM as part of a solvent mixture consisting
of this substance plus methanol and also acetone in a separate container. A
second fiber production plant utilizing only acetone, but similar to the first
in other respects, was selected as a referent population. The investigation
focused primarily on health effects occurring to the cardiovascular system stem-
ming from the metabolism of DCM to COHb in the body. In addition to a cohort
mortality study, the authors examined several other health endpoints in the
still-employed group. These involved clinical evaluations, electrocardiographic
monitoring, metabolism tests, and evaluation of oxygen half-saturation pressures.
Environmental exposure in the plant varied from 140 ppm in areas of low
DCM use to 475 ppm in areas of high DCM use, based on an 8-hour time-weighted
average (TWA). Methanol was present in smaller quantities by a factor of ten,
while acetone ranged from 100 to 1,000 ppm TWA in both plants (Ott et al.,
1983a). Industrial hygiene surveys were conducted from September 1977 to
February 1978.
To qualify for inclusion in the cohort, production employees (both men
and women) had to have worked a minimum of three months in the preparation or
extrusion areas of either plant during January 1, 1954 to January 1, 1977. In
the DCM plant, both cellulose diacetate (acetate) and cellulose triacetate
(CTA) fibers were made side by side. Although acetone was present in both
5-90
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plants, it was the solvent of choice for making acetate fibers in the referent
plant. The exposed cohort consisted of 1,271 persons versus 948 persons in the
referent plant, as follows:
Referent
PCM plant plant
White men 487 696
women 615 248
Nonwhite men 64 1
women 105 3
Total T72TT
Persons were followed through June of 1977. The authors noted that vital
status was not available for 226 (18 percent) of the DCM-exposed cohort and 112
(12 percent) of the companion plant. The authors commented that few deaths
would be added from this last group based on "previous experience with the
social security follow-up mechanism." Such a statement is subject to question,
however, without knowing the age distribution of this group.
The authors found no excessive mortality from any cause in the "exposed"
or the "referent" population, either when the group with unknown vital status
was presumed lost to follow-up at the time lost, or was presumed alive until
June of 1977; i.e., person-years would cease accumulating at the time the
person was lost to follow-up in the former instance, but in the latter case,
person-years would continue to accumulate until the end of the follow-up period,
as if the individual were still alive. Altogether in the exposed category for
white men, 37 observed deaths out of 487 were seen versus 34.9 expected based
on the latter definition, or 30.4 expected based on the former definition re-
garding the group with unknown vital status. For white women, only 11 deaths
were observed versus either 15.9 or 13.5, depending upon the choice of the first
or second definition above. For malignant neoplasms in white men, there were
five observed deaths versus either 6.3 or 5.6 expected. In white women, two ob-
served deaths were seen, versus either 5.2 or 4.5 expected. No cancer deaths were
reported in nonwhite males and females, probably due to the small size of these
select subgroups. Since the authors were mainly interested in cardiovascular
5-91
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effects, they decided to examine further only ischemic heart disease in terms
of duration of exposure and length of follow-up. Even when considering only
individuals with a minimum of 10 years of exposure who were followed for a
minimum of ten years, only two male deaths were observed versus 1.7 expected
based on this cause. Ischemic heart disease was not observed as a cause of
death in women. No corresponding data are available for any form of cancer by
latency or duration of employment.
Several deficiencies are to be noted with respect to the use of this study
as a sensitive indicator of mortality. Foremost among these is the relatively
low and unusual distribution of mortality among the members of the cohort.
Because of the excessive numbers of observed and expected deaths from external
causes compared to the numbers of observed and expected deaths from malignant
neoplasms, and the fact that deaths from accidents are the leading cause of
death in young males nationwide, it is quite likely that the cohort is a rela-
tively youthful group. This is further evidenced by the surprisingly few
deaths observed overall compared to the size of the cohort--!ess than 10 percent
in all instances (women, nonwhite women, and white females). Unfortunately,
no age breakdown is available to confirm this observation.
Additionally, only 310 of the exposed males have had a chance to be followed
at least 17.5 years. Some 241 exposed males entered the cohort after 1960 and
could not possibly have been followed 17.5 years. Because of the 15- to 20-year
latency period involved with most human cancers, cancer effects attributable to
DCM exposure would probably not have been expected to manifest themselves prior
to the 17th year. Thus, the power of this study to detect a statistically
significant elevated risk of cancer {as well as ischemic heart disease) is low.
Another possible problem deals with the extent of follow-up of the cohort.
Almost 18 percent (226) of the exposed cohort was lost to follow-up as of June
1977. Although the authors discount that as unimportant, it should be of
concern that if the ages of the lost-to-follow-up group are relatively advanced,
the likelihood is great that enhanced mortality will be in the higher age
groups. Whenever extraordinary means are employed to determine the vital status
of a subgroup of the cohort for which all other methods of follow-up have
failed, the residual deaths found as a result of this endeavor are usually
overly represented by sudden deaths due to heart failure or accidents. The
5-92
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opportunity for leaving a record of their deaths is minimized because of the
nature of the deaths; hence, it becomes less likely that the vital status can
be determined.
Still another problem with this study is the "healthy worker" effect. In
most studies of this kind, workers at the time of their employment and shortly
thereafter are generally somewhat healthier than the population from whence
they came. Ill or infirm persons do not usually choose jobs that may be detri-
mental to their health. This tendency usually results in as much as a 20 percent
deficit of mortality compared to that expected.
In summary, this study is inadequate to assess cancer mortality in the
described cohort for the reasons stated. The study focuses mainly on heart
disease as a consequence of DCM exposure.
5.3.3.3 Quantitative Estimation. This quantitative section deals with the unit
risk for DCM in air and water and the potency of DCM relative to other carcino-
gens that the CAG has evaluated. The unit risk estimate for an air or water
pollutant is defined as the lifetime cancer risk occurring in a hypothetical
population in which all individuals are exposed continuously from birth through-
out their lifetimes to a concentration of 1 yg/m^ of the agent in the air they
breathe or to a concentration of 1 yg/1 in the water they drink. This calcula-
tion provides a quantitative estimation of the impact of the agent as a carcino-
gen. Unit risk estimates are used to compare the carcinogenic potency of
several agents with each other and to give a crude indication of the population
risk that might be associated with air or water exposure to these agents, if
the actual exposures are known.
5.3.3.3.1 Procedures for the determination of unit risk for animals. The data
used for the quantitative estimate are taken from one or both of the following:
1) lifetime animal studies, and 2) human studies where excess cancer risk has
been associated with exposure to the agent. In animal studies it is assumed,
unless evidence exists to the contrary, that if a carcinogenic response occurs
at the dose levels used in the study, then responses will also occur at all
lower doses, with an incidence determined by an extrapolation model.
There is no solid scientific basis for any mathematical extrapolation
model that relates carcinogen exposure to cancer risks at the extremely low
concentrations that must be dealt with in evaluating environmental hazards.
5-93
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For practical reasons, such low levels of risk cannot be measured directly
either by animal experiments or by epidemiologic studies. We must, therefore,
depend on our current understanding of the mechanisms of carcinogenesis for
guidance as to which risk model to use. At the present time, the dominant view
of the carcinogenic process involves the concept that most cancer-causing
agents also cause irreversible damage to DMA and are mutagenic. There is
reason to expect that the quanta! type of biological response, which is charac-
teristic of mutagenesis, is associated with a linear non-threshold dose-response
relationship. Indeed, there is substantial evidence from mutagenicity studies
with both ionizing radiation and a wide variety of chemicals that this type of
dose-response model is the appropriate one to use. This is particularly true
at the lower end of the dose-response curve; at higher doses, there can be an
upward curvature, probably reflecting the effects of multistage processes on
the mutagenic response. The linear non-threshold dose-response relationship is
also consistent with the relatively few epidemiologic studies of cancer responses
to specific agents that contain enough information to make the evaluation
possible (e.g., radiation-induced leukemia, breast and thyroid cancer, skin
cancer induced by arsenic in drinking water, liver cancer induced by aflatoxins
in the diet). Also, some evidence from animal experiments is consistent with
the linear non-threshold model (e.g., liver tumors induced in mice by 2-acetyla-
minofluorene in the large scale EDgi study at the National Center for Toxicolog-
ical Research, and the initiation stage of the two-stage carcinogenesis model
in rat liver and mouse skin).
Because its scientific basis, although limited, is the best of any of the
current mathematical extrapolation models, the linear non-threshold model has
been adopted as the primary basis for risk extrapolation in the low-dose region
of the dose-response relationship. The risk estimates made with this model
should be regarded as conservative, representing the most plausible upper limit
for the risk; i.e., the true risk is not likely to be higher than the estimate,
but it could be lower.
The mathematical formulation chosen to describe the linear non-threshold
dose-response relationship at low doses is the linearized multistage model.
This model employs enough arbitrary constants to be able to fit almost any
monotonically increasing dose-response data, and it incorporates a procedure
5-94
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for estimating the largest possible linear slope (in the 95 percent confidence
limit sense) at low extrapolated doses that is consistent with the data at all
dose levels of the experiment.
5.3.3.3.1.1 Description of the low-dose animal extrapolation model. Let P(d)
represent the lifetime risk (probability) of cancer at dose d. The multistage
model has the form
P(d) = 1 - exp [-(q0 + qjd + q2d + q2d2 + ... + qkdk]
where
qj _> 0, i = 0, 1, 2, .... k.
Equivalently,
Pt(d) = 1 - exp [-(q^ + q2d2 + ... + qkdk)]
where
P (d) = P(d) - P(0)
t i - P(0)
is the extra risk over background rate at dose d or the effect of treatment.
The point estimate of the coefficents qj, i = 0, 1, 2, ..., k, and
consequently the extra risk function P^(d) at any given dose d, is calculated
by maximizing the likelihood function of the data.
The point estimate and the 95 percent upper confidence limit of the extra
risk, Pt(d), are calculated by using the computer program GLOBAL79, developed by
Crump and Watson (1979). At low doses, upper 95 percent confidence limits on the
extra risk and lower 95 percent confidence limits on the dose producing a given
risk are determined from a 95 percent upper confidence limit, q*, on parameter qj.
1
Whenever qi > 0, at low doses the extra risk Pt(d) has the approximate form
P-t(d) = qj x d. Therefore, q* x d is a 95 percent upper confidence limit on the
extra risk, and R/q* is a 95 percent lower confidence limit on the dose producing
an extra risk of R. Let LQ be the maximum value of the log-likelihood function.
The upper limit, q*, is calculated by increasing qj to a value q* such that
when the log-likelihood is remaximized subject to this fixed value, q*, for the
5-95
-------
linear coefficient, the resulting maximum value of the log-likelihood LI
satisfies the equation
2 (LQ - LI) = 2.70554
where 2.70554 is the cumulative 90 percent point of the chi -square distribution
with one degree of freedom, which corresponds to a 95 percent upper limit
(one-sided). This approach of computing the upper confidence limit for the
extra risk, Pt(d), is an improvement on the Crump et al . (1977) model. The
upper confidence limit for the extra risk calculated at low doses is always
linear. This is conceptually consistent with the linear non-threshold concept
discussed earlier. The slope, q*, is taken as an upper bound of the potency of
the chemical in inducing cancer at low doses. (In the section calculating the
risk estimates, P^fd) will be abbreviated as P.)
In fitting the dose-response model, the number of terms in the polynomial
is equal to (h-1), where h is the number of dose groups in the experiment,
including the control group.
Whenever the multistage model does not fit the data sufficiently, data at
the highest dose is deleted, and the model is refit to the rest of the data.
This is continued until an acceptable fit to the data is obtained. To determine
whether or not a fit is acceptable, the chi-square statistic
(1-Pj)
is calculated where N^ is the number of animals in the in dose group, X^ is
the number of animals in the i™1 dose group with a tumor response, P.,- is the
probability of a response in the i'th dose group estimated by fitting the
multistage model to the data, and h is the number of remaining groups. The
fit is unacceptable whenever X^ is larger than the cumulative 99 percent point
of the chi-square distribution with f degrees of freedom, where f equals the
number of dose groups minus the number of non-zero multistage coefficients.
5-96
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5.3.3.3.1.2 Selection of data. For some chemicals, several studies using
different animal species, strains, and sexes and run at several doses and
different routes of exposure are available. A choice must be made as to which
of the data sets from several studies to use in the model. The procedures used
in evaluating these data are consistent with the approach of making a maximum-
likely-risk estimate. They are listed as follows:
1. The tumor incidence data are separated according to organ sites or tumor
types. The set of data (i.e., dose and tumor incidence) used in the model is the
set where the incidence is significantly higher statistically than the control
for at least one test dose level or where the tumor incidence rate shows a
statistically significant trend with respect to dose level. The data set that
gives the highest estimate of the lifetime carcinogenic risk, q*, is selected
in most cases. However, efforts are made to exclude data sets that produce
spuriously high risk estimates because of a small number of animals. That is,
if two sets of data show a similar dose-response relationship and one has a
very small sample size, the set of data having the larger sample size is selected
for calculating the carcinogenic potency.
2. If there are two or more data sets of comparable size that are identi-
cal with respect to species, strain, sex, and tumor sites, the geometric mean
of q*, estimated from each of these data sets, is used for risk assessment.
The geometric mean of numbers Aj_, A2, ..., Am is defined as
(Ax x A2 x ...
3. If two or more significant tumor sites are observed in the same study,
and if the data are available, the number of animals with at least one of the
specific tumor sites under consideration is used as incidence data in the model
5.3.3.3.1.3 Calculation of human equivalent dosages. It is appropriate to
correct for metabolism differences between species and absorption factors via
different routes of administration. Following the suggestion of Mantel and
Schneiderman (1975), it is assumed that ing/surface area/day is an equivalent
dose between species. Since the surface area is approximately proportional to
the two-thirds power of the weight, as would be the case for a perfect sphere,
5-97
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the exposure in mg/day per two-thirds power of the weight is also considered
to be equivalent exposure. In an animal experiment, this equivalent dose is
computed in the following manner. Let
Le = duration of experiment
le = duration of exposure
m = average dose per day in mg during administration of the agent (i.e.,
during le), and
W = average weight of the experimental animal.
Then, the lifetime average exposure is
d- leXm
Le x W2/3
Inhalation—When exposure is via inhalation, the calculation of dose can be
considered for two cases where 1) the carcinogenic agent is either a completely
water-soluble gas or an aerosol and is absorbed proportionally to the amount of
air breathed in, and 2) where the carcinogen is a poorly water-soluble gas
that reaches an equilibrium between the air breathed and the body compartments.
After equilibrium is reached, the rate of absorption of these agents is expected
to be proportional to the metabolic rate, which in turn is proportional to the
rate of oxygen consumption, which in turn is a function of surface area.
Case 1—Agents that are in the form of particulate matter of virtually
completely absorbed gases, such as sulfur dioxide, can reasonably be expected
to be absorbed proportionally to the breathing rate. In this case the exposure
in mg/day may be expressed as
m = I x v x r
where I = inhalation rate per day in m3, v = mg/m3 of the agent in air and
r = the absorption fraction.
5-98
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The inhalation rates, I, for various species can be calculated from the
observations of the Federation of American Societies for Experimental Biology
(FASEB 1974) that 25 g mice breathe 34.5 liters/day and 113 g rats breathe
105 liters/day. For mice and rats of other weights W (in kilograms), the sur-
face area proportionality can be used to find breathing rates in m3/day, as
follows:
For mice, I = 0.0345 (W/0.025)2/3 m3/day
For rats, I = 0.105 (W/0.113)2/3 m3/day.
For humans, the value of 20 m3/day* is adopted as a standard breathing rate
(ICRP 1977).
The equivalent exposure in mg/W2/3 for these agents can be derived from
the air intake data in a way analogous to the food intake data. The empirical
factors for the air intake per kilogram per day, i = I/W, based upon the pre-
viously stated relationships, are tabulated as follows:
Species W i = I/W
Man 70 0.29
Rats 0.35 0.64
Mice 0.03 1.3
Therefore, for particulates or completely absorbed gases, the equivalent ex-
posure in mg/W2/3 is
m = Ivr = iWvr = iwl/3vr.
*From "Recommendation of International Commission on Radiological Protection,"
page 9. The average breathing rate is 10? cm3 per 8-hour workday and 2 x 10' cm3
in 24 hours.
5-99
-------
In the absence of experimental information or a sound theoretical argument to
the contrary, the fraction absorbed, r, is assumed to be the same for all species.
Case 2 — The dose in mg/day of partially soluble vapors is proportional to
the 02 consumption, which in turn is proportional to W2'3 and is also proportional
to the solubility of the gas in body fluids, which can be expressed as an absorp-
tion coefficient, r, for the gas. Therefore, expressing the Q£ consumption as
02 = k W2'3, where k is a constant independent of species, it follows that
m = k W2/3 x v x r
or
d = J" = kvr.
~
As with Case 1, in the absence of experimental information or a sound theoretical
argument to the contrary, the absorption fraction, r, is assumed to be the same
for all species. Therefore, for these substances a certain concentration in
ppm or yg/m3 in experimental animals is equivalent to the same concentration
in humans. This is supported by the observation that the minimum alveolar con-
centration necessary to produce a given "stage" of anesthesia is similar in man
and animals (Dripps et al . , 1977). When the animals are exposed via the oral route
and human exposure is via inhalation or vice versa, the assumption is made, unless
there is pharmacokinetic evidence to the contrary, that absorption is equal by
either exposure route.
5.3.3.3.1.4 Calculation of the unit risk from animal studies. The 95 percent
upper-limit risk associated with d mg/kg2/3/day is obtained from GLOBAL79, and
for most cases of interest to risk assessment, can be adequately approximated
by P(d) = 1 - exp (-q*d). A "unit risk" in units X is the risk corresponding
to an exposure of X = 1. This value is estimated by finding the number of
mg/kg2/3/day that corresponds to one unit of X and substituting this value into
the above relationship. Thus, for example, if X is in units of yg/m3 in the
air, we have for case 1, d = 0.29 x 7C-1/3 x 10~3 yg/kg2/3/day, and for case 2,
d = 1, when yg/m3 is the unit used to compute parameters in animal experiments.
5-100
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If exposures are given in terms of ppm in air, the following calculation
may be used:
1 ppm = 1.2 x molecular weight (gas) mg/m3.
molecular weight (air)
Note that an equivalent method of calculating unit risk would be to use
mg/kg/day for the animal exposures, and then to increase the jth polynomial
coefficient by an amount
(Wh/Wa)J/3 j = 1, 2, .... k
and to use mg/kg/day equivalents for the unit risk values. In the section
calculating the unit risk for animals, the final q* will always be the upper-
limit potency estimate for human risk based on animal data.
5.3.3.3.1.5 Interpretation of quantitative estimates. For several reasons, the
unit risk estimate based on animal bioassays is only an approximate indication
of the absolute risk in populations exposed to known carcinogen concentrations.
First, there are important species differences in uptake, metabolism, and organ
distribution of carcinogens, as well as species differences in target site
susceptibility, immunological responses, hormone function, dietary factors, and
disease. Second, the concept of equivalent doses for humans compared to animals
on a mg/surface area basis is virtually without experimental verification re-
garding carcinogenic response. Finally, human populations are variable with
respect to genetic constitution, diet, living environment, activity patterns,
and other cultural factors.
The unit risk estimate can give a rough indication of the relative potency
of a given agent as compared with other carcinogens. The comparative potency of
different agents is more reliable when the comparison is based on studies in
the same test species, strain, and sex, and by the same route of exposure,
preferably inhalation.
5-101
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The quantitative aspect of carcinogen risk assessment is included here
because it may be of use in the regulatory decision-making process, e.g.,
setting regulatory priorities, evaluating the adequacy of technology-based
controls, etc. However, with present technology, only imprecise estimations
are possible concerning cancer risks to humans at low levels of exposure. At
best, the linear extrapolation model used here provides a rough but plausible
estimate of the upper limit of risk, and while the true risk is probably not
much more than the estimated risk, it could be considerably lower. The risk
estimates presented in subsequent sections should not be regarded, therefore,
as accurate representations of the true cancer risks even when the exposures
are accurately defined. The estimates presented may, however, be factored into
regulatory decisions to the extent that the concept of upper risk limits is
found to be useful.
5.3.3.3.1.6 Alternative methodological approaches. Methods used by the CAG
for quantitative assessment are consistently conservative, i.e., they tend toward
high estimates of risk. The most important part of the methodology contributing
to this conservatism is the linear non-threshold extrapolation model. There
are a variety of other extrapolation models that could be used, all of which
would give lower risk estimates. These alternative models, the one-hit, Probit,
and Wei bull models, have not been used by the CAG in the following analysis, but
are included for comparison in the appendix. The CAG's position is that, given
the limited data available from these animal bioassays, especially at the
high-dose levels required for testing, almost nothing is known about the true
shape of the dose-response curve at low environmental levels and that the risk
estimates obtained by use of the linear non-threshold model represent plausible
upper limits only.
Extrapolation from animals to humans could also be done on the basis of
relative weight rather than on the basis of relative surface area. Although
the latter approach, used here, has more justification in terms of human pharma-
cological responses, it is not yet clear which of the two approaches is more
appropriate for the assessment of carcinogenicity. In the absence of informa-
tion on this point, it seems appropriate to use the more conservative basis for
extrapolation, or relative surface area.
5-102
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5.3.3.3.2 Unit risk potency estimates and relative potency. Neither of the
two available epidemiologic studies provided positive results from which to
derive a quantitative risk estimate for DCM exposure. With respect to animal
data, three chronic studies are relevant to this discussion: two inhalation
studies and one drinking water study. These are summarized in Table 5-30. In
the two inhalation studies (Dow Chemical Company 1980, 1982) only the salivary
gland regions in male Sprague-Dawley rats in the 1980 study showed statistically
significant increased cancers. These results are discussed in Section 5.3.3.1.1.2
and are presented in Tables 5-11 through 5-14. In the drinking water study (NCA
1982), the increase over untreated controls in combined neoplastic nodules and
hepatocellular carcinomas in female rats was the only statistically significant
finding (Table 5-24). However, when compared with historical controls, these
results lose their statistical significance.
5.3.3.3.2.1 Unit risk (yg/m3) for inhalation studies. The data used for esti-
mates of the unit risk for inhalation are presented in Table 5-31, which shows
the positive salivary gland region sarcomas for the inhalation bioassay.
Exposure was 6 hours/day, 5 days/week, for 2 years. Equivalent dosages were
determined for humans from the animal dosages utilizing the equivalent dosage
methodology presented previously. As described in the section on pulmonary
uptake and distribution, DCM is readily absorbed into the body following inhala-
tion and it equilibrates rapidly across the alveolar epithelium. Therefore,
the CAG considers it a virtually completely absorbed gas especially at low
doses and determines equivalent human exposure as explained under Case 1
of the inhalation section. As presented in Table 5-31, the nominal exposures
are nearly 15 times the human lifetime equivalent exposures. This difference,
24/6 x 7/5 = 5.6, is partly due to the use of a continuous equivalent dosage.
There is an additional factor of about 2.6, however, which is attributable
to the nature of the method used for determining human equivalent dosages
for inhalation studies. Put another way, if DCM had been determined to be a
partially soluble vapor, the unit risk slope would be less by a factor of about
2.6.
5-103
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TABLE 5-30. SELECTED DICHLOROMETHANE CHRONIC ANIMAL STUDIES
en
i
i—»
O
Author
Dow (1980)
Dow (1980)
Animal species
and strains
Sprague-Dawley rats
Syrian hamsters
Numbers
95 rats/sex/dose
107-109 hamsters
sex/dose
Routes
Inhalation
6/hours/day
5 days/week
for 2 years
Inhalation
6 hours/day
5 days/week
for 2 years
Doses
0, 500, 1500,
and 3500 ppm
0, 500, 1500,
and 3500 ppm
Resul ts
Salivary gland
sarcoma in males
"
Dow (1982)
NCA (1982)
Sprague-Dawley rats
90 rats/sex/
dose
Fischer 344 rats
85 rats/sex/
dose; 135 male
controls; 134
female controls
Inhalation
6 hours/day
5 days/week
for 20 (males)
or 24 (females)
months
Deionized
drinking water
for 24 months
0, 50, 200, and
500 ppm
0, 5, 50, 125,
250, 250
(recovery)
Borderline neo-
plastic nodule
response of the
liver in females
-------
TABLE 5-31. INCIDENCE ".ATES OF SALIVARY GLAND REGION SARCOMAS IN
MALE SPRAGUE-DAWLEY RATS IN THE
DOW CHEMICAL COMPANY (1980) DICHLOROMETHANE INHALATION STUDY
Continuous human equivalent
(animal nominal) exposures
ppm
and
ig/m3
Incidence rates
Number of rats with tumors/
total rats examined (%)
0
34
103
240
(0)
(500)
(1
(3
,500)
,500)
1
3
8
0 (0)
.2
.6
.4
x 105
x 105
x 105
(1
(5
(1
.8
.2
.2
x 106)
x 106)
x 107)
1/93
0/94
5/91
11/88
(1%)
(0%)
(5%)
(12%)
* 1 ppm x 1.2 x 103 yg/m3 x 84.9 = 3.5 x 103 yg/m3
"ZO"
An exposure of 500 ppm DCM in air expressed as yg/m3 is
500 ppm x 3.5 x 103 yg/m3 = 1.8 x 106 yg/m3
ppm
Since animal exposure was for 6 hours/day, 5 days/week, the animal continuous
lifetime exposure equivalent was
1.8 x 106 ug/m3 x 6 x 5 = 3.2 x 105 yg/m3
?4~ 7
The human equivalent dose for DCM is calculated first by determining the amount
actually breathed by the rats. As presented in the inhalation dose equivalence
section, 350 g rats breathe
I = 0.105 (0.350/0.113)2/3 = 0.223 m3 (air)/day.
Thus continuous animal dose was 3.2 x 105 yg/m3 x 0.223 m3/day = 7.15 x 104 yg/
day, or, for a 350 g rat, 7.15 x 104 yg/day/0.350 kg = 2.0 x 105 yg/kg/day.
The human equivalent dose is
2.0 x 105 yg/kg/day * (70/0.350)!/3 = 3.5 x io4 yg/kg/day
or
3.5 x 104 yg/kg/day x 70 kg = 1.2 x 10$ yg/m3.
20 m3
5-105
-------
When the above incidence data were fitted with the continuous human
equivalent exposures, the linearized multistage model yielded the following
value for the 95 percent upper limit of risk:
q* = 1.8 x ID'7 ( yg/m3)-!
5.3.3.3.2.2 Unit risk (mg/kg/day) and (yg/1) for oral studies. This unit risk
estimate should be used only under the assumption that DCM is a potential human
carcinogen. As discussed in the qualitative section, there is only limited
animal evidence and no human evidence to support that assumption.
Since publication of a final NTP report on gavage studies for rats and
mice on DCM has been cancelled, there is no suitable oral study from which to
estimate a unit risk.
5.3.3.3.2.3 Comparison of animal and human inhalation studies. The study of
Kodak employees, yielding negative cancer results, is compared with the positive
tumor results of the rat inhalation study (Dow, 1980). In the latter study, the
salivary gland region tumors in male Sprague-Dawley rats led to a 95 percent upper-
limit slope estimate of q* = 1.8 x 10"7 (yg/m3)"!. If this slope factor is applied
to the human inhalation study, in which time-weighted average exposure was esti-
mated as ranging from approximately 30 to 120 ppm, the expected impact of expo-
sure can be estimated. For this purpose, 1 yg/m3 of DCM is equivalent to 2.9 x
10~* ppm. Thus, the upper-limit slope in ppm is
q* = 1.8 x 10-7 (ug/n,3)-l x 3.45 x 103 yg/m3 = 6.2 x 10-4 ppm-l.
1 ppm
5-106
-------
Since this upper-limit slope is based on continuous lifetime equivalent exposure,
the exposure range of 30 to 120 ppm time-weighted average must be adjusted to
lifetime equivalence as follows:
30 ppm x 20 years x 240 days x 8 hrs = 1.88 ppm
Tff "353" "2T
and 7.52 ppm lifetime equivalence for the 120 ppm exposure group, assuming
20 years of exposure for the 252 long-term exposure workers. Based on this
upper-limit slope factor and the range of exposure, the group of 252 workers
could expect an additional lifetime risk of between
R = 6.2 x 10~4 ppm'1 x 1.88 ppm = 1.2 x 10~3 and 4.7 x 10~3.
For these 252 workers, this would translate to an upper limit of between
252 x 1.2 x 10-3 =0.3 and 1.2 excess lifetime cancer deaths. Based on the
total expected Kodak employee deaths of 65.9 (Table 5-28) for this 20 years
minimally exposed cohort, we would expect 26 percent of the cohort to die in
the 17-year follow-up period. Transposing this 26 percent to the expected
excess lifetime cancer deaths, an upper limit of between 0.1 and 0.3 excess
cancer deaths can be expected during the 17-year follow-up period. The power
to detect this increase from 17.77 to 18.1 cancer deaths is less than 10 percent.
If these excess cancer deaths were from cancers of one specific site, the power
would be greater, but not great enough to declare this a negative study. Even
for a rare cancer, such as a liver cancer, the expected number of cases would
be much less than 1. Since only deaths can be observed, the power to observe
one death from liver cancer in this cohort is quite small.
Based on the above analysis, the study of Kodak workers exposed to DCM,
showing no increase in cancer, cannot be judged as having negative results
because of its low power, which is related to low exposure from a weak animal
carcinogen.
5.3.3.3.2.4 Relative potency. One of uses of the unit risk concept is to com-
pare the relative potencies of carcinogens. To estimate relative potency on a
per mole basis, the unit risk slope factor is multiplied by the molecular weight,
5-107
-------
and the resulting number is expressed in terms of (mMol/kg/day)~!. This is
called the relative potency index.
Figure 5-1 is a histogram representing the frequency distribution of the
potency indices of 54 chemicals evaluated by the CAG as suspect carcinogens.
The actual data summarized by the histogram are presented in Table 5-32. Where
human data are available for a compound, they have been used to calculate the
index. Where no human data are available, animal oral studies and animal
inhalation studies have been used, in that order. Animal oral studies are
selected over animal inhalation studies because most of the chemicals have
been subjected to animal oral studies; this allows potency comparisons by route.
The potency index for DCM, based on salivary gland region tumors in male
Sprague-Dawley rats in the Dow inhalation study (1980), is 5.3 x 10~2 (mMol/kg/
day)-1. This is derived as follows: the slope estimate from the Dow study, 1.8 x
10"7 (ug/m3)-!, in converted units of 6.3 x 10~4 (mg/kg/day)"1, is multiplied by
the molecular weight of 84.9 to give a potency index of 5.3 x 10~2. Rounding
off of the nearest order of magnitude gives a value of 10~1, which is the scale
presented on the horizontal axis of Figure 5-1. The index of 5.3 x 10~2 is the
least potent of the 54 suspected carcinogens. Ranking of the relative potency
indices is subject to the uncertainty of comparing estimates of potency of
different chemicals based on different routes of exposure to different species
using studies of different quality. Furthermore, all the indices are based on
estimates of low-dose risk using linear extrapolation from the observational
range. Thus these indices are not valid for the comparison of potencies in the
experimental or observational range if linearity does not exist there. The
potency index for DCM, furthermore, is valid only under the assumption that
DCM is a potential human carcinogen. The evidence for that is limited.
5.3.3.3.2.5 Summary of quantitative estimation. No positive epidemiologic
studies exist from which to estimate a unit risk for exposure to DCM. Only one
animal data set has shown increased cancers from which a unit risk assessment
could be estimated. In the Dow Chemical Company (1980) rat inhalation study, there
were increased sarcomas in the salivary gland region of male rats; this yielded an
upper 95 percent limit of the potency estimate of q* = 1.8 x 10~7
5-108
-------
4th
quartile
-j
3rd
quartile
-h
4x10*2
2nd
quartile
+
2x10*3
1st
quartile
1x10*1
I
CN
'CO
I
I
1
I
-2
Jill
0246
Log of Potency Index
I
8
Figure 5-1. Histogram representing the frequency distribution of the
potency indices of 54 suspect carcinogens evaluated by the Carcinogen
Assessment Group.
5-109
-------
TABLE 5-32. RELATIVE CARCINOGENIC POTENCIES AMONG 54 CHEMICALS EVALUATED
BY THE CARCINOGEN ASSESSMENT GROUP AS SUSPECTED HUMAN CARCINOGENSl>2,3
Compounds
Acrylonitrile
Aflatoxin Bj
Aldrin
Ally! Chloride
Arsenic
B[a]P
Benzene
Benzidine
Beryllium
Cadmium
Carbon Tetrachloride
Chlordane
Chlorinated Ethanes
1,2-dichl oroethane
hexachloroethane
1 , 1 ,2 ,2-tetrachl oroethane
1 , 1 , 1- tri chl oroethane
1 , 1 ,2-tri chl oroethane
Chloroform
Chromium
DDT
Di chl orobenzi dine
1 , 1-di chl oroethyl ene
Dieldrin
Slope
(mg/kg/day)'1
0.24(W)
2924
11.4
1.19 x ID'2
15(H)
11.5
5.2 x 10-2(W)
234(W)
4.86
6.65(W)
1.30 x 10-1
1.61
6.9 x 10-2
1.42 x 10-2
0.20
1.6 x 10-3
5.73 x lO-2
7 x ID'2
41(W)
8.42
1.69
1.47 x 10-1(1)
30.4
Molecular
weight
53.1
312.3
369.4
76.5
149.8
252.3
78
184.2
9
112.4
153.8
409.8
98.9
236.7
167.9
133.4
133.4
119.4
104
354.5
253.1
97
380.9
Order of
magnitude
Potency (logio
index index)
1 x 10+1
9 x 10+5
4 x 10+3
9 x 10-1
2 x 10+3
3 x 10+3
4 x 10°
4 x 10+4
4 x 10+1
7 x 10+2
2 x 10+1
7 x 10+2
7 x 10°
3 x IQO
3 x 10+1
2 x 10-1
8 x 10°
8 x 10°
4 x 10+3
3 x 10+3
4 x 10+2
1 x 10+1
1 x 10+4
+1
+6
+4
0
+3
+3
+1
+5
+2
+3
+1
+3
+1
0
+1
-1
+1
+1
+4
+3
+3
+1
+4
5-110
-------
TABLE 5-32. (continued)
Compounds
Dinitrotoluene
Di phenyl hydrazine
Epichlorohydrin
Bis(2-chloroethyl )ether
Bis(chloromethyl ) ether
Ethyl ene Di bromide (EDB)
Ethyl ene Oxide
Formal dehyde
Heptachlor
Hexachlorobenzene
Hexachlorobutadiene
Hexachl orocycl ohexane
technical grade
alpha isomer
beta isomer
gamma isomer
Methyl ene Chloride
(dichloromethane)
Nickel
Nitrosamines
Dime thy! nitrosamine
Diethylnitrosamine
Di butyl ni trosami ne
N-ni trosopyrrol i di ne
N-ni troso-N-ethyl urea
N-ni troso-N-methyl urea
N-ni troso-di phenyl ami ne
Slope
(mg/kg/day)-l
0.31
0.77
9.9 x 10-3
1.14
9300(1)
8.51
0.63(1)
2.14 x 10-2(1)
3.37
1.67
7.75 x 10-2
4.75
11.12
1.84
1.33
6.3 x 10-4
1.15(W)
25.9{not by q*)
43.5(not by q*)
5.43
2.13
32.9
302.6
4.92 x ID'3
Molecular
weight
182
180
92.5
143
115
187.9
44.0
30
373.3
284.4
261
290.9
290.9
290.9
290.9
84.9
58.7
74.1
102.1
158.2
100.2
117.1
103.1
198
Order of
magnitude
Potency (logio
index index)
6 x 10+1
1 x 10+2
9 x 10-1
2 x 10+2
1 x 10+6
2 x 10+3
3 x 10+1
6 x 10-1
1 x 10+3
5 x 10+2
2 x 10+1
. o
1 x 10+3
3 x 10+3
i f\
5 x 10+2
4 x 10+2
5 x 10-2
7 x 10+1
2 x 10+3
4 x 10+3
9 x 10+2
, o
2 x 10+2
i O
4 x 10+3
i M
3 x 10+4
1 x 10°
+2
+2
0
+2
+6
+3
+1
0
+3
+3
+1
+3
+3
+3
+3
-1
+2
+3
+4
+3
+2
+4
+4
0
PCBs
4.34
324
1 x 10+3
5-111
-------
TABLE 5-32. (continued)
Slope Molecular Potency
Compounds (mg/kg/day)-1 weight index
Phenols
2,4,6-trichlorophenol 1.99 x 1Q-2 197.4
Tetrachlorodioxin 4.25 x 105 322
Tetrachloroethylene 6 x 10'2 165.8
Toxaphene 1.13 414
Trichloroethylene 1.26 x 10-2 131.4
Vinyl Chloride 1.75 x 10-2(I) 62.5
4 x 10°
1 x 10+8
1 x 10+1
5 x 10+2
2 x 10°
1 x 100
Order of
magnitude
Oog10
index)
+1
+8
+1
+3
0
0
Remarks:
1. Animal slopes are 95 percent upper-limit slopes
based on the 1
inearized
multi-stage model. They are calculated based on animal oral studies,
except for those indicated by I (animal inhalation), W (human occupational
exposure), and H (human drinking water exposure). Human slopes are point
estimates based on the linear non-threshold model.
2. The potency index is a rounded-off slope in (mMol/Kg/day)-1 and is cal-
culated by multiplying the slopes in (mg/kg/day)-1 by the molecular
weight of the compound.
3. Not all of the carcinogenic potencies presented in this table represent
the same degree of certainty. All are subject to change as new evidence
becomes available.
equivalent to q* = 6.3 x 10~4 (mg/kg/day)-1 by the oral route. However, no
positive data exist with which to provide a direct oral route estimate. In
total, there is only limited evidence that DCM is a potential human carcinogen.
The unit risk estimate is valid only if one accepts that limited evidence.
Further, if one chooses to express the potency of DCM relative to that of 54
chemicals evaluated as suspect carcinogens, DCM is the weakest, ranking last.
5-112
-------
5.3.3.4 Summary—Six chronic studies of DCM administered to animals have been
reported: four in rats, one in mice, and one in hamsters. The Dow Chemical
Company (1980) reported the results of chronic inhalation studies in rats and
hamsters. The rat study showed a small increase in the number of benign mammary
tumors compared to controls in female rats at all doses and in male rats at the
highest dose, as well as a statistically significant increased incidence of
ventral cervical sarcomas, probably of salivary gland origin, in male rats. The
response pattern of the salivary gland tumors is unusual, consisting of sarcomas
only and appearing in males but not in females. In hamsters, there was an
increased incidence of lymphosarcoma in females only, which was not statistically
significant after correction for survival. The second inhalation study in rats
by the Dow Chemical Company (1982) reported that there were no compound-related
increased incidences of any tumor type, but that the highest dose was far below
that of the previous Dow inhalation study in rats. The National Coffee Associa-
tion (NCA) drinking water study in Fischer 344 rats reported that the incidence
of neoplastic nodules and/or hepatocellular carcinomas in female rats was
increased significantly with respect to matched controls, but their incidence
was within the range of historical control values at that laboratory. The NTP
(1982) draft gavage study on rats and mice will not be published as a final
report due to data discrepancies. Selected information from the gavage studies
may be incorporated into the future NTP inhalation bioassay, pending the results
of the in-depth audit.
There are some other inadequate animal studies in the literature. One
study (Theiss et al., 1977) reported a marginally positive pulmonary adenoma
response in strain A mice injected intraperitoneally with DCM. 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 other carcinogenic!ty studies of DCM in animals are currently in
progress: an NTP 2-year bioassay by inhalation in Fischer 344/N rats and
B6C3F1 mice, and an NCA 2-year drinking water study in B6C3F1 mice.
Positive results in a rat embryo cell transformation study were reported by
Price et al. (1978). The significance of their findings with regard to carcino-
genicity is not well understood at the present time.
5-113
-------
The epidemiologic data consists of two studies: Friedlander et al. (1978),
updated by Hearne and Fried!ander (1981); and Ott et al. (1983a, b, c, d, e).
Although neither study showed excessive risk, both showed sufficient deficiencies
to prevent them from being judged negative studies. The Friedlander et al. study
(1978) lacked great enough exposure (based on animal cancer potency estimates)
to provide sufficient statistical power to detect a potential carcinogenic
effect. The Ott et al. study (1983a, b, c, d, e), among other deficiencies,
lacked a sufficient latency period for site-specific cancer.
Only one animal data set has shown increased cancers from which a unit risk
assessment could be estimated. In the Dow Chemical Company (1980) rat inhala-
tion study, there were increased sarcomas in the salivary gland region of male
rats; this yielded an upper 95 percent limit of the potency estimate of q* = 1.8 x
10~7 (ug/m3)-1, equivalent to q* = 6.3 x 10~4 (mg/kg/day)'1 by the oral route.
This unit risk estimate should be used only under the assumption that DCM is a
potential human carcinogen. As discussed previously, there is only limited
animal evidence and no human evidence to support that assumption.
5.3.3.5 Conclusions. Animal studies show a statistically positive salivary gland
sarcoma response in male rats (Dow, 1980) and a borderline hepatocellular neo-
plastic nodule response in the rat (NCA, 1982). There is also evidence that DCM
is weakly mutagenic. According to the criteria of the International Agency
for Research on Cancer (IARC), the weight of evidence for carcinogem'city in
animals is limited.
There was an absence of epidemiologic evidence for the carcinogem'city of
DCM in a well-conducted epidemiologic study having long-term exposure. However,
on the basis of animal data, the level of exposure to the individuals in the
study was too low to produce an observable increase in cancer. The overall
evaluation of DCM, based on IARC criteria, is group 3, meaning that the chemical
cannot be classified as to its carcinogem'city for humans.
The unit risk for DCM, based on a rat inhalation study, is estimated to be
1.8 x 10~7 for a lifetime exposure to 1 yg/m3 in air, but the above is true only
under the assumption that DCM is a potential human carcinogen. Even under that
assumption, the potency of DCM is the lowest of the 54 chemicals which the CAG
has evaluated as suspect carcinogens.
5-114
-------
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APPENDIX
COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS
The estimates of unit risk based on animal studies presented in the body
of this document are all calculated by the use of the linearized multistage
model. The reasons for its use have been detailed herein. Essentially, this
model is part of a methodology that estimates a conservative linear slope at
low extrapolation doses and is consistent with the data at all dose levels of
the experiment. It is a nonthreshold model which holds that the upper-limit of
risk predicted by a linear extrapolation to low levels of the dose-response
relationship is the most plausible upper limit for the risk.
Other models have also been used for extrapolation, and include the
three nonthreshold models presented here: the one-hit, the log-Probit, and
the Weibull. The one-hit model is characterized by a continuous downward
curvature, but is linear at low doses. It can be considered the linear form or
first stage of the multistage model because of its functional form. Because of
this and its downward curvature, the one-hit model will always yield estimates
of low-level risk that are at least as large as those of the multistage model.
Further, whenever the data can be fitted adequately by means of the one-hit
model, estimates from the two procedures will be comparable.
The other two models, the log-Probit and the Weibull, are often used to
fit toxicological data in the observable range, because of their general "S"
curvature. The low-dose upward curvatures of these two models usually yield
lower low-dose risk estimates than those of the one-hit or multistage model.
The log-Probit model was originally proposed for use in problems of
biological assay, such as the assessment of potency of toxicants and drugs,
and has usually been used to estimate such values as percentile lethal dose
or percentile effective dose. Its development was strictly empirical, i.e.,
it was observed that several log dose-response relationships followed the
cumulative normal probability distribution function. In fitting the cancer
bioassay data, assuming an independent background, this becomes:
P(D;a,b,c) = c + (1-c) * (a+blogioD) a,b > o £ c < 1
A-l
-------
where P is the proportion responding at dose D, c is an estimate of the
background rate, a is an estimate of the standarized mean of individual
tolerances, and b is an estimate of the log dose-Probit response slope.
The one-hit model arises from the theory that a single molecule of a
carcinogen has a probability of transforming a single noncarcinogenic cell
into a carcinogenic one. It has the probability distribution function:
P(D;a,b) = l-exp-(a+bd) a,b > 0
where a and b are the parameter estimates. The estimate a represents the
background or zero dose rate, and the parameter estimated by b represents
the linear component or slope of the dose-response model. In discussing the
added risk over background, incorporation of Abbott's correction leads to
P(D;b) = l-exp-(bd) b > 0
Finally, a model from the theory of carcinogenesis arises from the multihit
model applied to multiple target cells. This model has been termed here the
Weibull model. It is of the form
P(D;b,k) = l-exp-(bdk) b,k > 0
For the power of dose only, the restriction k > 0 has been placed on this model.
When k > 1, this model yields low-dose estimates of risks usually significantly
lower than either the multistage or one-hit models, which are linear at low
doses. All three of these models usually project risk estimates significantly
higher at the low exposure levels than those from the log-Probit.
The estimates of added risk for low doses for the above models are given
in Table A-l for the DCM inhalation study. Both maximum likelihood estimates
and 95% upper confidence limits are presented. All estimates incorporate
Abbott's correction for independent background rate.
A-2
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TABLE A-l. ESTIMATES OF LOW-DOSE RISK TO HUMANS BASED ON SALIVARY GLAND REGION SARCOMAS IN MALE RATS
IN THE DOW CHEMICAL COMPANY (1980) INHALATION STUDY DERIVED FROM FOUR DIFFERENT MODELS
(All estimates Incorporate Abbott's correction for Independent background rate.)*
Dose
ug/m3
Males
1 ug/m3
10 ug/m3
100 ug/m3
1000 ug/m3
10,000 ug/m3
* Response fit
Maximum likelihood estimates of
additional risks
Multistage One-hit Wei bull Log-Prob1t Multistage
model model model model model
2.2 x 10-8 1.3 x 10-7
2.2 x 10-7 1.3 x 10-6
2.2 x 10-6 1.3 x 10-5
2.2 x 10-5 1.3 x 10-*
2.2 x 10-* 1.3 x 10-3
against human equivalent dosages
Human equivalent dose ug/m3
0
1.2 x 105
3.6 x 105
8.4 x 105
2.6 x 10-11 o 1.8 x 10-7
1.1 x 10-9 o 1.8 x 10-6
5.0 x 10-8 1.1 x 10-16 1.8 x 10'5
2.2 x 10-6 5.0 x 10-H 1.8 x 10'*
9.5 x 10-5 1.7 x 10-6 1.3 x 10'3
as presented In Table 5-31.
Tumors/Total
1/93
0/94
5/91
11/88
95% upper confidence limit of
additional risks
One-hit Welbull Log-Prob1t
model model model
2.0 x 10-7 4.8 x 10-1° 3.5 x 10-31
2.0 x 10-6 1.7 x 10-8 1.6 x 10-22
2.0 x 10-5 6.1 x 10-6 2.5 x 10-15
2.0 x 10-* 2.0 x 10-5 1.3 x 10'9
2.0 x 10-3 6.1 x 10-* 2.4 x 10-1
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