vvEPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-600/8-82-006B
December 1983
External Review Draft
Research and Development
Health Assessment
Document for
Trichloroethylene
Review
Draft
(Do Not
Cite or Quote)
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.
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EPA-600/8-82-006B
Jan. 1984
External Review Draft
Health Assessment Document
for
Trichloroethylene
NOTICE
This document is a preliminary draft. It has not been formally
released by the U.S. Environmental Protection Agency 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.
,,.;on Agency
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.
Environ:;
ii
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PREFACE
The Office of Health and Environmental Assessment, in consultation with
an Agency work group, has prepared this health assessment to serve as a "source
document" for EPA use. Originally the health assessment was developed for use
by the Office of Air Quality Planning and Standards, however, at the request
of the Agency Work Group on Solvents, the Assessment scope was expanded to
address multimedia aspects.
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.
m
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TABLE OF CONTENTS
Page
LIST OF TABLES vii
LIST OF FIGURES x
1. SUMMARY AND CONCLUSIONS 1-1
2. INTRODUCTION 2-1
3. BACKGROUND INFORMATION 3-1
3.1 PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.2 ENVIRONMENTAL FATE AND TRANSPORT 3-3
3.2.1 Production 3-3
3.2.2 Use 3-4
3.2.3 Emissions 3-5
3.2.4 Persistence 3-6
3.2.5 Degradation 3-7
3.3 LEVELS OF EXPOSURE 3-8
3.3.1 Analytical Methodology 3-8
3.3.2 Calibration 3-12
3.3.3 Sampling and Sources of Error 3-12
3.4 ECOLOGICAL EFFECTS 3-23
3.5 CRITERIA, STANDARDS, AND REGULATIONS 3-25
3.6 REFERENCES 3-26
4. PHARMACOKINETICS AND METABOLISM 4-1
4.1 ABSORPTION AND DISTRIBUTION 4-1
4.1.1 Dermal Absorption 4-1
4.1.2 Oral Absorption 4-2
4.1. 3 Pulmonary Absorption 4-3
4.1.4 Tissue Distributions and Concentrations 4-9
4.2 EXCRETION 4-11
4.2.1 Pulmonary Elimination in Man 4-11
4.2.2 Urinary Metabolite Excretion in Man 4-14
4.2.3 Excretion Kinetics in the Rodent 4-16
4.3 MEASURES OF EXPOSURE AND BODY BURDEN 4-18
4.4 METABOLISM 4-19
4.4.1 Known Metabolites 4-19
4.4.2 Magnitude of TCI Metabolism: Evidence of Dose-
Dependent Metabol i sm 4-20
4.4.3 Enzyme Pathways of Biotransformation 4-24
4.4.4 Metabolism and Covalent Binding 4-30
4.4.5 TCI Metabolism: Drug and Other Interactions 4-37
4.4.6 Metabolism and Cellular Toxicity 4-40
4.5 REFERENCES ' 4-43
IV
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TABLE OF CONTENTS (continued)
5. TOXICOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS 5-1
5.1 INTRODUCTION 5-1
5.2- EFFECTS ON THE CENTRAL NERVOUS SYSTEM 5-1
5.2.1 Human Studies: Acute Inhalation Exposure 5-1
5.2.2 Human Studies: Chronic Exposure 5-3
5.2.3 Experimental Human Studies 5-3
5.2.4 Animal Studies 5-6
5.3 EFFECTS ON THE CARDIOVASCULAR AND RESPIRATORY SYSTEMS ... 5-9
5.3.1 Cardiovascular Effects 5-9
5.3.2 Respiratory Effects 5-11
5.4 HEPATIC AND RENAL TOXICITY 5-12
5.4.1 Human Studies 5-12
5.4.2 Animal Studies 5-14
5.5 IMMUNOLOGY 5-17
5.6 DERMAL EFFECTS 5-17
5.7 MODIFICATION OF TOXICITY: SYNERGISM AND ANTAGONISM 5-18
5.8 SUMMARY OF TOXICOLOGICAL EFFECTS 5-21
5.9 REFERENCES 5-22
6. TERATOGENICITY, EMBRYOTOXICITY, AND REPRODUCTIVE EFFECTS 6-1
6.1 ANIMAL STUDIES 6-2
6.1.1 Mouse 6-2
6.1.2 Rat 6-4
6.1.3 Rabbit 6-8
6.2 FETOTOXICITY IN HUMANS 6-10
6.3 SUMMARY 6-10
6.4 REFERENCES 6-11
7. MUTAGENICITY 7-1
7.1 GENE MUTATION STUDIES 7-1
7.1.1 Prokaryotic Test Systems (Bacteria) 7-1
7.1.2 Eukaryotic Test Systems 7-11
7.2 CHROMOSOME ABERRATION STUDIES 7-26
7.2.1 Experimental Animals 7-26
7.2.2 Humans 7-30
7.3 OTHER STUDIES INDICATIVE OF MUTAGENIC ACTIVITY 7-33
7.3.1 Gene Conversion in Yeast 7-33
7.3.2 Sister Chromatid Exchange (SCE) Formation 7-34
7.3.3 Unscheduled DNA Synthesis (UDS) 7-36
7.4 EVIDENCE THAT TCI REACHES THE GONADS 7-41
7.5 MUTAGENICITY OF METABOLITES 7-43
7.6 BINDING TO DNA 7-44
7.7 SUMMARY AND CONCLUSIONS 7-45
7.8 REFERENCES 7-53
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TABLE OF CONTENTS (continued)
Page
8. CARCINOGENICITY 8-1
8.1 ANIMAL STUDIES AND CELL TRANSFORMATION STUDIES 8-1
8.1.1 Oral Administration (Gavage): Rat 8-1
8.1.2 Oral Administration (Gavage): Mouse 8-20
8.1.3 Inhalation Exposure: Rats 8-41
8.1.4 Inhalation Exposure: Hamsters 8-47
8.1.5 Inhalation Exposure: Mice 8-47
8.1.6 Other Carcinogenicity Evaluations of
Trichloroethylene 8-54
8.1.7 Trichloroethylene Oxide 8-56
8.1.8 Cell Transformation Studies 8-60
8.1.9 Summary of Animal and Cell Transformation Studies. 8-68
8.2 EPIDEMIOLOGIC STUDIES 8-71
8.2.1 Axel son et al. (1978) 8-72
8.2.2 Tola et al. (1980) 8-74
8.2.3 Malek et al. (1979) 8-76
8.2.4 Paddle (1983) 8-77
8.2.5 Blair et al. (1979) 8-78
8.2.6 Katz and Jowett (1981) 8-79
8.2.7 Lin and Kessler (1981) 8-80
8.2.8 Asal (personal communication 1983) 8-81
8.3 QUANTITATIVE ESTIMATION 8-81
8.3.1 Procedures for Determination of Unit Risk 8-82
8.3.2 Unit Risk From Animal Studies 8-94
8.3.3 Relative Potency 8-99
8.4 SUMMARY AND CONCLUSIONS 8-105
8.4.1 Qualitative Assessment 8-105
8.4.2 Quantitative Assessment 8-113
8.5 REFERENCES 8-115
VI
004TC5/A 11/29/83
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LIST OF TABLES
Table Page
3-1 Physical properties of trichloroethylene 3-1
3-2 Stabilizers in trichloroethylene formulations 3-2
3-3 Partition coefficients of trichloroethylene and other
chlorinated hydrocarbons 3-3
3-4 U.S. production capacity for trichloroethylene 3-4
3-5 Miscellaneous uses of trichloroethylene 3-5
3-6 Estimated half-life of TCI in surface waters 3-7
3-7 Ambient air mixing ratios of trichloroethylene 3-15
3-8 Mean levels of TCI in surface waters 3-21
4-1 Partition coefficients of TCI for various body tissues and
components of rat and man 4-3
4-2 Pulmonary uptake of TCI for 25 volunteers exposed to TCI for
four consecutive 30-minute periods at rest and during
exerci se 4-6
4-3 Pulmonary uptake of TCI for 4 subjects at rest exposed to
70 and 140 ppm TCI for 4-hour periods and 3-hour periods ... 4-6
4-4 Recovery of radioactivity for 50-hour postinhalation
exposure of male B6C3F1 mice to 10 or 600 ppm 14C-TCI for
6 hours 4-9
4-5 Recovery of radioactivity for 50-hour postinhalation
exposure of male Osborne-Mendel rats to 10 or 600 ppm
14C-TCI for 6 hours 4-9
4-6 Organ contents of TCI after daily inhalation exposure of
200 ppm for 6 hours per day 4-12
4-7 Demonstrated metabolites of TCI 4-21
4-8 J_n vitro covalent binding of TCI 4-32
4-9 Microsomal bioactivation and covalent binding of aliphatic
halides to calf thymus ONA 4-33
4-10 Hepatic and renal macromolecular binding of 1,1,2 (UL-14C)
metabolite in male B6C3F1 mice and Osborne-Mendel rats
exposed to 10 or 600 ppm for 6 hours 4-36
4-11 ]_n vivo alkylation of hepatic DMA by 1,1,2 (UL-14C) TCI in
male B6C3F1 mice dosed with 1200 mg/kg (14C) TCI by gavage . 4-36
6-1 Summary of animal studies of fetotoxic and teratogenic
potential of TCI 6-3
6-2 Experimental design of Beliles et al. study (1980) on
Sprague-Dawley rats 6-5
6-3 Experimental design of Dorfmueller et al. study (1979) on
Long-Evans rats 6-7
6-4 Experimental design of Beliles et al. study (1980) on
New Zealand white rabbits 6-8
vn
004TC5/A 11/29/83
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LIST OF TABLES (continued)
Table Page
7-1 Mutagenicity of technical-grade TCI in Salmonella/mammalian
mi crosome assay 7-4
7-2 Mutagenicity of trichloroethylene and epichlorohydrin in
5>. pombe by means of the host-mediated assay in B6C3F1 mice. 7-12
7-3 Mutagenicity of technical-grade TCI at the hi s 1-7 locus of
Saccharomyces cerevisiae 7-16
7-4 Mutagenicity of reagent-grade TCI at the ilv locus in
Saccharomyces cerevisiae 7-19
7-5 Mutagenicity of TCI at the ilv locus in Saccharomyces
cerevisiae D7 7-21
7-6 Mutagenicity of TCI in the dominant lethal assay using NMRI
mice 7-29
7-7 Numerical chromosome aberrations in TCI workers 7-32
7-8 Unscheduled DNA synthesis in WI-38 cells 7-37
7-9 Summary of tests for mutagenicity of TCI 7-46
8-1 TCI carcinogenic!ty bioassays in animals 8-2
8-2 Analysis of primary tumors in male rats 8-8
8-3 Dosage and observation schedule-TCI chronic study on
Osborne-Mendel rats 8-13
8-4 Mean body weights, food consumption, and survival-TCI
chronic study-male rats 8-14
8-5 Mean body weights, food consumption, and survival-TCI
chronic study-female rats 8-15
8-6 Analysis of primary tumors in male mice 8-24
8-7 Analysis of primary tumors in female mice 8-25
8-8 Dosage and observation schedule: TCI chronic study on B6C3F1
mice 8-27
8-9 Mean body weights, food consumption, and survival: TCI
chronic study, male mice 8-29
8-10 Mean body weights, food consumption and survival: TCI
chronic study, female mice 8-30
8-11 Incidence of hepatocellular carcinomas in B6C3F1 8-34
8-12 Mouse skin bioassays of TCI and TCI oxide 8-38
8-13 Subcutaneous injection of TCI 8-39
8-14 Carcinogenicity of TCI by intragastric feeding in male
and female mice 8-40
8-15 Estimation of probability of survival of rats 8-42
8-16 Estimation of probability of survival of hamsters 8-48
8-17 Estimation of probability of survival of mice 8-49
8-18 Statistical analysis of the incidence of primary hepato-
cellular neoplasms in control and TCI-treated B6C3F1 mice .. 8-53
8-19 Summary of negative carcinogenicity data for TCI 8-55
8-20 Chronic skin application in mice 8-58
8-21 Chronic s.c. injection in mice 8-59
8-22 Jjn vitro transformation of Fischer rat embryo cell cultures
by TCI 8-62
vm
004TC5/A 11/29/83
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LIST OF TABLES (continued)
Table Page
8-23 Transformation and survival of Syrian hamster embryo cells
treated with diverse chloroalkene oxides 8-69
8-24 A cohort study of mortality among men exposed to TCI in the
1950's and 1960ls and followed through December 1975 8-73
8-25 Comparison of workers exposed to TCI with the total Finnish
population for mortality from all causes and for cancer
mortal ity 8-76
8-26 Incidence rates of hepatocellular carcinomas in male and
female mice in the NTP (1982) and NCI (1976) gavage
studies. Continuous human equivalent dosages and estimates
of the 95% upper-limit slope from the linearized multistage
model 8-95
8-27 Number of workers with cancer death 8-98
8-28 Relative carcinogenic potencies among 54 chemicals evaluated
by The Carcinogen Assessment Group as suspect human car-
cinogens 8-102
IX
004TC5/A 11/29/83
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LIST OF FIGURES
FIGURE PAGE
4-1 Predicted partial pressure of TCI in alveolar air and
tissue groups during and after an 8-h exposure of
100 ppm 4-4
4-2 The relationship between the concentration of TCI in
arterial blood and alveolar air at the end of inhalation
exposures 4-7
4-3 Predicted partial pressure of TCI in fatty tissue for a
repeated exposure to 100 ppm, 5 days, 6 h per day 4-10
4-4 Elimination curves of TCI and kinetic model for transfer
of TCI in the body 4-13
4-5 Relationship between environmental TCI concentration and
urinary excretion of 1CI metabolites in human urine 4-22
4-6 Postulated scheme for the metabolism of TCI to chloral 4-25
4-7 Metabolism of chloral hydrate 4-26
8-1 Growth curves for rats administered TCI in corn oil by
gavage 8-6
8-2 Survival curves for rats administered TCI in corn oil by
gavage 8-7
8-3 Product-limit estimates of probability of survival: TCI-
treated male rats 8-16
8-4 Product-limit estimates of probability of survival: TCI-
treated female rats 8-17
8-5 Growth curves for mice administered TCI in corn oil by
gavage 8-21
8-6 Survival curves for mice administered TCI in corn oil by
gavage 8-22
8-7 Product-limited estimates of probability of survival: TCI-
treated male mice 8-31
8-8 Product-limited estimates of probability of survival: TCI-
treated female mice 8-32
8-9 TCI histogram of chamber concentration measurements (100 ppm) 8-44
8-10 TCI histogram of chamber concentration measurements (300 ppm) 8-45
8-11 TCI histogram of chamber concentration measurements (600 ppm) 8-46
8-12 Incidence of malignant lymphomas in female mice 8-50
8-13 Cell transformation assays on vinyl chloride and TCI in
BHK-21/C1 13 in vitro 8-66
8-14 Structures and stability of chloroalkene oxides 8-70
8-15 Histogram representing the frequency distribution of the
potency indices of 54 suspect carcinogens evaluated by The
Carcinogen Assessment Group 8-101
004TC5/A 11/29/83
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The authors of this document are:
Larry D. Anderson, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
Steven Bayard, Carcinogen Assessment Group, U.S. Environmental Protection Agency,
Washington, D.C.
I. W. F. Davidson, Professor, Department of Physiology/Pharmacology, The
Bowman Gray School of Medicine, Wake Forest University, Winston-Sal em,
North Carolina.
John R. Fowle, III, Reproduction Effects Assessment Group, U.S. Environmental
Protection Agency, Washington, D.C.
Herman J. Gibb, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
Mark M. Greenberg, Health Scientist, Environmental Criteria and Assessment
Office, U.S. Environmental Protection Agency, Research Triangle Park,
North Carolina.
Jean C. Parker, Health Scientist, Office of Solid Waste, U.S. Environmental
Protection Agency, Washington, D.C.
xi
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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.
^ AeJLL°A4£LLvJL JL^ fJJtL5 _Asse_s_sment Group
John R. Fowle III, Ph.D.
Ernest R. Jackson, M.S.
K.S. Lavappa, Ph.D.
Sheila L. Rosenthal, Ph.D.
Carol N. Sakai, Ph.D.
Daniel S. Straus, Ph.D., Consultant
Lawrence R. Valcovic, Ph.D., Consultant
Vicki Vaughan-Dellarco, Ph.D.
Peter E. Voytek, Ph.D.
The Environmental Mutagen Information Center (EMIC) in Oak Ridge, Tennessee,
kindly identified literature bearing on the mutagenicity of TCI. Their initial
report and subsequent updates were used to obtain papers from which the
mutagenicity assessment was written.
A9^J-X^ArJLA^JiP_^_A°Jj(^JlLs
Elizabeth L. Anderson
Charles H. Ris
Jean C. Parker
Mark M. Greenberg
Cynthia Sonich
Steve Lutkenhoff
James A. Stewart
Paul Price
Wi 1 1 iam 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
xi i
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The following persons attended a review workshop to discuss EPA draft
documents on organics, which included an early draft of this document.
Dr. Mildred Christian
Argus Laboratories, Inc.
Perkasie, Pennsylvania 18944
Dr. Herbert Cornish
Dept. of Environmental and Industrial Health
University of Michigan
Ypsilanti, Michigan 48197
Dr. I. W. F. Davidson
Dept. of Physiology/Pharmacology
The Bowman Gray School of Medicine
300 S. Hawthorne Road
Winston-Sal em, North Carolina 27103
Dr. John Egle
Dept.. of Pharmacology
Virginia Comonwealth University
Richmond, Virginia 23298
Dr. Thomas Haley
National Center for Toxicological Research
Jefferson, Arkansas 72079
Dr. Rudolph J. Jaeger
Institute of Environmental Medicine
New York University Medical Center
New York, New York 10016
Dr. John G. Keller
P.O. Box 12763
Research Triangle Park, North Carolina 27709
Or. Norman Trieff
Dept. of Preventive Medicine
University of Texas Medical Branch
Galveston, Texas 77550
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center-
New York, New York 10016
Dr.. James Withey
Food Directorate
Bureau of Food Chem.
Tunney's Pasture
Ottawa, Canada
xiii
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1. SUMMARY AND CONCLUSIONS
Trichloroethylene (1,1,2-trichloroethylene) (TCI) is a solvent widely
used in the industrial degreasing of metals. It has no known natural sources.
Current U.S. production is estimated at about 130,000 metric tons per year.
Of the trichloroethylene used in the United States, 80 to 95 percent evaporates
to the atmosphere. In addition to the workplace, TCI is found in a variety of
urban and nonurban areas of the United States and other regions of the world.
It has been measured in ambient air and water. An average ambient air concen-
tration of about 1 part per billion (ppb) would be expected for some large
urban centers. Concentrations as high as 32 ppb have been measured in urban
centers in the United States, and 47 ppb has been measured in urban Tokyo.
Ambient air concentrations of TCI are greatly influenced by the rate and
geographic distribution of emissions and the rate of decomposition and trans-
position in the atmosphere. The average mixing ratio in the troposphere of
the northern hemisphere is 11 to 17 parts per trillion (ppt). Reaction with
hydroxyl radicals is the principal mechanism by which TCI is scavenged from
the atmosphere.
Trichloroethylene has been detected in both natural and municipal waters
in the United States. Up to 403 ppb has been measured in some surface and
subsurface waters, and, in finished drinking water, concentrations have ranged
from 1 to as much as 32 ppb. There is no direct evidence of bioaccumulation
of TCI in the food chain. Few studies have been made of the ecological conse-
quences of TCI in the environment.
The pharmacokinetics and metabolism of TCI have been studied in man as
well as in animals. As with other related halogenated aliphatic solvents, the
principal route of exposure to TCI is by inhalation of vapor in air followed
by lung absorption. Ingestion of drinking water contaminated with TCI is a
secondary source of exposure. Both exposure routes are important because TCI
contaminates air, water and food. Ingested TCI enters the portal system
first, perfusing the liver: thus, at low concentrations only a small propor-
tion of TCI would enter the general circulation, because uptake and metabolism
by the liver are expected to be nearly complete. In contrast, inhaled TCI
directly enters the systemic circulation and is widely distributed in the
body.
Because of its lipophilic nature, TCI readily crosses lipid-containing
cell membranes and portions selectively in adipose tissue upon chronic or
004TC1/C 1-1 11/10/83
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long-term exposures to even low ambient air concentrations, until steady-state
is reached. Physical activity increases uptake.
The amount of TCI absorbed after oral ingestion is virtually complete;
with air exposure, the amount of TCI absorbed increases in proportion to its
concentration in inspired air, the respiratory rate, and duration of exposure.
TCI is eliminated by two major processes, pulmonary excretion of unchanged TCI
and liver metabolism to urinary metabolites. At air concentrations of 500 ppm
or less, humans are estimated to metabolize between 60 to 90 percent of absorbed
TCI. There is no evidence that TCI metabolism in man is saturation-dependent,
at least up to about 300 ppm. Studies have not been made of metabolism after
oral exposure in man; however, an even higher proportion of an absorbed amount
of TCI is expected to be metabolized following exposure by this route. The
principal identified urinary metabolites of TCI are trichloroethanol (TCE) and
its glucuronide, and trichloroacetic acid (TCA). The evidence is strong that,
in the liver, TCI is first metabolized to a reactive epoxide (trichloroethylene
oxide) by a microsomal P4so system. This reactive epoxide covalently binds
extensively to cellular macromolecules. The epoxide is further converted to
chloral hydrate and thence to TCE and TCA. Metabolism of TCI is enhanced by
drugs and xenobiotics such as barbiturates, oral antidiabetic agents, and
PCBs, which induce the hepatic microsomal P450 metabolizing system. Heightened
toxicity can result from clinical drug interactions known to occur with ethanol,
barbiturates, disulfiram and Warfarin.
Toxicity testing in experimental animals, coupled with limited human data
derived principally from overexposure situations, suggests that long-term
exposure of humans to environmental (ambient) levels of TCI is not likely to
represent a health concern. Limited controlled human exposures indicate that
impairment of the performance of certain mental and physical tasks may occur
during acute exposure to levels in the range of 100 to 200 ppm. There is no
information that such impairment, representative of dysfunction of the central
nervous system (CNS), would or would not occur during chronic, low-level
exposures. At levels found or expected in the ambient environment, such an
effect would be unlikely. Similarly, dysfunction of the liver or kidneys
would not be likely during environmental exposures. Damage to renal tubules
and signs of liver dysfunction have been observed only in experimental animals
during exposures to excessively high levels (>1000 ppm).
Teratogenicity is another health endpoint for which available data from
experimental animals suggest that the conceptus is not uniquely susceptible to
004TC1/C 1-2 11/10/83
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TCI. Exposure of rats, mice, and rabbits, during gestation, to levels (300
ppm) greatly in excess of those generally found in the environment has not
been observed to result in any teratogenic effects. It should be noted,
however, that such results cannot be easily extrapolated to humans. The
teratogenic potential of TCI for humans is still unknown and represents a
basis for continued awareness and investigation.
With respect to the mutagenic potential of TCI, available data provide
suggestive evidence that commercial-grade TCI is a weakly active indirect
mutagen, causing effects in a number of different test systems representing a
wide evolutionary range of organisms. Based on this, commercial TCI may have
the potential to cause weak or borderline increases above the spontaneous level
of mutagenic effects in exposed human tissue. The observations that commercial
TCI causes adverse effects in the testes of mice suggest TCI may cause adverse
testicular effects in man too, provided that the pharmacokinetics of TCI in
humans also result in its distribution to the gonads. The data on pure TCI do
not allow a conclusion to be drawn about its mutagenic potential. However,
mutagenic potential cannot be ruled out. If it is mutagenic, the available
data suggest that TCI would be a very weak, indirect mutagen.
Trichloroethylene, both in epoxide-stabilized form and in epoxide-free
form, has been shown to be positive for the induction of malignant tumors of
the liver in both male and female B6C3F1 mice during lifetime bioassay under
the auspices of the National Toxicology Program. This constitutes a signal
that TCI might be a carcinogen for humans. It should be recognized, however,
that there is a substantial body of opinion in the scientific community to the
effect that the mouse liver overreacts to chlorinated organic compounds, in
contrast to the rat, and that the induction of liver cancer in the mouse
represents only a promoting action for spontaneous liver tumors, which normally
occur with substantial incidence. Furthermore, this promoting action might be
related to liver damage associated only with high-level exposures to chlorinated
agents such as TCI. However, the evidence is inconclusive either for this re-
strictive position on the mouse liver carcinogenic response to chlorinated
organics, or for the position that the mouse liver is as good as any other
mammalian indicator of carcinogenicity for these compounds.
Based on available animal cancer bioassay data, the classification of the
TCI results under the criteria of the International Agency for Research on
Cancer (IARC) could be either "sufficient or "limited," depending on the
004TC1/C 1-3 11/10/83
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differing current scientific views about the induction of liver tumors in mice
by chlorinated organic compounds. Because there are no adequate epidemiological
data in humans, the overall ranking of TCI under the criteria of IARC could be
either Group 2B or Group 3, i.e., the more conservative scientific sentiment
would regard TCI as a probable human carcinogen, but there is considerable
scientific sentiment for regarding TCI as an agent that cannot be classified
as to its carcinogenicity for humans.
RECOMMENDATIONS FOR FURTHER STUDIES
Areas in which incomplete information is available, and that should be
considered in formulating research needs, are presented below. The needs listed,
however, are not necessarily in the order of relative priority.
1. Teratogenicity and Reproductive Effects. There are few animal
studies via inhalation that adequately assess the teratogenic and reproductive
effects potential of TCI. Uncertainty about these biological endpoints in
humans should serve as a stimulus for future research.
2. Neurobehavioral Toxicity. These have been few animal studies of the
effects of TCI on the nervous system and behavior. Most endpoints studied
have been relative insensitive. Further studies are warranted on more sensitive
endpoints.
004TC1/C 1-4 11/10/83
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2. INTRODUCTION
Trichloroethylene (TCI) is a member of a family of unsaturated chlorinated
aliphatic compounds. Trichloroethylene, though a water and solid waste con-
taminant, is primarily of interest in situations involving ambient air exposure.
It is released into ambient air as a result of evaporative losses during pro-
duction, storage, and/or use. It has no known natural sources. It is photo-
chemical ly reactive in the troposphere and is removed by scavenging mechanisms,
principally via hydroxyl radicals.
The scientific data base concerning the effects of TCI on humans is limited.
Effects on humans have generally been ascertained from studies involving indivi-
duals occupationally or accidentally exposed. During such exposures, the concen-
trations associated with adverse effects on human health were either unknown or
far in excess of concentrations found or expected in ambient air. Controlled
TCI exposure studies have been directed toward elucidating the effects on the
CNS, effects on clinical chemistries, and pharmacokinetic parameters of TCI
exposure.
Since epidemiologic studies have not been able to assess adequately the
overall impact of TCI on human health, it has been necessary to rely greatly
on animal studies to derive indications of potential harmful effects. Although
animal data cannot always be extrapolated to humans, indications of probable
or likely effects in animal species increase confidence that similar effects
may occur in humans.
This document is intended to provide an evaluation of the scientific data
base concerning TCI. The literature has been comprehensively reviewed through
September 1983. The publications cited in this document represent a majority
of the known scientific references to TCI. Reports which had little or no
bearing on the issues discussed were not cited. Some topic areas, such as
effects on terrestrial ecosystems, are only briefly discussed. Such topics
reflect the nature of the scientific data base.
004TC2/C 2-1 11/5/83
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3. BACKGROUND INFORMATION
3.1 PHYSICAL AND CHEMICAL PROPERTIES
Trichloroethylene (TCI) is a colorless, highly volatile liquid that is
miscible with a variety of solvents. Some of its important physical properties
are shown in Table 3-1.
TABLE 3-1. PHYSICAL PROPERTIES OF TRICHLOROETHYLENE
Molecular weight 131.39
Boi1 ing point 87 C
Vapor pressure 94 mm Hg @ 30°C
Vapor specific gravity 4.55 at boiling point
(air = 1)
Solubility @ 20°C 0.107 gm percent
At standard temperature and pressure (25°C, 1 atmosphere), one part-per-
million (ppm) of the vapor is equivalent to 5.38 mg/m3.
Trichloroethylene is photochemically reactive and autooxidizes upon
catalysis by free radicals (McNeil!, 1979). Autooxidation is greatly acceler-
ated by high temperatures and exposure to ultraviolet radiation (McNeil!, 1979).
Some of its degradation products, e.g., hydrochloric acid (HC1), are corrosive
to metals. These decomposition products include dichloroacetyl chloride, phos-
gene, carbon monoxide, hexachlorobutene, and HC1 (McNeil 1, 1979.)
Decomposition is catalyzed when TCI comes in contact with aluminum metal.
The HC1 produced reacts with aluminum to yield aluminum chloride (A1C13) which
catalyzes formation of hexachlorobutene (McNeill, 1979). Sufficient quantities
of aluminum have been reported to cause violent decomposition of TCI (Metz and
Roedy, 1949). While TCI is nonflammable under ordinary conditions, mixtures of
TCI and oxygen will ignite at temperatures above 25.5 C when the TCI concentra-
tion is between 10.3 and 64.5 percent (Jones and Scott, 1942).
To inhibit its decomposition, stabilizers are added to commercial formula-
tions of TCI (McNeill, 1979). Commonly used stabilizers in vapor degreasing
grades of TCI are neutral inhibitors and free radical scavengers (McNeil!,
1979). Epoxides (including epichlorohydrin, a known carcinogen) are added to
004TC2/D 3-1 11/10/83
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scavenge free HC1 or A1C13. Antioxidants are usually added in quantities less
than 1 percent by weight (Hardie, 1964). Fuller (1976) reported that inhibited
grades of TCI are stable up to 130 C in the presence of air, moisture, light,
and common construction metals. At higher temperatures, inhibitors are less
effective and corrosion of metals can result. Stabilizers that have been used
in TCI formulations are shown in Table 3-2.
TABLE 3-2. STABILIZERS IN TRICHLOROETHYLENE FORMULATIONS (Beckers, 1972; U.S.
EPA, 1975; Hardie, cited in: Kirk Othmer, 1975; Schlossburg, 1980)
Amy! alcohol
Propanol
Diethyl amine
Triethyl amine
Dipropylamine
Di i sopropylami ne
Diethanolamine
Morpholine
N-Methylmorpholi ne
Ani1ine
Acetone
Ethyl acetate
Borate esters
Ethylene oxide
1,2 Propylene oxide
1,2 Epoxy butene
Cyclohexene oxide
Propylene oxide
Butadiene dioxide
Styrene oxide
Pentene oxide
2,3 Epoxy 1-propenol
3, Methoxy-1,2 epoxy propane
3, Ethoxy-1,2 epoxy
Stearates
2-Methyl-l,2 epoxy propanol
Epoxy cyclopentanol
Epichlorohydrin
Tetrahydrofuran
Tetrahydropyran
1,4-Dioxane
Dioxalane
Trioxane
Alkoxyaldehyde hydrazones
Methylethyl ketone
Nitromethanes
Nitropropanes
Phenol
o-Cresol
Thymol
p-tert-Butylphenol
p-tert-Amylphenol
Isoeuganol
Pyrrole
n-Methyl pyrrole
n-Ethyl pyrrole
(2-pyrryl) Trimethylsilane
Glycidyl acetate
Isocyanates
Thiazoles
Trichloroethylene decomposes at temperatures up to 700 C. Substances
found in the decomposition mixture include dichloroethylene, tetrachloroethylene,
carbon tetrachloride, chloroform, and methyl chloride (Hardie, 1964). Formation
of phosgene, a highly toxic gas, was observed when TCI came in contact with
iron, copper, zinc, or aluminum over the temperature range 250 C to 600 C
(Sjoberg, 1952). When TCI came in contact with zinc at 450°C, 69 mg phosgene
per gram of TCI was produced.
Trichloroethylene is not readily hydrolyzed by water (McNeil 1, 1979). In
the presence of strong alkali (e.g., sodium hydroxide) TCI can decompose to
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dichloroacetylene, an explosive, flammable, and highly toxic compound (Humphrey
and McClelland, 1944). Noweir et al. (1973) recommended that TCI be stored in
cans or dark glass bottles to minimize decomposition.
Because of its lipophilic properties, TCI can be expected to partition
selectively in the body's adipose tissue. The relationships between such parti-
tioning and manifestation of its toxic properties is discussed in Chapter 4.
The unsymmetrical nature of the molecule and the electron-withdrawing effect of
the chlorine substituents (leading to destabilization of the charge at the double
bond) have been proposed as factors responsible for the supposed mutagenicity of
TCI (Bonse and Henschler, 1976).
Measurements of the lipophilic potential of TCI have been made by a number
of investigators (Waters et al. , 1977; Mapleson, 1963; Powell, 1947; Sato and
Nakajima, 1979). Mapleson (1963) reported an oil/water partition coefficient of
960:1 at 37 C. Waters et al. (1977) reported the oil/water coefficient as 900:1.
Recently Sato and Nakajima (1979) reported an olive oil/water partition coeffi-
cient of 552:1 at 37 C and an olive oil/blood partition coefficient of 76:1.
The relationship of TCI to other compounds with respect to partition coefficients
is shown in Table 3-3 (Sato and Nakajima, 1979).
3.2 ENVIRONMENTAL FATE AND TRANSPORT
3.2.1 Production
Trichloroethylene has been declining in production in recent years owing to
restrictions on emissions (Federal Register, 1977), substitution by other solvents,
and other factors. According to statistics published by the U.S. International
Trade Commission (1980), 133,000 metric tons of TCI were produced in 1980.
TABLE 3-3. PARTITION COEFFICIENTS OF TRICHLOROETHYLENE AND OTHER
CHLORINATED HYDROCARBONS (Sato and Nakajima, 1979)
Solvent
Oil/water Oil/blood
Log P
(partition
coefficient)
Dichloromethane
Chloroform
Carbon tetrachloride
1,2-Dichloroethane (EDC)
Methyl chloroform
Tetrachloroethylene
Trichloroethylene
21
115
1444
40
383
4458
552
16
39
150
23
108
146
76
1.32
2.06
3.16
1.60
2.58
3.65
2.74
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Recent estimates of world production of TCI are not available. Such figures
for 1973 indicated 700,000 metric tons were produced (McConnell et al., 1975).
About two-thirds was produced outside the United States.
In 1977, TCI was produced by five American manufacturers (SRI, 1978). The
producers and production capacities are shown in Table 3-4.
TABLE 3-4. U.S. PRODUCTION CAPACITY FOR TRICHLOROETHYLENE (SRI, 1978)
Producer
PPG Industries
Dow Chemical
Diamond Shamrock
Occidental Petroleum
Ethyl Corporation
Site
Lake Charles, LA
Freeport, TX
Deer Park, TX
Taft, LA
Baton Rouge, LA
Annual capacity
(metric tons)
127,000
68,040
24,948
22,680
18,144
Most of the TCI produced commercially is derived from ethylene or
dichloroethane (EDC) (McNeil!, 1979). Dichloroethane, produced by
chlorination of ethylene, can be further chlorinated to TCI and
tetrachloroethylene at 280° to 450°C. Catalysts include potassium chloride,
aluminum chloride, fuller's earth, graphite, activated carbon, and activated
charcoal (McNeill, 1979). Maximum conversion to TCI (75% of EDC feed) is
achieved at a chlorine-to-EDC ratio of 1.7:1 (McNeill, 1979).
Oxychlorination of ethylene or EDC to TCI is achieved at 425°C and at 20
to 30 psi (McNeil!, 1979). Common catalysts are mixtures of potassium and
cupric chlorides. Conversion efficiency is between 85 and 90 percent.
3.2.2 Use
It has been estimated that from 80 to 95 percent of the TCI produced in
the United States is used in the degreasing of fabricated metal parts (SRI,
1978; McNeill, 1979; Chemical and Engineering News, 1975). The remaining 5 to
20 percent is divided equally between exports and miscellaneous applications.
Use in vapor degreasing is estimated to account for 87 percent of production,
according to industry trade statistics (Chemical Marketing Reporter, 1978).
In addition to its use as a vapor degreasing agent, TCI has a wide
variety of other uses (Table 3-5). About 4,500 to 6,800 metric tons are
consumed annually in the production of polyvinyl chloride, in which TCI is used
as a chain-transfer agent (McNeil!, 1979).
004TC2/D 3-4 11/10/83
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TABLE 3-5. MISCELLANEOUS USES OF TRICHLOROETHYLENE
(McNeil!, 1979; Waters et al. , 1977)
Adhesive formulations
Paint-stripping formulations
Low-temperature heat-transfer medium
Carrier solvent in industrial paint systems
Solvent in textile dyeing and finishing
Uses of TCI that have been discontinued in the United States because of
environmental and health restrictions include use as an inhalation anesthetic
(Page and Arthur, 1978; Waters et al., 1977), use in fumigant mixtures (Farm
Chemicals Handbook, 1976), and use as an extractant in the decaffeination of
coffee. Use in anesthetic procedures appears to be continuing to some extent
in other countries (Cundy, 1976).
Declining consumption of TCI from the early 1970's has been offset by the
increased use of substitute solvent, e.g., methyl chloroform and methylene
chloride (U.S. EPA, 1979).
3.2.3 Emissions
Because of its dispersive use pattern in degreasing operations, most of
the TCI used is expected to be emitted to the atmosphere. There are no known
natural sources of TCI. Graedel and Allara (1976) concluded that atmospheric
chemical production of TCI was negligible. Recent emissions estimates (U.S. EPA,
1979) for 1976, pertaining to vapor degreasing, cold cleaning, and miscellaneous
use categories suggest that 130,000 metric tons were discharged to the atmosphere.
Solvent emissions from vapor degreasing occur primarily during unloading
and loading of the degreaser (Bucon et al., 1978). Daily emissions of a
single spray degreasing booth may vary from a few pounds to 1,300 pounds (Bucon
et al. , 1978). In uncontrolled degreasing operations, as much as 2.8 kg/h
can be emitted, based on 200,000 pounds of metal cleaned per day (Bucon et al.,
1978).
McConnell et al. (1975) estimated that, for 1973, global emissions of TCI
amounted to about 1 million metric tons.
Singh et al. (1979) estimated global emissions of TCI for 1977 at 435,000
metric tons ± 50 percent. Estimates were derived from production figures and
usage patterns.
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3.2.4 Persistence
Many processes occur in the troposphere which can alter the atmospheric
levels of TCI. Once emitted into the troposphere, vertical and horizontal
mixing occurs. Transport is highly dependent upon the length of time TCI remains
in the troposphere, which is determined largely by the extent to which TCI
reacts with hydroxy free radicals (-OH), the principal scavenging mechanism for
TCI and many other halogenated compounds in the atmosphere.
Edney et al. (1983), on the basis of the observed rate of reaction of TCI
with hydroxy radicals in a reaction cell, calculated an atmospheric lifetime for
TCI of about 54 h. A hydroxy concentration in the troposphere of 106 molecules/
cm3 was assumed.
The average northern hemisphere background mixing ratio is between 11 and
17 ppt (Singh et al. , 1979), which suggests that TCI is short-lived in the
troposphere. Singh et al. (1979) estimated a residence time of about 2 weeks,
an estimate based on a seasonally-averaged OH concentration of 4 x 105
molecules cm 3 and a rate constant (National Bureau of Standards, 1978), at
265 K, of 2.30 x 10 12. Singh and coworkers (1979) estimated that, given an
•OH concentration of 1 x 106 molecules cm 3, 20 percent of the TCI in ambient
air can be destroyed each day. Crutzen et al. (1978) estimated a residence time
of 11 days for TCI. Derwent and Eggleton (1978) estimated the lifetime at 0.04
years (~ 15 days). The percentage of the ground level injection of TCI that was
estimated to survive free radical attack was 0.4 percent.
Seasonal variations in -OH concentrations, important for longer-lived
species, are not expected to play a significant role in the tropospheric
survival of TCI. Altshuller (1980) calculated that in January (when -OH levels
and solar flux are low) 0.6 days would be required for the photochemical decom-
position of 1 percent of the ambient TCI. In July (when -OH levels and solar
flux are high), only 0.07 days is required.
Trichloroethylene was observed to have a slow decomposition rate in dilute
aqueous solution (Dilling et al. , 1975). Its half-life (in the dark) was 10.7
months at ambient temperatures. When the solution was exposed to sunlight, 75
percent of the TCI decomposed in 12 months, compared with 54 percent for the
reaction in the dark. A correction for the amount of TCI that volatilized into
the air space above the solution was not employed. On the other hand, Pearson
and McConnell (1975), in their determinations of the hydrolytic decomposition,
extrapolated to zero volatilization. Their estimate was a half-life of 30
months.
004TC5/A 3-6 11/5/83
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The major route of removal of TCI from water is volatilization. Volatil-
ization of TCI from water has been investigated by several authors. Dilling et
al. (1975) have shown that the loss of TCI from an agitated dilute aqueous
solution occurs exponentially with an evaporative half-life of 21 ± 3 min.
Scherb (1978) measured the volatilization of TCI from an aeration channel in a
wastewater treatment plant and found the half-life to be 3 h. The rate of
volatilization of TCI from surface waters in the environment has been reported
by Zoeteman et al. (1980). TCI was found to have a half-life of 1 to 4 days in the
Rhine River and 30 to 40 days in a tidal estuary. Smith et al. (1980) have shown
that the rate of volatilization of TCI and other low-molecular-weight compounds
from various bodies of waters is a function of reaeration rates. From the work
of Smith et al. (1980), it is estimated that the half-life for TCI in surface
waters ranges from 3 h, for rapidly moving shallow streams, to 10 days or longer
for ponds and lakes (Table 3-&).
TABLE 3-6. ESTIMATED HALF-LIFE OF TCI IN SURFACE WATERS
Water Type TCI half-life (days)
Pond 11
Lake 4 to 12
River 1 to 12
Source: Calculated from information in Smith et al. (1980).
3.2.5 Degradation
The transformation products of the decay of TCI have been investigated in
chamber studies simulating atmospheric conditions. Two principal transformation
products have been observed, phosgene and dichloroacetyl chloride. Dahlberg
(1969) observed that both were produced when TCI was photooxidized in air. Gay
et al. (1976) also found formyl chloride in addition to phosgene and dichloro-
acetyl chloride, when TCI was photooxidized in the presence of nitrogen dioxide
(N02). A maximum observed consumption rate for TCI of 70 percent per hour was .
reported. In this system, dichloroacetyl chloride, identified by long-path in-
frared spectroscopy, was the principal product. In a smog chamber system
containing 2.5 ppm TCI and 0.5 ppm N02, TCI disappeared at the rate of about
25 percent per hour (Dimitriades et al., 1983). Ozone built up to 0.5 ppm. The
presence of equal amounts of tetrachloroethylene and TCI in a chamber containing
N02 did not alter the consumption rate of TCI above TCI controls.
004TC5/A 3-7 11/5/83
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Edney et al. (1983) observed a number of degradation products when hydroxy
radicals reacted with TCI in a 700 liter cell coupled to an FTIR spectrometer
and scanning interferometer. Reaction products observed were phosgene, dichloro-
acetyl chloride, formyl chloride, carbon monoxide, and nitric acid. The rate
constant of reaction was 3.6 x 10 12 cnrVsec.
Lillian et al. (1976), finding phosgene formation from the photooxidation
of the TCI congener, tetrachloroethylene, considered that phosgene formation also
was likely from other chloroethylenes. Singh (1978) reported that -OH-catalyzed
photodecomposition of TCI, as occurs in the troposphere, can result in phosgene
as the principal transformation product.
Under simulated atmospheric conditions in the presence of nitric oxide (NO)
and nitrogen dioxide (NO-), TCI (10 ppm) was very short-lived (less than 3.5 h)
(Dilling et al. , 1976). A consumption rate up to 30 percent per hour was
reported. The high reactivity of TCI and its catalytic role in the formation of
photochemical smog have been recently discussed (U.S. EPA, 1978a; National Acade-
my of Sciences, 1977). Free radicals (e.g., -R02), are formed when TCI is photo-
decomposed, and these in turn react with NO by oxidizing it to N02- According
to the following reaction sequence, N02 results in the formation of ozone (03):
(1) N02 —— » NO + 0
(2) 0 + 02 + M (third body) » 03 + M
Because chamber studies simulate some atmospheric reactions believed to
occur in the environment, further studies of greater complexity would be needed
to determine the extent and pathways by which TCI is photodecomposed. Phosgene
and dichloroacetyl chloride, though likely to be formed in the troposphere, also
are subject to transformation and scavenging processes.
3.3 LEVELS OF EXPOSURE
3.3.1 Analytical Methodology
Of the analytical methods and procedures available for detecting and quanti-
tating halogenated hydrocarbons present in ambient air, water, soil, foods, etc.,
two systems are commonly used: gas chromatography/mass spectrometry (GC-MS) and
gas chromatography with electron capture detection (GC-EC). Each system has the
capability of detecting TCI concentrations as low as a few parts-per-tri11 ion by
volume. The usefulness of GC-EC is that the apparatus can be used in the field
004TC2/D 3-8 11/10/83
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to provide quasi-continuous measurements by intermittent sampling (every 15 to
20 minutes). Such use can circumvent decomposition that results when samples of
TCI are stored for long periods before assay (Pellizzari, 1974; Lovelock, 1974b).
3.3.1.1 Gas Chromatography with Electron Capture Detection—The GOEC methodology
has been reviewed by Pellizzari (1974) and by Lovelock (1974b). The EC detector
is specific in that halogenated compounds are quantified while non-halogenated
hydrocarbons are not detected. Thus, high background levels of non-halogenated
hydrocarbons in ambient air or water samples do not interfere with measurements
of halogenated compounds. 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.
In the method developed by Rasmussen et al. (1977), the detection limit for
TCI was 0.3 ppt. With four samples of ambient air, the percent standard devia-
tion was 23.0 by electronic area integration (+ 5 percent by peak height measure-
ment). The system, which employs a freezeout concentration loop, has the capabil-
ity of measuring various halocarbons in 500-ml aliquots of air. Total analysis
time is 40 min. The GC column consisted of a 6-mm x 3-m stainless steel column
packed with 10 percent SF-96 on 100/120 meshrChromosorb W.
A similar analytical procedure was employed by Harsch and Cronn (1978) to
measure levels of halocarbons in 30-ml samples of low-pressure stratospheric air.
For TCI, the detection limit was 1 ppt and the precision (percent standard devia-
tion) for 10 samples with a mixing ratio of 22 ppt was 10 percent.
Gas Chromatography with electron capture detection also has been the method
of choice by Singh and coworkers for measuring the ambient air levels of a wide
variety of halocarbons. Singh et al. (1979) maintained the EC detector at 325 C.
The GC column used was 6-ft x 1/8-in. stainless steel, containing 20 percent
SP2100, 0.1 percent Carbowax 1500 on 100/120 mesh Supelcoport. Singh et al.
(1979b) recommended two columns for separation of TCI: (a) silicone oil 10 per-
cent DC 200 on 30/60 mesh Chromosorb W in a 6-ft x 1/4-in. stainless steel column
or (b) 20 percent SP-2100 on 80/100 mesh Supelcoport in a 15-foot x 1/8-inch
column.
The applicability of GC-EC to analysis of TCI and other halohydrocarbons
purged from water samples has been discussed by Bellar et al. (1979). Precon-
centration on a Tenax and silica gel trap was recommended.
Singh et al. (1979a) avoided the use of Tenax traps for preconcentrating
organics in air samples because of artifacts. They thought that oxygen or
004TC2/D 3-9 11/10/83
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ozone was oxidizing the Tenax monomer and producing electron-absorbing oxygenated
species, thus interfering with EC detection of the organics of interest.
Makide et al. (1980) used GC, coupled with constant current ECD, to measure
TCI in ambient air at sites in Japan. The reported detention limit was < 0.2
ppt (< 1.36 x 10 6 mg/m3). Sampling canisters were made of stainless steel and
were polished electrochemically. Preconcentration was carried out on a silicone
OV-101 column at -40°C. Column temperature was raised at a rate of 5°C per minute
up to 70°C for separation of components.
A direct headspace technique for use with EC detection has been reported
by Piet and coworkers (1978). This technique avoids the need for preconcentration
traps when water samples are purged. An overall detection limit below 1 ug/1
was reported. With isothermal operation of the GC, a detection limit for TCI of
0.05 (jg/1 was reported. An OV225 capillary column was used.
Dietz and Singley (1979) recently described a headspace method applicable
to EC detection of volatile organics from water samples. A standard deviation
of 5 percent was reported for routine analyses of drinking, natural, and
industrial waters. Concentrations ranging froip 0.1 ppb to several ppm of TCI
were detected. In this method, the water sample is sealed in a vial until
organics equilibrate between the water and vial headspace. A major advantage is
that only relatively volatile, water-insoluble compounds tend to partition in
the headspace. In a comparison of this method with the purge and trap technique,
Dietz and Singley (1979) obtained comparable results. However, the chromatograms
from the purge-trap method were of poorer quality. The headspace method was
recommended for routine sample analyses of volatile halohydrocarbons.
3.3.1.2 Gas Chromatography-Mass Spectrometry--While GC-EC relies on retention
time and ionization efficiency for compound identification, the equally sensi-
tive GC-MS system identifies compounds by their characteristic mass spectra.
However, the expense, bulk, and demands on the operator preclude routine use
of GC-MS in field measurements. Often, GC-MS is used to verify results obtained
by GC-EC.
Comparisons between the precision afforded by GC-MS versus GC-EC have been
made by Cronn and co-workers (1976). In these comparisons, the detection limit
was defined based on a peak height three times the noise level with a 75-ml sample
loop. With TCI at an average mixing ratio of 128 ppt, the detection limit (GC-MS)
was 23 ppt (with 3 doped samples of zero air). The percent standard deviation
was 10. More recent data from Cronn and Harsch (1979) show a slightly lower detection
limit (16 ppt) for a 100-ml freezeout sample. The sensitivity of the GC-MS
004TC5/A 3-10 11/5/83
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technique was achieved using direct coupling of the gas chromatograph and mass
spectrometer and selective ion monitoring (at mass 95 for TCI).
Pellizzari et al. (1976) used Tenax GC to absorb TCI and other halogenated
hydrocarbons from ambient air. Recovery was made by thermal desorption and
helium purging into a freezeout trap. The estimated detection limit when high
resolution GC-MS is used is 1.92 ppt (0.01 x 103 mg/m3). Accuracy was reported
at ±30 percent. Included among the inherent analytical errors were (1) the abil-
ity to accurately determine the breakthrough volume, (2) the percent recovery
from the sampling cartridge after a period of storage, and (3) the reproducibi1-
ity of thermal desorption from the cartridge and its introduction into the analy-
tical system. To minimize loss of sample, cartridge samplers should be enclosed
in cartridge holders and placed in a second container that can be sealed, protec-
ted from light, and stored at 0°C. The advantages reported for Tenax include
(1) low water retention, (2) high thermal stability, and (3) low background levels
(Pellizzari, 1974; Pellizzari, 1975; Pellizzari, 1977; Pellizzari et al., 1976).
In an analytical system using Tenax GC as the absorbent, Krost et al. (1982)
reported an estimated detection limit for TCI of 2 ppt. Analysis of the break-
through volumes at various temperatures suggests that Tenax GC is not a particu-
larly effective absorbent for TCI.
Resolution of TCI from other hydrocarbons was reported to be effective when
Carbopak C-HT, a graphitized thermal carbon black treated with hydrogen at 1,000
°C was used as the adsorbent in a GC column (Knoll et al., 1979). Flame ioniza-
tion was used in detection. Interference between TCI and ethylene dichloride
was reported with Porapak T.
Gas chromatography-mass spectrometry has been more widely used in measure-
ments of volatile organic compounds in aqueous samples. In this regard, gas
purging followed by trapping on an absorbent has been extensively used in com-
bination with GC-MS detection. A recent review of this system, as applied to
TCI and other volatile organics, has been made by Bellar et al. (1979). A
combination of Tenax GC (60/80 mesh) and silica gel was recommended. Purging was
accomplished at a water temperature of about 22 C. In general, the method is
applicable to compounds that have a low solubility in water and a vapor pressure
greater than water at ambient temperatures. For well-defined samples when the
probability of unexpected compounds is low, the EC detector could be used. The
detection limit of this system was reported to be in the 0.1 to 1 ug/1 range.
Desorption onto the head of the column can be accomplished in one step by heating.
004TC5/A 3-11 11/5/83
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3.3.2 Calibration
Both static and dynamic procedures have been used to calibrate the instru-
mentation used for measurement of TCI. Because of the very low levels of TCI
found or expected in ambient air, the generation of known low-level concentra-
tions of TCI is critical in instrument calibration.
Singh and coworkers (1979, 1977) employed permeation tubes to calibrate
their GC-EC instrumentation. A tube constructed of FEP Teflon (wall thickness
of 0.03 in.) was found to be satisfactory for TCI. Errors in the permeation
rate were less than 5 percent (Singh et a!., 1979). It was considered the best,
most accurate means of generating primary standards. Since primary standards
could not be used in field operations, secondary standards were generated and
compared to them. The permeation rate at the 95-percent confidence level was
246.0 + 20.1 mg/min (Singh et al., 1977).
Primary and secondary standards prepared by multiple dilutions were used by
Cronn and coworkers (1976, 1977a,b).
3.3.3 Sampling and Sources of Error
Common approaches used to sample ambient air for trace gas analysis are pre-
sented below (National Academy of Sciences, 1978).
(1) Pump-pressure samples: A mechanical pump is used Lo fill a stainless
steel or glass container to a positive pressure relative to the surrounding atmos-
phere.
(2) Ambient pressure samples: An evacuated chamber is opened and allowed
to fill until it has reached ambient pressure at the sampling location. If fill-
ing is conducted at high altitude, the container may easily become contaminated
when returned to ground level.
(3) Adsorption on molecular sieves, activated charcoal., or other adsorbents.
(4) Cryogenic samples: Air is pumped into a container and liquefied, and
a partial vacuum is created which allows more air to enter. This method allows
for the collection of several thousand liters of air.
Singh and coworkers (1979) employed electropoIished stainless steel vessels
in their sampling of tropgspheric air. Vessels were checked for background
contamination until it was reduced to less than 2 to 3 percent of the expected
background concentration of a given trace substance. Typically, vessels that
maintained positive pressure were not affected by room contamination.
Sampling and storage of ambient air collected at stratospheric altitudes
present more of a problem. Because of the low pressures in the sampling vessels,
004TC2/D 3-12 11/10/83
-------
contamination may easily result when vessels are returned to ground level.
Cronn and Robinson (1978) and Harsch and Cronn (1978) reported that contamina-
tion of low-pressure stainless steel vessels precluded use of data for TCI.
Whole-air samples were collected at stratospheric altitudes. Contamination
occurred during the sample-transfer procedure because of high mixing ratios of
halocarbons in laboratory air.
To overcome contamination of low-pressure samples, Harsch and Cronn (1978)
developed a low-pressure sample-transfer technique. A vacuum transfer procedure
employing a freezeout loop (Rasmussen et al., 1977) was found to yield results
with sufficient sensitivity and with minimal contamination problems. A leak-
tight system was considered essential. System components were silver-soldered.
Sample collection and handling and preservation of water samples have been
discussed by Kopfler et al. (1976) and by Bellar et al. (1974). Narrow-mouth
vials with a total volume in excess of 50 ml were considered acceptable (Bellar
et al., 1974). It was recommended that the vials be filled to overflowing and
each covered with a Teflon-faced silicone rubber septum. Because of the presence
of chlorine in some drinking water supplies and the chlorination of organic
materials, an increase in the concentration of some halohydrocarbons may occur
also during storage (Kopfler et al., 1976).
Bellar et al. (1974) reported that free chlorine can result in the forma-
tion of TCI and other halohydrocarbons. In measurements of TCI in water from a
sewage-treatment plant, they found an increase of 1.2 pg TCI/1 in the water after
chlorination. Dietz and Singley (1979) collected water samples in 125-ml serum
vials that had been cleaned by detergent wash, distilled water rinse, dichromic
acid wash, distilled water rinse, acetone rinse, hexane rinse, acetone rinse,
distilled water rinse, and oven-drying at 150°C. Vials were completely filled
and then sealed with Teflon-faced septa and aluminum crimp seals. Samples were
stored at 4 C. Minimum loss occurred during storage at 4°C up to 28 days.
The stability of stored samples of TCI present in ambient air or breath
samples from exposed individuals has been investigated by Pasquini (1978). The
mean percent loss of TCI in alveolar breath samples collected in glass tubes at
room temperature was 59.3 + 15.3 percent over 169 h. The mean decay rate of
TCI in dilution air samples was 4.7 + 3.2 percent over the same time interval.
Moisture, temperature, and tube surface and condition can greatly alter vapor
retention. For TCI, tubes stored at 37°C had high vapor retention rates. When
breath was exhaled into the tubes at 37 C, water vapor was not condensed from
004TC5/A 3-13 11/5/83
-------
the sample. Additional experiments with TCI indicated that si 1 iconized tubes
showed a lower decay rate than non-si 1 iconized tubes.
Renberg (1978) judged XAD-4 the most suitable of several possible adsorbents
for volatile halogenated hydrocarbons in water, including TCI. This method results
in an extract concentrated enough for both chemical determination and small-scale
biological tests.
Kummert et al. (1978) have suggested a direct aqueous injection high-pressure
liquid chromatography technique for analysis of TCI in natural waters. The tech-
nique has been successfully applied to an actual water pollution case for tetra-
chloroethylene. The detection limit for TCI was reported as 1 umol 1 1 for a
10-ml sample.
3.3.3.1 Air-Mixing Ratios—Measurements of ambient air mixing ratios of TCI have
been made at a variety of sites in the United States and abroad. As shown in
Table 3-7, urban areas have mixing ratios in the low ppb range.
Singh et al. (1979) reported an average mixing ratio in the northern hemi-
sphere of 11 to 17 ppt, with the average in the southern hemisphere of less than
3 ppt. Over the Pacific Northwest, an average tropospheric mixing ratio of 20 +
3.9 ppt was found (Cronn et al., 1977a). Harsch (1977) reported levels between
22 ppb and 8.7 ppb in more than 20 different indoor environments. Brodzinsky
and Singh (1982), in their review of monitoring data, reported that background
concentrations average about 30 ppt. A maximum level of 87 ppb was reported for
Newark, New Jersey.
3.3.3.2 Water--Trich1oroethy1ene has been detected in a number of raw water
sources, finished drinking waters, and subsurface waters in the United States.
The occurrence of TCI in waters of the United States has been recently reviewed
by the U.S. Environmental Protection Agency (1979).
(1) Drinking water
Dowty et al. (1975) detected TCI in finished drinking water in the New
Orleans area. It was reported to pass through the water treatment plant without
alteration. It was also reported to be incompletely removed after passage of
water through a charcoal filtering unit. Detection was made by GC-MS.
The most comprehensive surface water monitoring effort for TCI to date
(Ewing et al., 1977) has shown TCI to be a common low-level (ppb) contaminant of
surface waters in the vicinity of urban/industrial areas. TCI was reported at
concentrations of 1 ug/1 (ppb) or greater in 72 of 179 surface water samples from
U.S. urban/industrial areas from 23 states (Table 3-8). Ewing also reported samples
of drinking water which were not included in this total.
004TC5/A 3-14 11/5/83
-------
TABLE 3-7. AMBIENT AIR-MIXING RATIOS OF TRICHLOROETHYLENE
CO
I
Location
ARIZONA
Phoenix
Grand Canyon*
ARKANSAS
Helena
El Dorado*
CALIFORNIA
Stanford Hills
Los Angeles
Los Angeles
Point Reyes
Palm Springs
Yosemite
Mill Valley
Riverside
Menlo Park
Upland
Badger Pass
Date of
measurements
04/23-05/06 1979
11/28-12/05 1977
11/30 1976
06/13-07/08 1978
11/23-11/30 1975
04/28-05/04 1976
04/09-04/21 1979
12/01-12/12 1975
05/05-05/11 1976
05/12-05/18
01/11-01-27 1977
04/25-05/04 1977
08/13-09/23 1977
05/05-05/13 1977
Mixing ratio (ppb)
Maximum Minimum Average
3.0696
0
< 0.03
13
3.09
1.77
1.702
0.064
0.446
0.022
0.090
0.476
0.64
0.016
0.0117
0
< 0.03
0.13
0.006
0.026
0.363
0
0.006
0.008
0.009
0.057
0
0.001
0.4835 ± .5869
0
< 0.03
3.1 ± 3.8
0.093 ± 0.32
0.315 ± .310
0.399 ± .302
0.024 ± 0.014
0.035 ± 0.050
0.015 ± 0.003
0.026 ± 0.094
0.266 ± 0.094
0.080 ± 0.22
0.012 ± 0.002
Reference
Singh et al .
Pellizzari ,
, 1981
1979a
Battelle, 1977
Pel 1 izzari ,
Singh et al .
Ibid.
Singh et al .
Singh et al .
Ibid.
Ibid.
Ibid.
Ibid.
Singh et al.
Pellizzari ,
Ibid.
1979a
, 1979a
, 1981
, 1979
, 1979
1979a
-------
TABLE 3-7. (continued)
CO
I
Location
Point Arena
Point Arena
San Jose
Oakland
La Joll a*
DELAWARE
Delaware City
LOUISIANA
Baton Rouge*
Lake Charles
MARYLAND
Baltimore
NEW JERSEY
Rutherford*
Somerset *
South Amboy
Date of
measurements
05/23-05/30 1977
08/30-09/05 1978
08/21-08/27 1978
06/28-07/10 1979
07/08-07/10 1974
11/18 1976
05/20 1977
12/06 1976
06/26 1978
07/11-07/12 1974
05/01 1978
12/29 1979
07/18-07/26 1978
01/27 1979
12/29 1979
Mixi
Maximum
0.012
0.034
2.192
1.5582
3.9
0.56
0.65
2.1
< 0.05
8.6
0.28
8.5
ng ratio
Minimum
0.010
0.008
0.048
0.0141
0
< 0.05
0
0.073
< 0.05
0
0
0
(ppb)
Average
0.011 ± 0.001
0.017 ± 0.007
0.417 ± 0.425
0.1876 ± 0.2697
1.3 ± 1.2
0.35
0.4 ± 0.23
1.6 ± 0.79
1.15
0.17 ± 0.17
0.08 ± 0.28
0.4 ± 1.4
Reference
Ibid.
Ibid.
Ibid.
Singh et al. , 1981
Su and Goldberg, 1976
Lillian et al. , 1975
Pellizzari et al. , 1979;
Battell e, 1977
Batten e, 1977; Pellizzari,
1979b
Pellizzari et al . , 1979
Bozzelli and Kebbekus, 1983
Bozzelli and Kebbekus, 1979
Bozzelli and Kebbekus, 1983
-------
TABLE 3-7. (continued)
CO
I
Location
Batso
Seagirt
Newark
Sandy Hook
Elizabeth
Fords*
Middlesex*
Edison*
Carlstadt*
Camden
Bayonne
Boundbrook*
Bridgewater*
Date of
measurements
02/26 1979
12/29 1979
06/18-06/19 1974
03/23 1976
12/29 1979
07/02-07/05 1974
09/15
12/29
03/26
09/27
1978
1979
1976
1978
07/23-07/28 1978
03/24
09/24
1976
1978
09/28-09/30 1978
04/03-10/24 1979
March-Dec
03/26
09/20
07/17
08/05
. 1973
1976
1978
1978
1978
Mixing ratio (ppb)
Maximum Minimum Average
1.
2.
1.
0.
6.
3.
0.
6.
9
3.
8.
4.
6.
3
8
9
8
4
1
36
1
5
8
1
9
0
< 0.05
0
< 0.05
0
0
0
0.54
3
0
< 0.05
0
0
0.
0.
0.
0.
0.
1.
0.
2.
6.
0.
0.
2.
0.
08 ± 0.28
26
25
34
76
9 ± 1.1
22 ± 0.11
7 ± 2.1
4 ± 3.1
42 ± 0.87
92
4 ± 1.8
35 ± 0.29
Reference
Ibid
Lillian et al . , 1975
Bozzelli and Kebbekus,
Lillian et al., 1975
Bozzelli and
Pell
Pell
Kebbekus,
1983
1983
izzari, 1977;
izzari et al. , 1979
Bozzelli and
Pell
Pell
Pell
Pell
izzari ,
izzari ,
izzari ,
Kebbekus,
1978;
1979;
1977b
1979
izzari et al . , 1979
Bozzelli and
Ibid
Pell
Pell
Kebbekus,
1983
izzari , 1977;
izzari et al . , 1979
Bozzelli and
Kebbekus ,
1979
Rahway*
09/20-09/22 1978
18
3.4
11 ± 7.8
Pellizzari et al., 1979
-------
TABLE 3-7. (continued)
Location
NEW YORK
NYC
Whiteface Mtns.
Niagara Falls/Buffalo
OHIO
Wilmington
,., NEVADA
i
00 Reese River
KANSAS
Jetmar
WASHINGTON
Pul "I man
Pacific Northwest
PACIFIC OCEAN
(lat 37°N)
PANAMA CANAL ZONE
Date of
measurements
06/27-06/28 1974
09/16-09/19 1974
Fall, 1978
07/16-07/26 1974
05/14-05/20 1977
06/01-06/07 1978
December 1974-
February 1975
March 1976
April 1977
Mixing ratio (ppb)
Maximum Minimum Average
1.1 0.11 0.71
0.35 < 0.05 0.10
0.11 0.05 0.10
0.63 < 0.05 0.19
0.016 0.001 0.012 ± 0.002
0.021 0.007 0.013 ± 0.004
< 0.005
0.020 + 0.0039
i
0.0124
Reference
Lillian et al . , 1975
Ibid.
Pellizzari et al. , 1979
Ibid.
Singh et al. , 1979
Ibid.
Grimsrud and
Rasmussen, 1975
Cronn et al. , 1977a
Cronn et al . , 1977b
(lat 9 N)
July 1977
0.015
Cronn and Robinson,
1978
-------
TABLE 3-7. (continued)
CO
I
Location
NORTH ATLANTIC OCEAN
WESTERN IRELAND
ENGLAND
JAPAN
Tokyo
Various sites
WESTERN EUROPE
COLORADO
Denver*
MISSOURI
St. Louis*
TEXAS
Aldine
El Paso
Freeport
Houston
Date of
measurements
October 1973
June-July 1974
March 1972
09/10-10/27 1975
Summer, 1979
1976
06/16-06/26 1980
05/30 1980
06/08 1980
06/22 1977
10/20 1977
04/05 1978
05/01 1978
08/09 1976
11/10 1976
07/27 1976
05/24/1980
Mixing
Maximum Mi
.005 4
47.1 0.
0.038 0.
8.5 <
0.41 0.
0.24 0.
0.029 0
0.3 0
0.029 0
0.33 0
ratio (ppb)
nimum Average
< 0.005 ± 0.0657
0.015 ± 0.0121
x 10"4 .002
8 3.5 ± 3.7
005 0.014 ± 0.010
0.02
028 0.2 ± 0.11
019 0.11 ± 0.063
0.009 ± 0.017
0.15 ± 0.11
0.001 ± 0.004
0.12 ± 0.098
Reference
Lovelock,
Ibid.
Murray and
Tada et al
Makide, et
1974a
Riley, 1973a
. , 1976
al . , 1980
Correia et al . , 1977
Singh et al . , 1980
Ibid.
Pellizzari
Pellizzari
Batten e,
Pellizzari
et al . , 1979
, 1979a
1977b;
et al. , 1979
Pellizzari et al. , 1979
Singh et al. , 1980
-------
TABLE 3-7. (continued)
CO
ro
o
Location
WASHINGTON
Auburn*
WEST VIRGINIA*
Nitre
West Belle
VIRGINIA*
Front Royal e
Date of
measurements
01/10-01/11 1977
09/27 1977
11/18 1977
09/27 1977
11/18 1977
09/29 1977
11/16 1977
Mixing ratio
Maximum Minimum
0.83 0.19
0.067 0
0 0
0.009 0
(ppb)
Average
0.64 ± 0.29
0.032 ± 0.035
0
0.003 ± 0.004
Reference
Battell e, 1977b
Pellizzari, 1978
Ibid.
Ibid.
*Data obtained from summary report of Brodzinsky and Singh, 1982.
-------
TABLE 3-8. MEAN LEVELS OF TCI IN SURFACE WATERS
Surface water samples
State
Alabama
Delaware
11 1 inois
Indiana
Kentucky
Louisiana
Michigan
Minnesota
New Jersey
New York
Pennsylvania
Tennessee
Texas
West Virginia
All Sites
Positive
for TCI
1
4
17
1
1
1
1
2
14
10
14
1
4
1
72
Total
7
10
25
4
3
7
8
9
15
20
21
6
8
3
179
Percent
positive
for TCI
14
40
68
25
33
14
13
22
93
50
67
17
50
3
40
Mean levels
(ug/1)
All .
samples '
0.5
1.2
0.6
0.7
0.6
23.9
1.9
4.0
1.6
2.3
0.9
1.2
0.7
2.7
Positive
samples only
1.0
2.3
3.2
1.0
1.0
1.0
188
7
4.3
2.6
3.2
3.0
1.8
1.0
5.9
Source: Ewing et al. (1977); drinking water samples not reported in these data.
Mean levels were calculated by substituting a value of one-half the reporting
limit of 1 ppb (1 ug/1) for samples reported as having non-detectable (i.e.,
less than 1 ug/1).
"Levels of TCI in sample from the following states (number of sites per state
in parentheses) were reported as not detectable (i.e., less than I ug/1):
California (9)
Iowa (2)
Mississippi (1)
Missouri (3)
North Carolina (1)
Oregon (3)
Washington (2)
Wisconsin (6)
004TC5/A
3-21
11/5/83
-------
A concentration of 8.5 ppb was found in the drinking water of the town of
Grand Island, New York (Coleman et al., 1976) using headspace GC-EC. In 10 lake
water samples from this area, an average concentration of 11.9 ppb +2.5 percent
was found.
Coleman et al. (1976) detected TCI in the drinking water of four cities in
1975. The concentrations were: 0.1 ppb (Cincinnati); 0.3 ppb (Miami); <0.1 ppb
(Ottumwa, Iowa); and 0.5 ppb (Philadelphia). No TCI was detected in drinking
water samples for Seattle. Identification and quantification were made by GC-MS
using the purge/trap technique.
As evidenced by the differences reported in the individual studies above,
the frequency of contamination and the amount of TCI found in potable waters
depend in part on the source of water being monitored.
The surface water supplies of 133 cities using surface water supplies have
been sampled during federal surveys. Thirty-two percent of the finished waters
serving these cities were found to contain TCI. The concentration of TCI in
these water supplies ranged from 0.06 to 3.2 ug/1. The average concentration in
positive water supplies was 0.47 ug/1. Thirty percent of all supplies contained
a level of TCI which was below 1 ug/1. The remaining two percent of the supplies
contained a concentration between 1 to 4 ug/1.
In 25 cities that draw their water from underground sources, water supplies
were sampled for TCI during the NOMS, NORS, SRI and EPA Region V surveys. Thirty-
six percent of these finished waters contained detectable concentrations of this
chemical. The median level detected was 0.31 ug/1 with a range from 0.11 to 53.0
ug/1.
The Community Water Supply Survey examined an additional 330 ground water
systems selected randomly from across the United States. Four percent of these
systems contained a detectable level of TCI. Seventy percent of these detectable
levels of TCI were between 0.5 to 5 ug/1. TCI was found in finished water from
small and large systems alike.
In analyses of drinking waters from sites near producer and user facilities
and from a background site, Battelle (1977) found the following concentrations in
1977: 19 ppb (Freeport, Texas); 0.4 ppb (Baton Rouge, Louisiana); 0.1 ppb
(Lake Charles, Louisiana); 32 ppb (Des Moines, Iowa); and 22 ppb (Helena, Arkansas).
(2) Rivers, treatment plant effluents
Up to 403 ppb TCI was detected by Battelle in surface waters near specific
manufacturing facilities (1977). Trichloroethylene was detected in both
industrial wastewater and river water receiving the waste (Jungclaus et al. ,
1978).
004TC2/D 3-22 11/10/83
-------
Murray and Riley (1973a) detected TCI in surface sea water in the eastern
Atlantic ocean.
Bellar et al. (1974) found an increased concentration of TCI after chlori-
nation treatment of the effluent from a sewage-treatment facility. Before chlori-
nation, the concentration detected by GC-MS was 8.6 ppb; after chlorination, the
concentration increased to 9.6 ppb.
Case reports describing TCI contamination of well waters and water supplies
have been reviewed elsewhere (U.S. EPA, 1979). Concentrations as high as 330 ppb
have been reported.
Correia et al. (1977) measured TCI in river, canal, and sea waters that re-
ceive effluent from production and user sites. Values ranged from 0.02 to 74 ug/1
(ppb w/w), showing the effect of these additions on background levels. Dilution
effects and losses to the surrounding air were noted.
3.3.3.3 Soil and Sediment—Battene (1977) reported the presence of TCI in soil
samples in the vicinity of producers and users at levels up to 5.6 ppb. Tri-
chloroethylene was detected in the soil in the Love Canal area in Niagara Falls,
New York (U.S. EPA, 1979).
Pearson and McConnell (1975) found up to 9.9 ppb in sediment from Liverpool
Bay, England. Murray and Riley (1973b) found TCI in river mud.
In freshwater sediment taken near the Hooker Chemical facility at Taft,
Louisiana, up to 300 ppb TCI was detected (Battelle, 1977).
Rogers and McFarlane (1981) reported that aluminum (Al 3)-saturated montmo-
rillonite clay sorbed about 17 percent of the TCI applied (100, 500, and 1,000 ppb).
The organic carbon content was assumed to be zero. There was no apparent absorp-
tion when calcium-saturated montmori 1 lonite clay was used. With two silty cla'y
loams (organic carbon content = 2 percent), less than 6 percent of the TCI availa-
ble was sorbed. When soil sorption was normalized, based on the soil organic
carbon contents, a correlation was found between water solubility, sorption con-
stant, and the partitioning coefficient.
3.4 ECOLOGICAL EFFECTS
Trichloroethylene has been tested for acute toxicity in a limited number of
aquatic species. This section presents observed levels reported to result in
adverse effects under laboratory conditions. Such parameters of toxicity are not
easily extrapolated to environmental situations. Test populations themselves may
not be representative of the entire species in which susceptibility of various
lifestages to the test substance may vary considerably.
004TC5/A 3-23 11/5/83
-------
Guidelines for the utilization of these data in the development of criteria
levels for TCI in water are discussed elsewhere (U.S. EPA, 1980).
The toxicity of TCI to fish and other aquatic organisms has been gauged
principally by flow-through and static testing methods (Committee on Methods for
Toxicity with Aquatic Organisms, 1975). The flow-through method exposes the or-
ganism(s) continuously to a constant concentration of TCI while oxygen is con-
tinuously replenished and waste products are removed. A static test, on the
other hand, exposes the organism(s) to the added initial concentration only.
Both types of tests are commonly used as initial indications of the potential of
substances to cause adverse effects.
Alexander et al. (1978) used both flow-through and static methods to
investigate the acute toxicity of several chlorinated solvents, including TCI,
to adult fathead minnows (Pimephales promelas). Studies were conducted in
accordance with test methods described by the U.S. Environmental Protection
Agency (1975). The lethal concentration of each solvent tested to produce 50
percent mortality (LC50) was lower in the flow-through than in the static-
type experiment. The flow-through method was considered the better of the two
for volatile solvents such as TCI. The 96-h LC50 concentration for TCI was
40.7 mg/1 in the flow-through test. The 95 percent confidence band was between
31.4 and 71.8 mg/1. Of four solvents tested (perchloroethylene, methylene chloride,
methylchloroform, and TCI), TCI had the second highest toxicity (after perchloro-
ethylene).
In acute static experiments with bluegills, TCI was reported to have a 96-h
LC50 value of 44.7 mg/1 (U.S. EPA, 1978b). With Daphnia magna as the test organ-
ism in a 48~h static experiment, the LC^ for TCI was 85.2 mg/1. In a chronic
test with this organism, no adverse effects were found at the highest test
concentrations of 10 mg/1.
Investigation of the bioconcentration potential of TCI for bluegill indicated
that the bioconcentration factor (BCF) was 17. The half-life of this compound
in tissues was less than 1 day (U.S. EPA, 1980; U.S. EPA, 1978b). These data sug-
gested the unlikelihood of residues at exposure concentrations below those directly
toxic to aquatic life (U.S. EPA, 1980).
Few data are available relating to effects of TCI on saltwater aquatic life.
Borthwick (1977) exposed sheepshead minnows and grass shrimp to 20 and 2 mg/1,
respectively, and noted erratic swimming, uncontrolled movement, and loss of
equilibrium after several minutes.
004TC5/A 3-24 11/5/83
-------
3.5 CRITERIA, STANDARDS, AND REGULATIONS
The current occupational standard for TCI established by the Occupational
Safety and Health Administration in 1972 (General Industry Standards, OSHA, 1978)
is 100 ppm (525 mg/m3) as a time-weighted average (TWA) over an 8-h period.
More recently, the National Institute for Occupational Safety and Health (NIOSH)
(Page and Arthur, 1978) has noted that the current standard appears to be inade-
quate for protection of workers from the carcinogenic potential of TCI. A level
of 25 ppm, as a TWA, was the recommended standard proposed by NIOSH, based on
engineering control capabilities.
004TC5/A 3-25 11/5/83
-------
3.6 REFERENCES
Alexander, H. C. , W. M. McCarty, and E. A. Bartlett. Toxicity of perchloro-
ethylene, trichloroethylene, 1,1,1-trichloroethane, and methylene chloride
to fathead minnows. Bull. Environ. Contam. Toxicol. 20:344-352, 1978.
Altshuller, A. P. Lifetimes of organic molecules in the troposphere and lower
stratosphere. Adv. Environ. Sci. Techno!. 10:181-219, 1980.
Battelle Columbus Laboratories. Environmental Monitoring Near Industrial
Sites: Trichloroethylene. Prepared for the U.S. Environmental Protection
Agency, PB 273203, August 1977.
Beckers, N. L. Stabilization of Chlorinated Hydrocarbons. Patent No. 3,682,830,
U.S. Patent Office, Washington, DC, August 8, 1972.
Bellar, T. A., W. L. Budde, and J. W. Eichelberger. The Identification and
Measurement of Volatile Organic Compounds in Aqueous Environmental Samples.
Chapter 4. In: Monitoring Toxic Substances. D. Schvetzle, ed. , ACS
Symposium Series 94, American Chemical Society, 1979.
Bellar, T. A., J. J. Lichtenberg, and R. C. Kroner. The occurrence of organo-
halides in chlorinated drinking waters. J. Am. Water Works Assoc. 66:
703-706, 1974.
Bonse, G. , and D. Henschler. Chemical reactivity, biotransformation, and
toxicity of polychlorinated aliphatic compounds. CRC Crit. Rev. Toxicol.
4:395-409, 1976.
Borthwick, P. W. Results of toxicity tests with fishes and macroinvertebrates.
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004TC1/E 3-33 11/5/83
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4. PHARMACOKINETICS AND METABOLISM
4.1 ABSORPTION AND DISTRIBUTION
Trichloroethylene (1,1,2-trichloroethylene or TCI), a chloroalkene, is a
highly volatile liquid (94 torr, 30°C) whose vapors exert pharmacologic effects
common to general inhalation anesthetics. Although it is no longer widely used
as an anesthetic or analgesic agent, the highly effective fat-solvent properties
of TCI are the basis of its numerous industrial uses and its use as an extrac-
tant in the food processing industry (Chapter 3.1). At work sites where this
compound is manufactured or used, the principal mode of entry into the body is
inhalation and lung absorption of TCI vapor. Dermal absorption occurs with
direct liquid contact in some degreasing applications and consumer uses (e.g.,
paint stripping). Oral absorption is also an important route of absorption.
Although sparingly soluble in water (1.0 mg/1, 25°C), TCI is present in the
water supplies of many of our cities and in their atmospheres, and it has been
identified in measurable amounts in the food chain. Hence, ordinary sources in
the nonindustrial environment include air, food, and drinking water. Indeed,
TCI has been detected in notable amounts in the breath of healthy humans and in
postmortem human tissue samples (Conkle et al., 1975; McConnell et al., 1975;
Barkley et al., 1980).
4.1.1 Dermal Absorption
Skin absorption from TCI vapor exposure is negligible, although direct skin
contact with the liquid by immersion of the hands, or partial body immersion,
may result in significant absorption. Uptake through the skin is related to the
type of skin exposed and the area of skin exposure as well as to the duration of
skin exposure. Tsuruta (1978) estimated skin absorption of TCI in mice by i_n
vivo and in vitro techniques. The percutaneous absorption rate of TCI in mice
2
was reported to be between 59.8 and 92.4 umol/min/cm or between 7.82 and 12.1
•2
pg/min/cm . Tsuruta concluded that direct contact with TCI, even if the percu-
taneous absorption rate of TCI is different between human skin and mouse skin,
may have the potential to contribute importantly to the overall exposure to TCI.
However, Stewart and Dodd (1964) have experimentally estimated skin absorption
of TCI in volunteers by noting its appearance and concentration in lung alveolar
air after thumb immersion. The mean peak concentration of TCI in expired air
30 min after immersion was only 0.5 ppm, indicating that the rate of absorption
through the skin is very slow, even after allowing for the possibility of lipid
004TC5/B 4-1 11-5-83
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storage and for metabolism to urinary metabolites (see below). In addition, Sato
and Nakajima (1978) demonstrated that for skin exposure in man, a large propor-
tion of absorbed TCI is eliminated unchanged through the lungs. These authors
also concluded that TCI would rarely be absorbed through the human skin in toxic
amounts during normal industrial use.
Recently Jakobson et al. (1982) carried out dermal absorption studies on
•2
TCI with guinea pigs. Liquid contact (skin area, 3.1 cm ) was maintained for up
to 6 h and solvent concentration monitored in blood during and following dermal
applications. Blood concentration (reflecting absorption rate) increased rap-
idly, peaking at 0.5 h (0.8 M9/ml blood), and then decreased despite continuing
exposure (0.46 ug/ml blood after 6 h). This pattern was characteristic of halo-
genated hydrocarbon solvents of low water solubility. Of 12 solvents tested,
TCI produced the second lowest blood concentrations from dermal absorption.
4.1.2 Oral Absorption
TCI is an uncharged, nonpolar, and highly lipophilic compound (Table 4-1)
and consequently can be expected to readily cross the gastrointestinal mucosal
barrier by passive diffusion. In man, absorption through the gastrointestinal
mucosa is extensive, as documented by the numerous cases of poisoning by oral
ingestion reported over the years (Defalque, 1961; Vyskocil and Polak, 1963;
Wiecko, 1966; Vignoli et al., 1970; Migdal et al., 1971). In animals, the oral
LD50 in mice, rats and dogs are similar, being 3.2, 4.9 and 2.8 g/kg respec-
tively (Klaasen and Plaa, 1966, 1967; Smyth et al., 1969).
Daniel (1963) dosed rats by stomach-tube with 36Cl-labeled TCI liquid (40
to 60 mg/kg b.w.) and recovered 90-95 percent of the label in expired air and
urine, suggesting virtually complete absorption by the route. Recently, Withey
et al. (1983) demonstrated in rats that gastrointestinal absorption of TCI is
considerably more rapid in water solution than when given in corn oil as a ve-
hicle. Following an 18 mg/kg intragastric dose in 5 ml water or corn oil to
fasting rats (400 g), the peak blood concentration (5.6 min for aqueous solu-
tion) averaged 15 times higher for water than for corn oil solution (14.7 vs <
1.0 |jg/ml); moreover, the peak blood concentration was reached faster for water
solution than for oil solution, which exhibited a second delayed peak 80 min post
absorption. Comparison of the area under the blood concentration curve (AUC)
during the absorptive phase reflects the rate and extent of absorption. The
ratio of the AUCs for 5 h after dosing was 218, water:corn oil. These results
illustrate the effect of solution medium on TCI intestinal absorption; the much
004TC5/B 4-2 11-5-83
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TABLE 4-1. PARTITION COEFFICIENTS OF TCI FOR VARIOUS BODY TISSUES AND
COMPONENTS OF RAT AND MAN
Rat
Blood/air
Lung/blood
Heart/blood
Kidney/blood
Liver/blood
Muscle/blood
Brain/blood
Testes/blood
Spleen/blood
Fat/blood
Man
Blood/ai r
Fat/air
Lecithin/ai r
Cholesterol/air
Cholesterol oleate/air
Triolei n/ai r
Mean values
25.82
1.03
1.10
1.55
1.69
0.63
1.29
0.71
1.15
25.59
9.92
674.4
387.9
52.15
261.93
848.24
Adapted from Sato et al., 1977.
Conversion factors:
1 ppm vapor in air = 5.38 mg/m3 at 25°C, 760 torr.
slower absorption of TCI dissolved in corn oil is probably due to the high lipid
solubility (and poor aqueous solubility) of this compound which remains as a de-
pot in the oil. Hence, absorption is limited to the rate of absorption of the
corn oil itself, or the corn oil effectively acts as a slow-release dosage form.
4.1.3 Pulmonary Absorption
4.1.3.1 Man--Inha1ed TCI rapidly equilibrates across the lung alveolar endothe-
lium. The blood/gas partition coefficient of 9.92 at 37°C is comparable to the
anesthetic gases—chloroform, diethylether, and methoxyfluorane--but its lipid
solubility is considerably higher (olive oil/gas coefficient, 960 at 37°C)
(Powell, 1947; Eger and Larson, 1964). Thus, pulmonary uptake of TCI is rapid
and extensive with considerable distribution into lipid of body tissues (Table
4-1).
004TC5/B 4-3 11-5-83
-------
Pulmonary uptake is proportional to the magnitude of the gas partial pres-
sure difference between alveoli and pulmonary venous blood. This difference di-
minishes when all body tissues achieve equilibrium with the alveolar (arterial)
partial pressure. Initially, uptake is rapid, but about 8 h exposure is re-
quired for complete tissue equilibrium to be achieved (Fernandez et al. , 1977;
Sato et al.; 1977; Monster et al., 1976, 1979; Muller et al., 1974). Figure 4-1
shows that equilibrium is achieved most rapidly (4 to 6 h) with the rich blood-
perfused tissues. The poorly perfused fat tissues (FG) require longer than 8 h
for equilibrium.
MG
VRG
ALV. AIR AND ART. BLOOD
246
EXPOSURE, hours
246
POST-EXPOSURE, hours
Figure 4-1.
004TC5/B
Predicted partial pressure of TCI in alveolar air and tissue
groups during and after an 8-h exposure of 100 ppm. From
Fernandez &t al. (1977).
4-4
11-5-83
-------
At equilibrium with a given concentration of TCI in inspired air, the dif-
ference between the inspired air concentration of TCI and alveolar concentration
of TCI provides a measure of gas uptake. With appropriate accounting for venti-
lation (about 4-8 1/min at rest) and duration of exposure, the amount taken up
by the body, or retention (dose; body burden), can be calculated. At alveolar
and tissue equilibrium, the retention reflects disposition within the body by
tissue storage, metabolism, or excretion by routes other than the lungs. Re-
tention of 36 to 75 percent has been reported for TCI by various investigators
(Soucek and Vlachova, 1960; Bartonicek, 1962; Nomiyama and Nomiyama, 1971, 1974a,b),
with higher values (70 to 75 percent) reported from more recent studies (Fernandez
et al., 1975; Monster et al., 1976, 1979) (Tables 4-2 and 4-3). The retention
value is independent of the inspired TCI concentration. In contrast, the amount
(dose) of TCI absorbed into the body is directly proportional to the TCI concen-
tration in the inspired air (Fernandez et al., 1975; Monster et al., 1976, 1979;
Muller et al., 1974). The body burden of TCI also increases with duration of
exposure, repeated daily exposures and exercise (increased ventilation) at a
given inhaled air concentration (Astrand and Ovrum, 1976; Monster et al., 1976,
1979). These relationships between inspired air concentration pulmonary uptake
and body burden are illustrated by the data of Astrand and Ovrum and of Monster
et al. in Tables 4-2 and 4-3. Astrand and Gamberale (1978) exposed volunteers to
100 and 200 ppm (ventilation volume 9.3 1/min) for 70 min and observed a linear
relationship between pulmonary uptake (Q) and alveolar concentration (A) divided
by inspired air concentration (I)
(f)
Q = 0.72 I Y/ + 74.91.
However, as about eight or more hours are required for complete body equilibra-
tion, this relationship only approximates steady-state inhalation conditions.
The blood concentration of TCI during inhalation and in the elimination
phase after exposure closely parallels alveolar gas concentration (Figure 4-1
and Figure 4-2) (Muller et al., 1974; Monster et al., 1976, 1979; Stewart et
al., 1974; Sato et al., 1977; Fernandez et al. , 1977). The kinetics of tissue
uptake, accumulation, and equilibration of TCI is a function of blood concen-
tration, the blood flow/tissue mass, and the relative solubilities of TCI in
004TC5/B 4-5 11-5-83
-------
TABLE 4-2. PULMONARY UPTAKE OF TCI FOR 25 VOLUNTEERS EXPOSED TO TCI FOR FOUR
CONSECUTIVE 30-MIN PERIODS AT REST AND DURING EXERCISE (50-150 WATTS).
FROM ASTRAND AND OVRUM (1976).
Exposure
concentration
(mg/m3)
540
1080
540
1080
Conditions
rest
rest
Exercise (50w)
Exercise (50w)
Amount inhaled
mg
149 ± 6
313 ± 15
395 ± 6
803 ± 12
Amount taken up*
mg
79 ± 4
156 ± 9
160 ± 5
308 ± 16
540 and 1080 mg/m3 s 100 and 200 ppm respectively.
^Calculated from difference in inspired air and expired air x ventilation volume
x time. Retention value averaged = 45 percent.
TABLE 4-3. PULMONARY UPTAKE OF TCI FOR 4 SUBJECTS AT REST EXPOSED TO
70 AND 140 PPM TCI FOR 4-HOUR PERIODS AND 3-HOUR PERIODS, PLUS TWO 30-
MIN PERIODS OF WORK (100 WATTS). FROM MONSTER (1976).
Exposure concentration
and conditions
Dose (mg)
Subject
Mean
mg
70 ppm, rest
70 ppm, rest + work
140 ppm, rest
140 ppm, rest + work
A B C D
320 430 330 470
500 450 470 660
740 790 710 790
1050 1100 970 1100
390
520
755
1055
Retention value average = 38 percent.
004TC5/B
4-6
11/10/83
-------
tissues (Table 4-1). Therefore, TCI most rapidly attains equilibrium by passive
diffusion into the vessel-rich group (VRG) of tissues (brain, heart, kidneys,
liver, and endocrine and digestive systems), more slowly with the lean mass (MG)
(muscle and skin), and last with adipose tissue, i.e., fat groups (FG). As
determined from elimination kinetics following exposure, TCI distributes from
blood into these three major compartments at approximate rate constants of
17 hr"1 (tj , 2.4 min) for VRG, 1.7 hr" (t^, 25 min) for MG, and 0.2 hr" (t^,
3.4 hr) for FG (Fernandez et al., 1975, 1977; Sato et al., 1977). Though the
lean muscle mass is 50 percent of the body volume and adipose tissue is 20 per-
cent, saturation and desaturation proceed more rapidly from the MG compartment
(t, , 25 min) than from the FG compartment (tj , 3.4 hr) because of the consi-
% *i
derably greater solubility of TCI in lipid (Table 4-1). Interperson variations
in TCI uptake (dose) are therefore influenced primarily by lean body mass and
secondarily by adipose tissue mass (Monster et al., 1976, 1979).
4.1.3.2 Animals--Stott et al. (1982) have determined the pulmonary uptake of TCI
3
in mice and rats exposed to 10 and 600 ppm (54 and 3228 mg/m ). The animals were
14
exposed in a 30-1 glass inhalation chamber to C-TCI, the concentration of which
was maintained within 10 percent of target. At termination of exposure (6 h),
the cumulative pulmonary uptake was estimated by determining the radioactivity
12
10
I 8
u
§ 6
g '
<
I I
riii
TRICHLOROETHYLENE
I I
I I I I
100
300
500
700
900
Figure 4-2.
AVERAGE CONC. mg/m3
The relationship between the concentration of TCI in arterial
blood and alveolar air at the end of inhalation exposures (30-
min periods, for 15 male subjects). Symbols stand for one ex-
posure period for a subject at rest, and at various levels of
exercise. The regression line is y = -0.489 + 0.014 X. From
Astrand and Ovrum (1976).
004TC5/B
4-7
11/10/83
-------
of carcass, and of TCI and metabolites in expired air, urine and feces. The data
for mice and rats are given in Tables 4-4 and 4-5, respectively. For mice, the
3
body burden for 6-h exposure to 10 and 600 ppm (54 and 3228 mg/m ) were 78.5 and
3138 umol equivalents/kg (103.0 and 412.3 mg/g) respectively. For rats, amounts
were 35.8 and 1075 umol equivalents/ kg (4.7 and 141.3 mg/kg) respectively. For
these species the 6-h pulmonary uptake was not directly proportional to inspired
air concentration level, as observed in man. Furthermore, the resultant body
burden to a given TCI-inspired concentration on a kilogram body weight basis is
highest for mouse (69 mg/kg/h/600 ppm), intermediate for rat (24 mg/kg/h/600 ppm)
and lowest for man (12 mg/kg/h/600 ppm). Presumably, these differences are pri-
marily related to the differences of metabolic and ventilation rates, and to
metabolism of TCI by these species. Stott et al. (1982) estimate that man metab-
olizes 20 times less TCI on a weight basis than rats.
4.1.4 Tissue Distribution and Concentrations
TCI distributes to all body tissues and appears in sweat and saliva
(Bartonicek, 1962). It crosses the blood-brain barrier (anesthetic effect) and
the placenta! barrier. Helliwell and Hutton (1950), investigating the distri-
bution in pregnant sheep and goats, found that TCI appeared in the fetal circu-
lation within 2 min of exposure; at equilibrium, the fetal/maternal blood con-
centration ratio was unity. The partition coefficients for various tissues of
rat and man are given in Table 4-1 (from Sato et al., 1977).
4.1.4.1 Man—There is little direct analytical information of tissue levels after
various exposure concentrations of TCI. Tissue concentrations are expected to
be proportional to exposure concentrations and to duration of exposure.
Fernandez et al. (1977), from their mathematical model of TCI uptake, dis-
tribution, and excretion, based on experimental data, have predicted the partial
pressure of TCI in the three major compartmental tissue groups, vessel-rich group
(VRG), muscle group (MG), and adipose tissue (FG), during and after an 8-h inhala-
3
tion exposure of 100 ppm (538 mg/m ). Figure 4-1 shows that VRG and MG are
nearly always in equilibrium with arterial blood concentration, but the concen-
tration of TCI in FG increases only very slowly and continues to increase beyond
the 8-h exposure. During desaturation, TCI in FG exhibits a long residence
(about 40 h) for disappearance with detectable FG concentrations remaining even
after 70 h. Because of the long residence of TCI in FG, repeated daily exposure
004TC5/B 4-8 11/10/83
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TABLE 4-4. RECOVERY OF RADIOACTIVITY FOR 50-HOUR POSTINHALATION EXPOSURE OF
MALE B6C3F1 MICE TO 10 OR 600 PPM 14C-TCI FOR 6 HOUR1
(From Stott et al., 1982).
10 ppm
umol-eq TCI/ ^
kg body weight %
Expired
1,1,2-TCI
CO^;
Urine
Feces a
Cage wash
Skin
Liver
Kidney
Carcass
Total body burden
Total metabolized
0.617
7.38
58.1
2.92
1.10
2.33
2.40
0.310
1.50
78.5
77.9
(0.182)
(0.383)
(18.8)
(0.542)
(0.446)
(1.13)
(0.401)
(0.310)
(0.316)
(17.9)
(18.1)
0.79
9.4
74.0
3.7
1.4
3.0
3.1
0.39
1.9
99.2
600 ppm
umol-eq TCI/
kg body weight
76.2
299
2278
115
85.5
56.3
72.2
11.1
146
3138
3062
(27.3)
(37.5)
(505.0)
(24.6)
(65.5)
(11.9)
(10.4)
(3.18)
(57.9)
(616.0)
(592.0)
2
2.4
9.5
72.6
3.7
2.7
1.8
2.3
0.35
4.6
97.6
TT
Average of four animals (±SD)
z
Percentage of recovered radioactivity.
a
Primarily due to urine.
TABLE 4-5. RECOVERY OF RADIOACTIVITY FOR 50-HOUR POSTINHALATION EXPOSURE OF
MALE OSBORNE-MENDEL RATS TO 10 OR 600 PPM 14C-TCI FOR 6 HOUR1
(From Stott et al., 1982).
Expired
1,1,2-TCI
CO.,
Urine
Feces 3
Cage wash
Skin
Liver
Kidney
Carcass
Total body burden
Total metabolized
10 ppm
umol-eq TCI/
kg body weight
0.778 (0.103)
1.71 (0.295)
22.6 (5.96)
2.52 (0.253)
0.357 (0.204)
3.78 (1.21)
1.01 (0.085)
0.131 (0.009)
2.82 (0.898)
35.8 (5.64)
35.0 (5.64)
%
2.1
4.8
63.1
7.0
1.0
10.6
2.8
0.37
7.9
97.8
600 ppm
umol-eq TCI/
kg body weight
227 (6.40)
31.2 (2.10)
594 (79.8)
38.3 (3.47)
15.1 (10.7)
76.2 (30.3)
16.3 (1.13)
1.72 (0.083)
75.1 (27.7)
1075 (87.2)
847 (81.6)
%
21.1
2.9
55.3
3.6
1.4
7.1
1.6
0.16
7.0
78.9
1
Average of four animals (±SD)
z
Percentage of recovered radioactivity.
3
Primarily due to urine.
004TC5/B 4-9 11/10/83
-------
(6 h/day; 100 ppm) results in an accumulated concentration, as TCI from new ex-
posures adds to residual concentration from previous exposures, until steady-
state is reached. This accumulation of TCI in FG is shown in Figure 4-3.
Adipose tissue levels of TCI reach a steady-state "plateau" (dependent on the
inhalation concentration and daily period of exposure) and thereafter remain
a
relatively constant. For a 6-h per day, 100 ppm (538 mg/m )-exposure, Fernandez
et al. (1977) predict that FG peak plateau concentration occurs 5 to 7 days after
initial daily exposure.
REPEATED EXPOSURE 100 ppm
(5 days, 5 hr. day)
Mo
Th Fr
EXPOSURE, days
Figure 4-3.
004TC5/B
Predicted partial pressure of TCI in fatty tissue for
a repeated exposure to 100 ppm, 5 days, 6 h per day.
From Fernandez et al. (1977).
4-10 11/10/83
-------
4.1.4.2 Rodents--Fo1lowing intravenous administration of TCI to Wistar rats,
Withey and Collins (1980) found a whole-body half-time of disappearance from the
rat of 2 to 6 min (dependent on dose) but a disappearance half-time of 215 min
for adipose tissue, indicative of the propensity of TCI to remain in adipose
tissue as compared to other tissues. Pfaffenberger et al. (1980), after dosing
rats by gavage for 31 days (1 and 10 mg/d), found that blood serum levels of TCI
were not detectable (analyzed 2 h after last dosing); but adipose tissue levels
averaged 0.28 and 20.0 M9 TCI/g, respectively. Three days after last dosing,
1 ug TCI/g fat still remained in adipose tissues.
Savolainen et al. (1977) analyzed tissue levels of TCI after exposure of
3
rats 6 h per day for 5 days to 200 ppm (1076 mg/m ) TCI. Table 4-6 gives the
values found. Seventeen hours after exposure on day four, blood and adipose
tissue levels were 0.35 and 0.23 moles/g, respectively, indicating long storage
in adipose tissue, though other tissues contained virtually no TCI. With expo-
sure on day five, tissue levels in brain, lungs, liver, fat and blood reached a
steady-state within 2 to 3 h. Partition coefficients were adipose tissue/blood,
10.4; brain/blood, 1.3; lungs/blood, 0.8; and liver/blood, 0.5. The lower value
for liver possibly reflects metabolism occurring in this organ, while the high
value for adipose tissue reflects high solubility of TCI in lipids.
4.2 EXCRETION
The total elimination of absorbed TCI involves two major processes, pulmo-
nary excretion of unchanged TCI and hepatic biotransformation to urinary metabo-
lites. The details of hepatic biotransformation of TCI are reviewed below. In
man, the principal metabolites are trichloroethanol (TCE), TCE-glucuronide,
and trichloroacetic acid (TCA). These metabolites are excreted in urine, and in
less significant quantities by other excretion routes, such as the bile
(Bartonicek, 1962).
4.2.1 Pulmonary Elimination in Man—Pulmonary elimination of unchanged TCI after
exposure occurs nearly as an inverted reproduction of its uptake. Alveolar con-
centration parallels blood concentration; both follow exponential concentration
decay curves showing three major components of approximate half-times of 2 to
3 min, 30 min, and 3.5 to 5 h (Figures 4-1 and 4-4), usually represented by VRG,
MG, and FG compartments, respectively (Fernandez et al., 1977; Sato et al., 1977;
Muller et al., 1974; Nomiyama and Nomiyama, 1971; Monster et al., 1979).
004TC5/B 4-11 11/10/83
-------
TABLE 4-6. ORGAN CONTENTS OF TCI AFTER DAILY INHALATION EXPOSURE OF 200 PPM
FOR 6 HOUR PER DAY. MEASUREMENTS MADE ON FIFTH DAY.
From Savolainen et al. (1977).
Time (h of
exposure) on
fifth day
0 [17 h after
day 4-expo-
sure]
2
3
4
6
Cerebel lum
0
11.7
± 4.2
8.8
± 2.1
7.6
± 0.5
9.5
± 2.5
Cerebrum
0
9.9
± 2.7
7.3
± 2.2
7.2
±1.7
7.4
± 2.1
Lungs Li
nmol/g
0.08
4.9
± 0.3
5.5
± 1.4 ±
5.8
± 1.1 ±
5.6
± 0.5 ±
ver
0.04
3.6
5.5
1.7
2.5
1.4
2.4
0.2
Peri renal
fat
0.23
± 0.09
65.9
±1.2
69.3
± 3.3
69.5
± 6.3
75.4
± 14.9
Blood
0.35
±0.1
7.5
+ 1.6
6.6
± 0.2
6.0
± 0.9
6.8
± 1.2
Values are mean of 2 determinations ± range.
Sato et al. (1977) developed a physiological kinetic model for TCI excre-
tion in man, based on experimental findings from controlled exposures of volun-
3
teers. Four male medical students (av. b.w. 61.6 kg) inhaled 100 ppm (538 mg/m )
for 4 h. After cessation of exposure, the blood and exhaled air concentrations
of TCI and the concentration of urinary metabolites were determined during the
time-course of 10-h postexposure. Figure 4-4 shows the results obtained. The
time-course of concentration decay (C) TCI in blood and exhaled air is the sum of
three exponentials:
-23.6+ -1.4&4- _0.18f
C . = 0.213E + 0.029E + 0.0126E
3 1 IT
_16.7t
C. , . = 0.115E + 0.449E
blood
0.255E
_0.20f
These reflect first-order excretion from 3 compartments (VRG, MG, and FG, re-
spectively) of the model (Figure 4-4). As indicated in the model, C . repre-
ai r
sents pulmonary clearance only, but C., , represents whole-body clearance by
004TC5/B
4-12
11/10/83
-------
z
g
\
UJ
U
O
U
OS
0.1
0.06
0.01
0.005
0.001
1
I—'
CO
1
0.6
01
0.05
0.01
x" = 0.0298a-1477t
I I I
I I I.
JAax
10
I I I I I I I
01234 5678
HOURS
10
Figure 4-4. Elimination curves of TCI and kinetic model for transfer of TCI in the body.
A. a x = pulmonary excretion (ventilation x air/blood partition coefficient
x blood concentration); bx = metabolic clearance (urine) x blood concentra-
tion; vt, V2, V3 volumes of VRG, MG, and FG compartments, respectively. From
Sato et al. (1977).
-------
the pulmonary system and metabolism. The kinetic parameters were determined as
follows: V (volume of FRG, MG, and FG compartments), 8, 31 and 10/1, respective-
ly; metabolic clearance, 104 1/hr; partition coefficients (blood/air, VRG/ blood,
MG/blood, FG/blood), 10, 1.5, 1.0, 67; t, of FG, 3.4 h. Similar tri-exponential
curves for decay of blood and exhaled air concentrations of TCI have been reported
by other investigators (Stewart et al., 1970, 1974; Nomiyama and Nomiyama, 1974;
Fernandez et al., 1975, 1977; Monster, 1976).
The long half-time of elimination from adipose tissue (3.5 to 5 h) explains
why, after a single short exposure (e.g., 4 to 6 h), exhaled air contains a not-
able concentration of TCI 18 h later, and also why accumulation of TCI (body burden)
occurs with repeated, fluctuating daily exposures until steady-state is reached
(Section 4.1.4). Several investigators (Fernandez et al., 1977; Monster et al.,
3
1979) have shown that, for volunteers exposed to 70 to 100 ppm (377 to 538 mg/m )
4 h daily for 5 days, the concentration of TCI in blood and exhaled air was two
to three times higher 18 h after the last exposure than at the comparable time
after a single exposure. TCI accumulation was highest in those volunteers with
a proportionally larger ratio of adipose to lean muscle mass.
4.2.2 Urinary Metabolite Excretion in Man
The urinary metabolites of TCI (total amount, excretion ratios, and excre-
tion time-course) have always been of considerable interest over the years as
quantitative indices of exposure and body burden. Recent investigations have
determined that the half-time for renal elimination of TCE and of TCE-glucuronide
approximates 10 h (range 7 to 14 h) after TCI exposure (Nomiyama and Nomiyama,
1971; Muller et al., 1974; Sato et al., 1977; Monster et al., 1976, 1979). This
value is in agreement with that observed after ingestion of TCE itself (Breimer
et al., 1974; Muller et al., 1974). As might be predicted from this relatively
short half-time of renal elimination, blood concentrations of TCE and TCE-
glucuronide after repeated daily TCI exposures reach a "plateau concentration"
(with peaks for each daily exposure) that is dependent on the inhaled air concen-
tration of TCI (Monster et al., 1979; Muller et al., 1974; Fernandez et al.,
1977). Consequently, the same daily amount of TCE and TCE-glucuronide is excreted
into the urine during repeated daily exposures to the same air concentration of
TCI.
In contrast to the short half-life of TCE, the half-time of renal elimina-
tion of TCA produced during TCI exposure approximates 52 h (range 35 to 70 h)
004TC5/B 4-14 11/10/83
-------
(Soucek and Vlachova, 1960; Nomiyama and Nomiyama, 1971; Muller et al. , 1974;
Sato et al. , 1977; Monster et al., 1976). Similar values have been observed
when TCA is directly administered to men (82 h, Paykoc and Powell, 1945; 51 h,
Muller et al., 1974). Distribution of TCA in the body is restricted to the
extracellular water space (Paykoc and Powell, 1945); it is very tightly and
extensively bound to plasma protein and excluded from erythrocytes, so that
plasma concentration is twice as high as whole blood (Soucek and Vlachova, 1960;
Monster et al., 1979). As a consequence of the long half-life of TCA, and of
the constant metabolic formation of TCA from TCE, blood concentrations of TCA
smoothly rise during a single TCI exposure, reaching a maximum 24 to 48 h post-
exposure before declining exponentially for 10 to 15 days. The blood concentra-
tion of TCA during the rising phase and at maximum is proportional to the TCI
inspired air concentration and the duration of the exposure, i.e., to the re-
tained TCI dose (Soucek and Vlachova, 1960; Monster et al., 1976; Sato et al.,
1977; Fernandez et al., 1977). During repeated daily exposures, TCA accumulates
in the blood exponentially, and declines only 24 to 48 h after the last exposure
(Muller et al., 1974; Fernandez et al., 1977; Monster et al., 1979). Conse-
quently the daily amount of TCA excreted into the urine increases during re-
peated exposure and then decreases slowly over a period of 15 to 20 days.
Several investigators have documented an apparent increase in the half-time of
renal elimination of TCA (to 100+ h) with repeated daily exposures to TCI. This
phenomenon is explained by continuous formation of TCA from accumulated tissue
stores of TCI and TCE, and by the very tight binding of TCA to plasma protein.
The renal elimination kinetics of TCE and TCA readily explain the urinary
excretion ratio time-course of these metabolites. The ratio of daily urinary
metabolites, TCE/TCA (mg/day), is highest (2:1) 24 h after a single TCI exposure
or after repeated daily exposures (Soucek and Vlachnova, 1960; Muller et al.,
1972, 1974; Fernandez et al., 1975; Nomiyama and Nomiyama, 1971, 1977; Sato
et al., 1977; Monster et al., 1976, 1979). Thereafter, the relationship of
TCE/TCA progressively decreases from day to day and becomes less than unity at
about the sixth day following exposure. Several additional factors are known to
influence the excretion ratio, TCE/TCA. The ratio is reported to be markedly
greater in males than females for the first 24 h after exposure (Nomiyama and
Nomiyama, 1971, 1977), and TCA urinary excretion is reported to vary diurnally
(Soucek and Vlachova, 1960; Monster et al., 1979). There is no evidence in man
004TC5/B 4-15 11/10/83
-------
that the elimination of TCI by metabolism is saturable, at least up to 300 ppm
(1584 mg/m ) inhalation concentrations, but saturation may occur at higher levels
(Section 4.4.2).
4.2.3 Excretion Kinetics in the Rodent
Withey and Collins (1980) determined the kinetics of distribution and eli-
mination of TCI from blood of Wistar rats after intravenous administation of 3,
6, 9, 12, or 15 mg/kg of TCI given in 1 ml water interjugularly. For the three
lower doses, the blood decay curves exhibited two components of exponential dis-
appearance and best fitted a first-order two-compartment model, with average kine-
tic parameters of Kg, 0.18 min" ; Vd, 158 ml; k12 and k21) 0.059 and 0.39, respec-
tively. For the highest dose (15 mg/kg), these investigators found that their
data best fitted a first-order three-compartment model. They suggest that the
shift from two- to three-compartment kinetics may have occurred either because of
a dose-related alteration in the kinetic mechanisms of" uptake, distribution, meta-
bolism, and elimination, or that three-compartment kinetics were followed at all
dose levels, but with lower doses, the third exponential component, representing
the third compartment, was obscured by the limit of analytical sensitivity. The
kinetic values for the three-compartment model (using a dose of 15 mg/kg) were
kg, 0.174 min ; Vd, 111 ml; k12, k21, k13: 0.183, 0.164, and 0.051, respec-
tively. The half-time, tj , for disappearance of TCI from perirenal fat was 217
min. Withey and Collins (1980) state that they found no evidence in their kine-
tic analysis of the disposition of intravenous bolus injections of TCI into the
rat of nonlinear or dose-dependent Michaelis-Menten kinetics, although the first-
order kinetic parameters obtained were clearly dose-dependent. These workers
suggested that a dose of 15 mg/kg TCI to the rat is below hepatic metabolism
saturation.
Other investigators have found evidence of dose-dependent Michaelis-Menten
kinetics in rats. Stott et al. (1982) exposed rats and mice to 10 and 600 ppm
3
(54 and 3228 mg/m ) TCI for 6 h. For exposed mice, the body burden (dose) from
the 6-h exposure was estimated as 78.5 Mm°les of TCI/kg body weight (10 mg/kg)
and 3138 umoles of TCI/kg (412 mg/kg), respectively. These inhalation doses
were virtually completely metabolized (Table 4-4). In contrast, for rats
(Osborne-Mendel), the inhalation doses were 35.8 pmoles/kg (4.7 mg/kg) and
1075 (jmoles/kg (141 mg/kg), respectively, of which 98 percent at the lower dose
3
was metabolized, but only 79 percent at 600 ppm (3228 mg/m ) (Table 4-5). These
004TC5/B 4-16 11-5-83
-------
results indicate a saturation of metabolism in the rat. Support for this con-
clusion is found also in the observation that pulmonary elimination of unchanged
TCI was only 2 percent of the dose from 10 ppm (54 mg/m3) exposure but 21 per-
cent of the dose at 600 ppm (3228 mg/m3) (Table 4-5).
Filser and Bolt (1979) exposed rats (Wistar) to TCI in a closed system
(desiccator jar chamber) and determined, from disappearance of TCI from the
chamber the inhalation pharmacokinetics, in accordance with the following model:
metabolism.
v,
1 2
2 1
V2
Chamber rat
atmosphere
Km
Vm
They found that the metabolism of TCI was dose-dependent in the rat, with the
saturation point occurring at 65 ppm (350 mg/m3). Zero order V was 210 [jmol/
ITI3X
h/kg and first-order (below 65 ppm) clearance was 77 1/hr/kg b.w.
Andersen et al. (1980) also analyzed the pharmacokinetics behavior of TCI
in rats (Fisher 344) using a Michaelis-Menten approach to demonstrate dose-
dependent metabolism. Exposures were conducted in a 31-1 battery jar chamber,
and the rate of depletion of atmosphere TCI was determined for 30, 100, 1000,
3500, and 8000 ppm (161, 538, 5380, 18830, and 43040 mg/m3). TCI uptake was
fitted to a four-compartment model as follows:
TCI
atmosphere
Blood,
liver, etc.
\
Fat, bone,
etc.
l<32
Metabolites
The metabolism of TCI was found to be dose-dependent, with a saturation point of
about 1000 ppm (5380 mg/m3) (K , 463 ppm). The kinetic parameters, calculated
as molar equivalents TCI, were Vm . 185 umoles/kg/h; K , 292 umoles/1 plasma;
ITidX HI
and first-order rate constant of 0.41 (jmoles/h/kg for uptake from the 31-1 chamber
A "whole animal'Vair partition coefficient of 15.4 (as compared to q for blood/air)
was calculated and suggested to be a more accurate estimate of microsomal-to-gas
distribution than a blood/gas constant.
004TC5/B
4-17
11-5-83
-------
4.3 MEASURES OF EXPOSURE AND BODY BURDEN
Trichloroethylene is principally absorbed by the lungs during industrial or
ambient-air exposure. However, as worker exposure periods and air concentrations
are likely to vary, air analysis does not necessarily provide a reliable indica-
tion of true exposure. An index of a worker's current exposure and total body
burden can be estimated in accordance with the amount of TCI absorbed and re-
tained as predicted by the known pharmacokinetics and metabolism of TCI. There-
fore, monitoring TCI levels in blood or expired air and measuring urinary metabol-
ites are the most common methods for determining the body burden (Stewart et al. ,
1962, 1970, 1974; Morgan et al., 1970; Nomiyama, 1971; Ogata et al. , 1971; Ertle
et al., 1972; Ikeda et al., 1972; Muller et al., 1972, 1974; Pfaffli and
Blackman, 1972; Kimmerle and Eben, 1973a,b; Lowry et al., 1974; Vesterberg et
al., 1976; Fernandez et al., 1975, 1977; Ikeda, 1977; Monster et al., 1976,
1979). These methods, however, are subject to high interpersonal variations in
the pharmacokinetics and metabolism of TCI that limit their predictive value.
Some of the factors now known to contribute to these variabilities are pulmonary
dysfunctional states, interindividual differences in intrinsic liver metabolism
capacity for TCI, modification of metabolism by drugs and environmental xenobio-
tics, age, sex, exercise or work load, and body anthropometry. Stewart and his
associates (1974) have been vigorous proponents of the value of postexposure
alveolar concentrations of TCI as measures of recent exposure and body burden.
However, this method is highly dependent on time of sampling and influenced by
individual variations in metabolism of TCI. Muller et al. (1974) have concluded
that neither TCA excretion nor the sum of urinary metabolites can be taken as
representative parameters of previous exposure, except possibly for 24-h daily
collections. These workers favor either alveolar TCI concentrations or blood
analysis of TCA or TCI as the best present indicators of exposure and body
burden. Monster et al. (1979) concluded from their studies that the most
"promising parameter for biological monitoring in repeated exposure to TCI" is
blood TCA concentration level, because of the ease of sampling and GC analysis,
the greater independence of time after exposure, accumulation with chronic ex-
posure and increased body burden, and the smaller interindividual variation of
this parameter.
Greater and more precise information than is now available on the absorp-
tion, distribution and metabolism kinetics of TCI is required for better means
of estimating or predicting body burdens with exposure. To this end, many in-
vestigators have developed mathematical models formulating the pharmacokinetics
004TC5/B 4-18 11-5-83
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of inhaled TCI and its metabolism (Fernandez et al., 1977; Droz and Fernandez,
1978; Feingold and Holaday, 1977; Gobbato and Mangivachhi, 1979; Sato et al. ,
1977; Sato, 1979).
4.4 METABOLISM
4.4.1 Known Metabolites
It has been known for 50 years that TCI is extensively metabolized in the
body to trichloroethanol (TCE), TCE-glucuronide, and trichloroacetic acid (TCA).
Following the introduction in the 1930's of TCI as a general anesthetic, Barrett
and co-workers (1939) isolated TCA from the urine of dogs and human patients
anesthetized with TCI. Powell (1945, 1947) demonstrated the presence of TCA in
both plasma and urine. She also observed that TCA continued to be excreted for
more than 2 days after a single anesthetic exposure. Butler (1949), who had pre-
viously discovered that the hypnotic, chloral hydrate, was metabolized to TCE,
TCE-glucuronide, and TCA (Butler, 1948), hypothesized that this widely used hypno-
tic was an intermediate metabolite of TCI. His experiments with dogs exposed to
TCI clearly demonstrated that the principal metabolites of TCI and TCE were TCE-
glucuronide ("urochloralic acid") and TCA (Figure 4-1). However, Butler could not
demonstrate the presence of chloral hydrate in either plasma or urine. Nonethe-
less, this demonstratation of biotransformation of TCI to TCE as well as to TCA
was the first proof that TCI anesthetic was converted to a metabolite with hypno-
tic properties. Trichloroethanol has a hypnotic potency comparable to that of
chloral hydrate (Marshall and Owens, 1954). Since that time, the intermediate
metabolite, chloral hydrate, has been found in the plasma of rats (Kimmerle and
Eben, 1973a) and humans inhaling TCI (Cole et al., 1975), and has been shown to
be produced in the isolated rat liver upon perfusion with TCI (Bonse et al. , 1975)
as well as with rat liver microsomal preparations (Byington and Liebman, 1965;
Ikeda et al., 1980; Miller and Guengerich, 1982). Defalque (1961) has reviewed
extensively the early literature on TCI.
The principal site of metabolism of TCI is the liver, although Dalbey and
Bingham (1978) have demonstrated that the rat and guinea pig lung also metabolize
TCI to TCE, and TCA. Other tissues, such as kidney, spleen and small intestine,
may also metabolize TCI, as these tissues have been shown to be sites of cellular
protein binding of metabolites from TCI (Bolt and Filser, 1977; Banerjee and Van
Duuren, 1978; Stott et al., 1982).
004TC5/B 4-19 11-5-83
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In addition to the major end-product metabolites of TCI, chloral, TCE, TCE-
glucuronide and TCA, a number of other metabolites have been demonstrated in
urine or exhaled breath (CO, C02, monochloro- and dichloroacetic acids, TCA-
glucuronide, chloroform) or i_n vitro with microsomal systems (TCI-epoxide,
glyoxylic acid, CO). These metabolites are listed in Table 4-7. Except for
dichloroacetic acid and TCA-glucuronide, these metabolites have been established
by more than one investigator. However, chloroform, which may be an artifact of
analytical methodology, needs further confirmation.
TCI-epoxide (1,1,2-trichloroethene oxide) is known to decompose in aqueous
physiological conditions (Table 4-7) to formic acid, dichloroacetic acid,
glyoxylic acid and CO. These metabolites may therefore be formed at cellular
microsomal sites of TCI-epoxide formation.
4.4.2 Magnitude of TCI Metabolism: Evidence of Dose-Dependent Metabolism
4.4.2.1 Man—Estimates of the extent of metabolism in man, as a percentage of
retained TCI, have been made from balance studies, i.e., accounting for a retained
dose after vapor exposure by measuring pulmonary elimination of TCI and metabo-
lites excreted into the urine. Balance studies with isotopically labeled TCI
have not been reported in man. Man appears to metabolize inhaled TCI extensively
(between 40 to 75 percent of the retained dose) as reported by various investigators
over the last three decades (Barrett et al., 1939; Powell, 1945; Forssman and
Holmqvist, 1953; Soucek and Vlachova, 1960; Bartonicek, 1963; Ogata et al. , 1971;
Ertle et al., 1972; Muller et al., 1972, 1974, 1975; Kimmerle and Eben,
1973a,b; Stewart et al., 1974; Fernandez et al., 1975, 1977; Vesterberg et al.,
1976; Nomiyama and Nomiyama, 1971, 1974a,b, 1977; Sato et al., 1977; Monster et al.,
1976, 1979). These studies do not present any evidence to indicate that a
saturation of metabolism occurs in man. The data of Nomiyama and Nomiyama (1977)
(Figure 4-5) and of Ikeda (1977), which correlate urinary metabolite excretion
with increasing levels of TCI exposure, indicate that liver capacity for
metabolism of TCI is nonlimiting, at least up to an exposure of 315 ppm (1695 mg/m3)
for 3 h (equivalent to 25 mg TCI/kg b.w.). Furthermore, the alveolar to inspired
air concentration ratio (25 percent; retention 75 percent) at exposure equilibrium
approximates the cardiac output flow through the liver (25 percent) and suggests
that TCI is completely removed from blood by the liver in a single pass. However,
Feingold and Holaday (1977) predicted from mathematical simulation models of TCI
at anesthetic concentration in man (2000 ppm; arterial concentration, 9.9 mg/dl)
that metabolism was rate-limited by saturation to 49 percent of uptake for an
8-h exposure.
004TC5/B 4-20 11/10/83
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TABLE 4-7. DEMONSTRATED METABOLITES OF TCI (OTHER THAN CHLORAL AND CHLORAL
DERIVATIVES; TCE, TCE-GLUCURONIDE, AND TCA)
Metabolite
System
Investigator
TCI-epoxide
Glyoxylic acid
Carbon monoxide
Carbon dioxide
Monochloroacetic acid
Dichloroacetic acid
TCA-glucuronide
Chloroform
rat, mouse
microsomes
rat, mouse
microsomes
rat, mouse
microsomes
exhaled air
rat and mouse
human and rabbit
urine
mouse urine
chimpanzee urine
rat adipose tissue,
serum, urine
Miller and Guengerich, 1982
Miller and Guengerich, 1982
Miller and Guengerich, 1982
Traylor et al. , 1977
Fetz et al., 1978
Parchman and Magee, 1982
Stott et al., 1982
Soucek and Vlachova, 1960
Ogata and Saeki, 1974
Hathaway, 1980
Muller et al., 1982
Pfaffenberger et al., 1980
Muller et al., 1974
Products from TCI-epoxide decomposition in aqueous solution, at 37°C
Formic acid
Dichloroacetic acid
Carbon monoxide
Glyoxylic acid
Henschler et al., 1977, 1979
Kline and Van Duuren, 1977
Bonse et al., 1975
Miller and Guengerich, 1982
004TC5/B
4-21
11-5-83
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The problems encountered with balance studies in man are the difficulties
associated with accurate measurement of the retained dose of TCI from vapor
exposure, and the imprecision of the older methodologies for determinations of
urinary metabolites by extraction and colorimetry using modifications of the
Fujiwara reaction. More recent careful studies, using GC methods, show that after
single or repeated daily exposures from 50 to 380 ppm, an average of 11 percent
O
oc
O
600
500
400
Si300
H ~*~
2 s 200
oc
o
X
UI
>
100
0 100 200 300
ENVIRONMENTAL TRICHLOROETHYLENE CONCENTRATION, ppm
Figure 4-5. Relationship between environmental TCI concentration and urinary ex-
cretion of TCI metabolites in human urine. Urine was collected for 6
days after exposure. (Adapted from Nomiyama ad Nomiyama, 1977).
of retained TCI is eliminated unchanged by the lungs (ti , 5 h), 2 percent of
'i
the dose is eliminated as TCE by the lungs (t^, 10 to 12 h), and 58 percent is
eliminated as urinary metabolites (Fernandez et al . , 1975, 1977; Muller et al . ,
1977; Monster et al., 1976, 1979). Unaccounted for is nearly 30 percent of the
TCI dose. These investigators suggest additional pathways or routes of elimination
of one or more unknown metabolites. Other reported but unconfirmed minor urinary
metabolites of TCI, such as chloroform (1 percent in rats, Muller et al . , 1974)
and monochloroacetic acid (4 percent in man, Soucek and Vlachova, 1960) do not
account for the discrepancy. On the other hand, early studies in dogs by Owens
and Marshall (1955) show that TCE-glucuronide is concentrated and secreted in
004TC5/B
4-22
11/10/83
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bile. Several studies also show^that TCE or TCA given orally or intravenously
is not fully recovered in the urine (Marshall and Owens, 1954; Owens and Marshall,
1955; Paykoc and Powell, 1945; Muller et al., 1974). Bartonicek (1962) found
that 8.4 percent of a TCI dose is excreted in the feces as TCE and TCA. Part of
the unaccounted TCI dose may also be explained by gastrointestinal excretion;
TCI can be expected to be rapidly distributed into the GI lumen because of its
high lipid solubility and ubiquitous distribution. In any case, these studies
indicate at least 60 percent and possibly as much as 90 percent of retained TCI
from vapor exposures of less than 500 ppm (2690 mg/m_) is metabolized in the body.
4.4.2.2 Animals — Early studies by Butler (1948) demonstrated the extensive metabo-
lism of TCI in dogs for which he estimated the extent of metabolism (as a per-
centage of the administered dose) to be 60 to 90 percent. Muller et al. (1982)
recently compared the metabolism of TCI in chimpanzees, baboons and rhesus monkeys.
14C-TCI was administered by intramuscular injection at a dose level of 50 mg/kg.
Radioactivity excreted in urine and feces (as an index of metabolism) ranged from
40 to 60 percent of the dose in chimpanzees, 11 to 28 percent in baboons, and 7
to 40 percent in rhesus monkeys. Their results showed a significant difference
in excretion route between man/chimpanzee and rhesus/baboon: while man and chim-
panzee excreted only from 2 to 5 percent of the totally eliminated TCI metabolites
in the feces, baboon and rhesus monkey excreted 18 to 35 percent of the radio-
activity in the feces.
For rodents (rat and mouse), the extent of metabolism after single doses of
TCI has been estimated by balance studies using isotopically labeled TCI. Daniel
(1963) carried out the earliest study in rats. The rats were dosed by stomach
tube with 36Cl-labeled TCI liquid (40 to 60 mg/kg b.w.); 80 percent of the dose
was eliminated via the lungs with a half-time of elimination of about 5 h; uri-
nary metabolites TCE, TCE-glucuronide and TCA accounted for only 15 percent of
the dose. More recent studies show considerably different results.
Stott et al. (1982) exposed rats and mice to 10 and 600 ppm (54 and
3228 mg/m3) 14C-TCI for 6 h. For exposed mice, virtually 100 percent of the body
burden was metabolized with no evidence of metabolic saturation. For rats,
however, 98 percent of the body burden from 10 ppm exposure was metabolized but
only 79 percent at 600 ppm (3228 mg/m3) exposure, suggesting a saturation of meta-
bolism in this species. In absolute amounts, mice metabolized 2.2 times more TCI
per kg body weight than rats after exposure to 10 ppm, and 3.6 times more after
exposure to 600 ppm (3228 mg/m3) (Tables 4-4 and 4-5).
004TC5/B 4-23 11-5-83
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Filser and Bolt (1979) and Anderson et al. (1980) have also presented strong
evidence of saturation of metabolism in the rat (Section 4.2).
4.4.3 Enzyme Pathways of Biotransformation
Current knowledge of the details of the enzymatic biotransformation of TCI
derives mainly from J_n vitro studies, principally with liver cell fractions.
Figures 4-6 and 4-7 summarize the probable pathways involved in the metabolism
of TCI as determined from those studies. Among the halogenated hydrocarbons,
TCI biotransformation appears to be unusual, since neither dehalogenation nor
removal of carbon atoms occurs in the first step. The initial biotransformation
of TCI involves metabolism to chloral hydrate and to formation of trichloroethy-
lene epoxide (Figure 4-6). The formation of chloral/epoxide is the rate-limiting
step in the metabolism of TCI (Ikeda et al., 1980; Nomiyama and Nomiyama, 1979).
Intramolecular rearrangement with migration of a chlorine atom is presumed to
occur in the formation of chloral hydrate, in order to explain the genesis of TCE
and TCA. Conversion of chloral hydrate to trichloroethanol (TCE) and trichloro-
acetic acid (TCA) is shown in Figure 4-7.
4.4.3.1 Formation of chloral — Byington and Leibman (1965) conclusively identified
chloral hydrate as the product of TCI when incubated with rat liver microsomes
fortified with NADPH and 02 (P450 mono-oxygenase system), thereby verifying the
hypothetical chloral hydrate formation proposed by Butler nearly 20 years earlier.
These workers also postulated an intermediate formation of an epoxide (as did
Powell in 1945), analogous to the known microsomal oxidation to epoxides of
dihydronaphthalene, aldrin, heptachlor and isodrin. Daniel (1963) provided evi-
dence that chloride migration must occur in chloral formation since no dilution
of 36C1 atoms with chlorine atoms from other body pools was observed in the
conversion of 36C1-TCI to its end products TCE and TCA. Microsomal P450-mediated
chloral formation from TCI has been repeatedly confirmed (Ikeda et al., 1980,
Miller and Guengerich, 1982) and the formation of the epoxide (1,1,2-trichloro-
ethene oxide) has also been directly demonstrated in liver microsomal preparations
by trapping with p-nitrobenzyl pyridine (Miller and Guengerich, 1982). In addition,
a specific P450 binding spectrum (Type I) in liver microsomes of rats and rabbits
has been observed after addition of TCI or TCI-epoxide (Kelley and Brown, 1974;
Uehleke et al., 1977b; Pelkonen and Vaino, 1975). TCI also competitively inhibits
the metabolism of hexobarbital and aniline by microsomal fractions (Kelley and
Brown, 1974), and the magnitude of the binding and chloral formation is enhanced
004TC5/B 4-24 11/10/83
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Cl
H
NADPH, O2
H —C—(C
O
Cl
Cl
H
CHLORIDE I
^ C
MIGRATION //
O
.C — Cl
•Cl
FORMIC ACID AND
CARBON MONOXIDE
GLYOXYLIC ACID
Figure 4-6. Postulated scheme for the metabolism of TCI to chloral (see
Figure 4.7 for chloral metabolism) and to trichloroethylene
epoxide and its metabolites. (From Miller and Guengerich,
1982).
004TC5/B
4-25
11-5-83
-------
CHLORAL
C CI3 CHO
MITOCHONDRIA
CYTOSOL
NAD +
ALDEHYDE
DEHYDROGENASE
CYTOSOL
NADH
ALCOHOL
DEHYDROGENASE
NADPH
ALDEHYDE
REDUCTASE
MICROSOMES
NADPH. 02
C CI3 COOH
rrcAi
C CI3 CH2OH
rrcEi
GLUCURONYL
TRANSFERASE
C CI3 CH2O C6H9O6
[TCE-GLUCURONIDE]
Figure 4-7. Metabolism of chloral hydrate. Modified from Ikeda et al., 1980.
004TC5/B
4-26
11-5-83
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with microsomes from animals pretreated with phenobarbital but not 3-methyl-
cholanthrene (Pelkonen and Vaino, 1975; Leibman and McAllister, 1967). However,
the question arises whether the epoxide is an obligatory intermediate in the formation
of chloral (Miller and Guengerich, 1982).
Attempts have been made to demonstrate chloral as a metabolite of TCI-oxide.
TCI-oxide has been synthesized and its reactions studied under a variety of con-
ditions (tj -v 1.3 min in water at pH 7.4, 37°C) Bonse et al., 1975; Kline and
Van Ouuren, 1977; Henschler et al., 1979; Miller and Guengerich, 1982).
Henschler et al. (1979) reported that, under physiological conditions (Tris buffer,
pH 7.4), TCI-oxide rearranged to dichloroacetic acid, CO, formic acid and
glyoxylic acid, but not to chloral. Entirely similar results were observed by
Miller and Guengerich (1982). However, Bonse and Henschler (1976) demonstrated
that the TCI oxide can be forced to rearrange to chloral by the catalytic action
of Lewis acids (FeCl3, A1C13, BF3), and Henschler et al. (1979) proposed that
the formation of chloral ijn vivo could therefore be due to catalytic action of
the iron of P450 in the trivalent form at the microsomal site of formation of
the oxide. Although Miller and Guengerich (1982) found that excess ferric iron
salts catalyzed the rearrangement of TCI-oxide to chloral in nonaqueous solutions
(CHi;Cl2 or CH3CN), chloral was not formed in aqueous media even when iron salts,
ferriprotoporphyrin IX or purified cytochrome P4bo, were present.
In studies with rat or mouse liver microsome fortified with NADPH and 0^
(or a reconstituted P45U enzyme system), Miller and Guengerich (1982) found that
TCI was metabolized to chloral, TCI-oxide, glyoxylic acid and CO. Traylor et al.
(1977) also found TCI converted to CO in microsomal incubations. Addition of
TCI-oxide to these systems did not produce chloral, but the oxide disappeared with
at, ~ 17 sec or, in the presence of rat liver epoxide hydro!ase, 9 sec. On the
^
basis of the reaction kinetics of formation of chloral and disappearance of TCI-
oxide in microsomal preparations, Miller and Guengerich (1982) proposed that TCI-
oxide was not an obligatory intermediate step in the formation of chloral. Alter-
natively, these investigators postulated an oxygenated TCI-P45U transition state
leading to two products: chloral (with chloride migration) and separate epoxide
formation, as shown in Figure 4-6. The working scheme for the metabolism of TCI
as presented in Figure 4-6 accounts for all the microsomal metabolites of TCI.
Miller and Guengerich (1982) excluded the likelihood of a rearrangement of TCI-
oxide or of TCI glycol to chloral, because although rearrangement to chloral
occurred in nonaqueous media in the presence of excess ferric iron salt, the
004TC5/B 4-27 11/10/83
-------
incubation of TCI-oxide with purified P450 did not result in rearrangement to
chloral. Since J_n vivo chloral is the primary metabolite of TCI metabolism, then
in view of Miller and Guengerich's (1982) postulate, the P450-med1ated reaction
to chloral may be preferential. The possibility of TCI-epoxide being a secondary,
minor product in the metabolism of TCI may account for the lesser mutagenic and
oncogenic potential of TCI in comparison to other halogenated ethylenes (Bolt et
al., 1982).
Van Dyke (1977) and Van Dyke and Chenoweth (1965) also proposed a direct
microsomal oxidation to chloral. TCI conversion to chloral was postulated to
involve a chloronium ion transition intermediate state in concert with oxidation,
presumably by cytochrome P450 system. As illustrated, the mechanism does not
require an epoxide intermediate. The oxidative attack forces chloride migration
to the adjacent carbon if possible, as in the case of TCI; if not, then the chlo-
rine bonded to the carbon, which is oxidatively attacked, is released (dechlorina-
tion).
Cl+ 0"
dx /Cl Cl ,,' \4 ^ 0
c = c -> c - c -» ci_c— t
^' ^ H W' VH - H
This mechanism also results in "reactive" intermediates which can covalently bind
to cell constituents.
4.4.3.2 Metabolism of chloral hydrate—The enzymatic pathways for the biotransfor-
mation of chloral hydrate formed from TCI exposure, to the plasma and urinary
metabolites TCE, TCE-glucuronide, and TCA, have received less attention in recent
years than the mechanism of chloral formation. Current knowledge of the enzymatic
pathways of chloral hydrate biotransformation is outlined in Figure 4-7. Chloral
hydrate is very rapidly metabolized i_n vivo with a half-life of only a few minutes
(Marshall and Owens, 1954; Breimer et al., 1974; Cole et al., 1975).
A number of investigators have concluded from jjn vitro studies that chloral
hydrate is a substrate for alcohol dehydrogenase purified from horse liver and
rat liver cytosol fractions (Butler, 1948, 1949; Marshall and Owens, 1954;
Friedman and Cooper, 1960; Sellers et al., 1972b). By this reaction, with NADH
as the required coenzyme, chloral hydrate is reduced to TCE (Km> 2.7 x 10 3M,
horse enzyme). The reaction is essentially unidirectional, since conversion back
to chloral hydrate has not been observed with this enzyme (Sellers et al., 1972b;
004TC5/B 4-28 11-5-83
-------
Owens and Marshall, 1955; Friedman and Cooper, 1960). Tabakoff et al. (1974)
studied the reduction of chloral to TCE and postulated the presence of enzymes
in rat liver cytosol and the presence of enzymes other than alcohol dehydrogenase
that are capable of reducing chloral. Ikeda et al. (1980) demonstrated at least
two NADPH-dependent enzymes to be present in rat liver cytosol (in addition to
NADH-dependent alcohol dehydrogenase) with a K of 6.0 x 10 3M. These investi-
gators also found that rat liver microsomes in the presence of NADPH and Og oxi-
dized TCE to chloral similarly to the oxidation of methanol and ethanol to ace-
taldehyde (Liebler and DiCarli, 1969).
Human red cells also reduce chloral hydrate to TCE (K , 4.0 x 10 4M)
(Sellers et al., 1972b). The efficiency of red cells and their relatively large
mass may explain the nearly undetectable levels of chloral hydrate in plasma and
urine after exposure to TCI, while TCE is prominent in plasma and urine. TCE-
glucuronide present in plasma and urine is accepted as resulting from conjugation
by hepatic microsomal glucuronyl transferase.
The origin of TCA as a plasma and urinary metabolite of chloral hydrate is
not as clearly defined. The most likely reaction for the oxidation of chloral
hydrate to TCA would involve the well-known enzyme acetaldehyde dehydrogenase.
However, chloral hydrate has been reported not to be a substrate for human ace-
taldehyde dehydrogenase (Kraemer and Deitrich, 1968; Blair and Bodley, 1969;
Sellers et al., 1972b). Cooper and Friedman (1958) obtained from rabbit liver
acetone powder a partially purified form of an NAD-dependent "chloral hydrate
dehydrogenase" which was substrate-specific for chloral hydrate. Acetaldehyde
was not a substrate but rather a markedly effective inhibitor. Chloral dehydro-
genase has not been identified in any other species even though the rabbit, in
comparison with the rat or man, poorly converts TCI to TCA. Furthermore, Cooper
and Friedman (1958) reported that their enzyme was not inhibited by disulfiram.
However, orally administered disulfiram is known to markedly decrease excretion
of TCA and TCE and TCI exposure to man (Bartonicek and Teisinger, 1962) and to
dogs and rats (Forssman, 1953, cited in Soucek and Vlachova, 1960).
In contrast to earlier studies on aldehyde dehydrogenase that found most of
this enzyme activity in the cytosol (Buttner, 1965), more recent studies show
that about 80 percent of total activity resides in the mitochondrial and microsomal
cellular fractions. Tottmar et al. (1973) described at least two different
aldehyde dehydrogenases (NAD and NADP-dependent) in rat liver, localized respec-
tively in mitochondria and microsomes. Grunett (1973) found the NAD-dependent
004TC5/B 4-29 11-5-83
-------
mitochondria! enzyme to have a broad substrate specificity, but chloral hydrate
was not a substrate. However, Ikeda et al. (1980) found that an aldehyde dehy-
drogenase prepared from rat liver mitochondria (NAD-dependent) converted chloral
to TCA (K , 62.5 x 10 3M). Furthermore, rat liver mitochondria had the highest
specific activity for TCA formation, cytosol less, but the microsomal fraction
contributed little to the formation of TCA.
Present evidence indicates that the liver is the principal organ contributing
to the metabolism of TCI to TCE and TCA. For example, Bonse et al. (1975) showed
that TCI perfused through the isolated rat liver led to the appearance of TCA,
TCE and chloral in the post perfusate. However, other tissues may also contribute
significantly. Dalbey and Bingham (1978) have demonstrated the metabolism of
TCI by the isolated perfused lungs of rats and guinea pigs to TCE and TCA, and
pretreatment of rats with phenobarbital increased the lung metabolism to TCE.
Tabakoff et al. (1974) found that rat brain cytosol was capable of reducing
chloral to TCE. The conversion of chloral in the presence of NADPH was 5 times
faster than with NADH, the co-factor for alcohol dehydrogenase. As noted above,
human red blood cells actively reduce chloral to TCE.
4.4.4 Metabolism and Covalent Binding
The extent of irreversible or covalent binding of reactive intermediates
from the metabolism of TCI to cellular macromolecules (protein, RNA, DMA) has
been extensively investigated, both i_n vitro and i_n vivo, as a theoretical basis
for evaluating the mutagenic and carcinogenic potential of TCI (Bolt et al., 1982).
The microsomal oxidative metabolism of TCI to chloral, as outlined in Figure 4-6,
suggests the possible reactive metabolites that may result in covalent binding
include chloral, TCI-epoxide, dichloroacetyl chloride and formyl chloride.
Chloroform, a putative metabolite as yet of indeterminate importance in TCI
metabolism, is known to generate covalent binding (Davidson et al., 1982). The
derivative metabolites of chloral (TCE, TCE-glucuronide, TCA) have not been
implicated in covalent binding. Primary concern accrues to the highly reactive
epoxide formed in TCI metabolism.
Henschler and coworkers (Bonse et al., 1975; Greim et al., 1975; Bonse and
Henschler, 1976; Henschler and Bonse, 1979) suggested that all chlorinated
ethylenes are initially transformed by microsomal P450 systems to epoxide interme-
diates which are subsequently "detoxified" by molecular rearrangement (in the
case of TCI to chloral). Further, reactivities, and hence, toxicities of
004TC5/B 4-30 11-5-83
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individual epoxide intermediates depend on the type of chlorine substitutions,
in that symmetric substitution renders the epoxide relatively stable and not
mutagenic, but asymmetric substitution causes unstable and therefore mutagenic
epoxides. To explain the lower-than-predicted mutagenic and carcinogenic poten-
tial of TCI (Chapter 7), these investigators suggest that TCI is an exception to
this general rule, because though its epoxide is asymmetrically substituted and
reactive, it immediately upon formation rearranges within the hydrophobic pre-
mises of the P450 site to the less reactive chloral; thus the epoxide is isolated
from reaction with cellular macromolecules and protected from aqueous spontaneous
decomposition to other reactive metabolites (dichloroacetyl chloride, formyl chlo-
ride) (Henschler et al., 1979). Alternatively, Miller and Guengerich (1982) have
provided evidence that the epoxide is not an obligatory intermediate for chloral
formation, and that, indeed, the two are separate P45o oxidative products with
chloral production four- to five-fold greater than the epoxide. Furthermore, they
observed a four- to five-fold greater production of chloral plus epoxide from mouse
microsomes than from rat microsomes.
4.4.4.1 In vitro Binding—Table 4-8 lists investigators who have demonstrated co-
valent binding of TCI to microsomal protein nucleic acids and lipids KI vitro.
In many of these experiments, jji vitro covalent binding from TCI microsomal
metabolism was decreased by animal pretreatment with SKF-525A (which inhibits
P450 metabolism) or enhanced by phenobarbital and other inducers of the P450
system. Binding was also decreased by addition of an inhibitor of epoxide
hydratase (trichloropropane epoxide) and by the addition of competing nucleophiles
such as reduced glutathione, imidazole or mercaptoethanol, indicating the impor-
tance of cellular SH groups and glutathione specifically for providing a
"detoxification mechanism" for reactive intermediates from TCI metabolism.
These experiments provide ample evidence that TCI is metabolized i_n vitro to
reactive products by P450 systems. Metabolism and covalent binding of reaction
products occurs with liver microsomes, the major site of metabolism, but also
with microsomes from kidney, stomach, and lung (Banerjee and Van Duuren, 1978).
Mice microsomes are two- to four-fold more active in metabolizing TCI to reactive
intermediates than rat microsomes, and males greater than females (Uehleke and
Poplawski-Tabarelli, 1977; Banerjee and Van Duuren, 1978). DiRenzo et al.
(1982a) found that aging rats (27 mo. old) have less capacity for microsomal
metabolism, as reflected by covalent binding of TCI, than either adult (11 mo.
old) or young (4 mo.) rats. These investigators have also compared the rat
004TC5/B 4-31 11-5-83
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TABLE 4-8. IN VITRO COVALENT BINDING OF TCI
Investigator
System
Macromolecules
covalently bound
Additions
Van Duuren and Banerjee
(1976)
Bolt and Filser (1977)
Bolt et al. (1977)
Uehleke and Poplawski-
Tabarelli (1977)
T Allemand et al. (1978)
Banerjee and Van Duuren
(1978)
rat liver microsomes
microsomal protein
Laib et al. (1979)
DiRenzo et al. (1982a)
rat liver microsoroes
rat and mice liver
microsomes
rat liver microsomes
Mice (B6C3F) and rat
(Osborne-Mendel)
microsomes
rat liver microsomes
rat liver microsomes
rat liver microsomes
young (4 mo) vs adult
(11 mo) vs aged
(27 mo)
SKF-525A or 7,8 benzoflavone- i binding; GSH,
methylmercaptoimidazole, mercaptoethanol, urea-i
binding; epoxide hydratase inhibitor (trichloro-
propane epoxide) t binding; pretreatment with
phenobarbital- t binding
albumin, polylysine GSH, i binding
microsomal protein
1 i p i d
microsomal protein
microsomal protein
Salmon sperm DNA
yeast RNA micro-
somal protein
calf thymus DNA
microsomal protein
Mouse > rat; pretreatment with phenobarbital,
t binding
GSH or piperonyl butoxide - 4- binding; pretreat-
ment of animals with phenobarbital - t binding
Mice > rats, males > females; microsomes from all
organs bind equally
Male > female enhanced by phenobarbital and 3-
methylcholanthrene admin; enhanced by addition
of epoxide hydratase inhibitor (trichloropropane
epoxide)
VC > TCI
TCI > VC
Rats pretreated with phenobarbital
4. P450 system in aged; i binding; pretreatment
with phenobarbital abolishes difference
-------
hepatic microsomal covalent binding to added DMA for a series of aliphatic halides,
including TCI. The degree to which TCI and other aliphatic halides form a DNA-
adduct is summarized in Table 4-9. TCI was bioactivated and covalently bound to
DNA at a level of 0.36 nmol/mg DMA, or fifth in the series of 10 compounds. Bind-
ing was confirmed by sedimentation of the DNA-adduct in a cesium gradient and
Sephadex LH-20 chromatography of the nucleotides, although structural identifi-
cation of modified DNA bases was not made.
4.4.4.2 In vivo binding: A number of investigators have determined the extent
of irreversible binding to cellular macromolecules following the administration
of TCI to rats and mice. Bolt and Filser (1977) exposed rats (male Wistar) for
TABLE 4-9. MICROSOMAL BIOACTIVATION AND COVALENT BINDING OF ALIPHATIC
HALIDES TO CALF THYMUS DNA
Binding to DNA
Aliphatic halides (nmol/mg/h ± SO)
1,2-Dibromoethane 0.52 ± 0.14 (6)
Bromotrichloromethane 0.51 ± 0.18 (6)
Chloroform 0.46 ± 0.13 (6)
Carbon tetrachloride 0.39 ± 0.08 (6)
Trichloroethylene 0.36 ± 0.14 (7)
1,1,2-Trichloroethane 0.35 ± 0.07 (7)
Dichloromethane 0.11 ± 0.05 (5)
Halothane 0.08 ± 0.01 (6)
1,2-Dichloroethane 0.06 ± 0.02 (6)
1,1,1-Trichloroethane 0.05 ± 0.01 (3)
14C-labeled aliphatic halides (1 mM) were incubated with hepatic microsomes.
Carbon tetrachloride, bromotrichloromethane and halothane were incubated under
an N2 atmosphere, and all other incubations were under an 02 atmosphere. From
DiRenzo et al., 1982b.
5 h in a closed system to initial concentrations of 100 and 1000 ppm (538 and
3 14
5380 mg/m ) C-TCI and determined protein-bound metabolites in various organs.
The irreversibly bound metabolites were calculated as a percentage of that amount
of TCI that was taken up by the animals in 5 h. Radioactivity following exposure
was mainly concentrated in the liver (0.77 - 0.88 percent dose), the primary
organ of metabolism, and in kidneys (0.37 - 0.39 percent dose), the organ of pri-
mary excretion. Lesser amounts were found in lung, spleen, small intestine, and
muscle. No major differences in percentage of the dose bound for the different
initial atmospheric concentrations of TCI. However, the extent of binding in
liver and kidney for TCI was less than for vinyl chloride and approximated that
observed with carbon tetrachloride.
004TC5/B 4-33 11-5-83
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Uehleke and Poplawski-Tabarelli (1977) determined j_n vivo binding for mice
(NMRI) following intraperitoneal injections of 14C-TCI (0.2 uCi/10 umole in 5 pi
of peanut oil/g b.w.). Binding to live microsomes, mitochondria, and cytosol
protein was measured at 2, 6, 12, and 24 h after injection. Peak binding occurred
after 6 h (8, 4 and 1.8 nmoles/mg protein) respectively, and thereafter declined
slowly. When the same dose of 14C-TCI was given by gavage at the 6-h time, bind-
ing to microsomal protein was found to be only 5.4 nmole/mg protein. 14C-TCI was
also bound to hepatic lipids; similarly to protein binding, 6 h after i.p. dosage,
the highest concentrations were 9.4 nmole/mg of endoplasmic fractions of livers
and 5.6 nmole/mg in mitochondria! lipids of the liver.
Allemand et al. (1978) investigated the metabolic activation of TCI and
irreversible binding in Sprague-Dawley rats after 14C-TCI (100 umol, 100 (jCi)
was administered i.p. in 200 |jl of methanol to nonpretreated and to phenobarbital-
pretreated rats; the rats were sacrificed 4 h later. Trace amounts of irre-
versibly bound radioactivity were found on muscle proteins (3.1 mmole equiv./g
tissue), while 40 times more was found on liver proteins (117 nmole equiv./g
tissue). Pretreatment of the animals with phenobarbital did not significantly
increase muscle binding but increased liver binding by 46 percent. Furthermore,
TCI reduced hepatic glutathione levels 61 percent 4 h after administration, con-
firming similar observations by Moslen et al. (1977b).
Parchman and Magee (1982) compared i_n vivo covalent binding of TCI metabo-
lites to liver protein and DMA in B6C3F1 male mice and Sprague-Dawley male rats.
Rats were injected i.p. with 10, 100, 500, and 1000 mg/kg b.w. of 14C-TCI in corn
oil (each animal received 160 uCi/kg of radioactivity). After 6 h, liver
"cytoplasm" and "nucleus" fractions contained 2.5 to 6.1 and 0.3 to 3.9 percent
of dose radioactivity, respectively. Radioactivity was detected in DMA isolated
from the nucleus fraction and purified by cesium chloride centrifugation and
extraction with phenol, isoamyl alcohol and chloroform. However, the level of
radioactivity was extremely low (35 cpm/mg DMA) and contamination with labeled
protein could not be entirely ruled out. Identification of DMA adducts by
hydrolysis followed by HPLC of nucleotides was not successful. Similar results
were obtained with mice injected i.p. with 10 and 250 mg/kg 14C-TCI (160 uCi/kg
b.w.) and sacrificed 6 h later. Hepatic DMA contained only 51 cpm/mg for the
lower dose and 283 cpm/mg for the higher dose. Compared to DNA binding by
dimethylnitrosamine (DMN) under identical conditions, the carcinogen-binding
index (Lutz, 1979) calculated for TCI was only 2, as compared to 2045 for DMN.
004TC5/B 4-34 11-5-83
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These investigators concluded that the ability of TCI to interact with DMA iji
vivo was very slight.
Stott et al. (1982) investigated covalent binding of TCI in male B6C3F1 mice
and male Osborne-Mendel rats, the rodent strains used for carcinogenicity testing
of TCI by the National Cancer Institute (NCI) (NCI, 1976). The animals were ex-
posed to 10 or 600 ppm (54 or 3228 mg/m3) of 14C-TCI (11.3 mCi/nmol) for 6 h. The
mice metabolized more inhaled TCI than rats at low and high exposure levels (123
and 260 percent, respectively). The amount of hepatic and renal covalent binding
was three-fold higher in mice than for the high dose (Table 4-10) and four-fold
greater for liver than kidney. Mice (4) were also dosed with 1200 mg/kg of 14C-
TCI with relatively high specific activity (2.4 mCi/nmol) and a maximum estimate
of a liver DNA alkylation level of 0.62 ± 0.42 alkylations/106 nucleotides was
observed after isolation and separation by HPLC (Table 4-11). However, no chemi-
cal identification of possible nucleotide adducts was made. Maximum covalent
binding index observed was 0.12, or one-twentieth of that estimated by Parchman
and Magee (1982), and many orders of magnitude less than that for known carcino-
gens such as DMN, methylnitrosurea, etc. (footnote, Table 4-11). These results
are in accord with the conclusion of Parchman and Magee (1982) that the ability
of TCI to interact with DNA ui vivo is very slight.
Stott et al. (1982) also chronically administered to male B6C3F1 mice (by
gavage) 2400 mg TCI/kg/day for 3 days. Localized cell necrosis, enhanced DNA
synthesis, and centrilobular hepatocellular swelling was observed. With pro-
longed exposure (3 weeks), the primary response observed was dose-related cen-
trilobular hepatocellular swelling and the occurrence of mineralized cells. In
Osborne-Mendel male rats, a maximum tolerated dose of 1100 mg/kg/day led to in-
creased liver weight and elevated DNA synthesis not coupled to histopathology,
but consistent with a simple increase in the number of normal hepatocytes. In
view of the weak genotoxic potential of TCI, as indicated by only slight DNA
covalent binding and weak hepatotoxicity in mice, these investigators suggest an
epigenetic mechanism for the tumorigenic action'of TCI observed in this species
only in the NCI carcinogenicity bioassay (NCI, 1976). They implicate also the
greater covalent protein binding (three-fold) that occurs in the mouse versus the
rat from the three-fold greater total metabolism of TCI per kilogram body weight.
004TC5/B 4-35 11-5-83
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TABLE 4-10. HEPATIC AND RENAL MACROMOLECULAR BINDING OF 1,1,2 (UL-14C)
METABOLITE IN MALE B6C3F1 MICE AND OSBORNE-MENDEL RATS
EXPOSED TO 10 OR 600 PPM FOR 6 HOURS. From Stott et al. (1982)
Exposure
(ppm)
10
600
Tissue
Liver
Kidney
Liver
Kidney
Test
animal
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Binding (pmol-
eq(14C) TCI/M9
protein) ± SO
0.318 (0.004)
0.268 (0.013)
0.168 (0.014)
0.155 (0.007)
20.4 (2.49)
4.72 (0.415)
5.06 (0.667)
1.77 (0.200)
Ratio
(mouse/rat)
1.2
1.1
4.3
3.0
TABLE 4-11. IN VIVO ALKYLATION OF HEPATIC DNA BY 1,1,2(UL-14C) TCI
IN MALE B6C3F1 MICE DOSED WITH 1200 MG/KG (14C) TCI BY GAVAGE.
From Stott et al. (1982)
Animal
1
2
3
4
Detection limit
(alk/104)
0.12
0.52
0.15
0.18
Non-Ci dpma
> 2 Sigma error
18.0
NDd
7.1
26.6
Maximum
alkylations 104
nucleotides
0.50
0.27
1.1
Maximum
CBIC
0.055
0.030
0.120
adpm not associated on HPLC analysis with normal DNA bases from normal metabolic
incorporation and not attributable to counting error (i.e., mean background
dpm + 2 SD).
b"Maximum" alkylations possible due to the relatively large amount of dpm
associated with protein binding and metabolic incorporation into normal bases.
cCovalent binding index (Lutz, 1979). Comparative hepatic CBI for dimethyl-
nitrosamine, diethylnitrosamine, methylnitrosourea, and aflatoxin B-l are
approximately 5500, 125, 640, 17,000, respectively. CBI = (|jmol adduct/mol dN)
(nmol/kg dose).
None detected.
004TC5/B 4-36 11/10/83
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4.4.5 TCI Metabolism: Drug and Other Interactions
A number of commonly used drugs might be expected to modify the extent of
metabolism of TCI during human exposure. The converse may also occur, resulting
in important modifications of the therapeutic action of drugs. Although largely
undocumented in man, the induction of hepatic microsomal mixed-function oxidate
system by drugs taken for therapeutic reasons or by exposure to certain environ-
mental chemicals (e.g., barbiturates, PCBs, etc.), may bring about an increased
rate of TCI metabolism. Numerous J_n vivo and i_n vitro experimental studies with
animals pretreated with microsomal inducers have demonstrated an enhanced meta-
bolism of TCI (Leibman and McAllister, 1967; Carlson, 1974; Moslen et a!.,
1977a,b; Pessayre et al., 1979). Conversely, TCI competitively inhibits the
metabolism of barbiturates, producing exaggerated effects of these drugs (Kelley
and Brown, 1974).
4.4.5.1 Disulfiram--A drug interaction has been shown to occur in persons exposed
to TCI who are also on a therapeutic regimen of disulfiram for alcoholism. Disul-
firam inhibits the oxidation of acetaldehyde by competing with NAD for active
sites on aldehyde dehydrogenase. Bartonicek and Teisinger (1962) showed that
disulfiram markedly inhibits the terminal enzymatic steps of TCI metabolism in
man. Since these steps, which involve the conversion of chloral hydrate to TCE
and TCA (Figure 4-7), can be considered detoxification mechanisms, disulfiram
has the potential of enhancing TCI and chloral hydrate toxicities.
4.4.5.2 Alcohol — Sellers et al. (1972b) investigated the interaction of ethanol
and chloral hydrate metabolisms. These investigators observed that concomitant
administration of alcohol and chloral hydrate in man exacerbated the side effects
of chloral hydrate (vasodilatation, tachycardia, facial flushing, headache and
hypotension). Their patients also exhibited impairment of auditory function and
of performance of complex motor tasks. Similar effects have been observed during
acute and chronic TCI exposure and concomitant ingestion of alcohol (Muller et al.,
1975). Stewart et al. (1974) have described the phenomenon of "degreasers" flush
(facial and upper extremity skin vasodilatation) and determined that this effect
of alcohol interaction with TCI persists after cessation of exposure. This in-
tolerance to alcohol syndrome is ascribed to mutual inhibition of TCI and alcohol
metabolisms producing increased plasma ethanol and TCE concentrations from
competitive inhibition of aldehyde dehydrogenase (Sellers et al., 1972a,b;
Stewart et al., 1974).
004TC5/B 4-37 11-5-83
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Muller et al. (1975) specifically investigated the interaction of TCI and
alcohol in human volunteers. After the volunteers inhaled TCI (50 ppm) for 6 h
per day for 5 days, ethanol ingestion inhibited TCI metabolism to TCE and TCA by
40 percent. Blood TCI concentration increased more than two-fold. This latter
observation suggested that alcohol also inhibited, by competition, the microsomal
oxidation of TCI to chloral hydrate. These investigators proposed that the intol-
erance syndrome from combined exposure to TCI and ethanol was due to increased
accumulation of TCI in the CMS, resulting from depression of TCI oxidation. From
experiments in rats, Nakanishi et al. (1978) found that ethanol ingested after
TCI exposure increased blood acetaldehyde levels three- to four-fold. They
suggest that TCI inhibits acetaldehyde dehydrogenase, and the resulting increase
of blood levels of acetaldehyde is responsible for the intolerance syndrome.
White and Carlson (1981), working with rabbits, observed that acute ethanol
administration (1 g/kg i.v. or p.o. 30 mm prior to TCI exposure, 6000 ppm; 32280
mg/m3) decreased TCI metabolism, as indicated by increased peak blood levels of
TCI (22 percent), decreased peak blood levels of TCE (50 percent) and decreased
blood levels of TCA (99 percent). Also, the zero-order metabolism of alcohol
itself was decreased 25 percent by TCI exposure (blood ethanol disappearance
rate, -0.707 control vs. -0.539 exposed, mg/dl/min). Furthermore, rabbits
treated with ethanol exhibited markedly increased susceptibility to the develop-
ment of TCI-induced cardiac arrhythmias, provoked by epinephrine. The results
of this ethanol study in rabbits confirm those of the human studies by Muller et
al. (1975).
In contrast to these studies, Sato et al. (1981) found that an acute ethanol
dose to rats (4 g/kg, 40 percent solution intragastric) significantly lowered
the blood concentration decay curve of TCI resulting from a subsequent (16 h later)
400 ppm (2152 mg/m3) for 4-h exposure to TCI. Furthermore, ethanol-treated rats
excreted in urine much greater amounts (three-fold) of total trichloro compounds
(TCE and TCA) than untreated rats. These findings would indicate that a single
large dose of ethanol can accelerate the J_n vivo metabolism of TCI. When ethanol
(4 g/kg) was given to rats and liver microsomes were prepared and tested for
their rate of metabolism of TCI, an enhancement of TCI metabolic rate was found,
with the greatest increase (two-fold) occurring 16 h after ethanol dosing. At
this time, almost no blood ethanol was detected and no increase occurred in micro-
somal protein and P450 liver contents. Ethanol addition directly to the TCI micro-
somal incubations inhibited TCI metabolism, thus demonstrating that ethanol was a
competitor of TCI metabolism. These investigators suggest that ethanol, when
004TC5/B 4-38 11-5-83
-------
taken Jji vivo, can exert a dual effect on drug-metabolizing enzymes, i.e., both
inhibition and stimulation of TCI metabolism. When present in an early period
at high blood levels, ethanol inhibits metabolism of TCI; but ethanol also
increases metabolic potential of the enzymes so that 16 to 18 h later, when
ethanol blood level has dissipated, the microsomal enzymes have an increased
capacity to metabolize TCI.
4.4.5.3 Warfarin: Sellers and Koch-Weser (1970) observed a potentiation of the
anticoagulant effect of Warfarin in patients after chloral hydrate ingestion.
This increased potential for bleeding appeared to result from displacement of
Warfarin from plasma protein binding sites by the chloral hydrate metabolite,
TCA. Considering the significant amounts of TCA formed after TCI exposure and
the long half-time of excretion of TCA, it is likely that TCI may potentiate the
effects of many other drugs which normally bind to the same plasma protein sites
as TCA.
4.4.5.4 Diet and age: Nakajima et al. (1982) have explored the effect of diet
on the hepatic microsomal metabolism of TCI and other chlorinated hydrocarbons.
These investigators found that hepatic microsomes from rats fed for 3 weeks on
an isocaloric diet deficient in carbohydrate (sucrose) had an increased capacity
(two and one half-fold) to metabolize TCI and other chlorinated hydrocarbons.
Varying protein content of the diet produced only a negligible influence on
enzyme activity. Microsomal protein and P450 hepatic content (per g liver) were
not significantly changed, although liver weight was slightly decreased. They
suggest from their findings that a low-carbohydrate diet produces enhanced P450
system activity. This concept, according to these investigators, is not in con-
flict with the widely held belief that a high-protein diet enhances this activity,
because high-protein diets are usually low in carbohydrate to maintain isocaloric
conditions. Thus, a diet rich in carbohydrate may offer significant protection
against the hepatic toxicity of chlorinated compounds by decelerating their
metabolism.
DiRenzo et al. (1982a) have recently shown that the hepatic microsomal capa-
city to metabolize TCI and other chlorinated hydrocarbons is influenced by chrono-
logical age. These investigators isolated hepatic microsomes from young (4 mo.),
adult (11 mo.) and aging (27 mo.) Fischer 344 rats. Biotransformation and subse-
quent covalent binding of labeled TCI and other compounds was studied. Levels
of microsomal protein and lipid binding increased slightly between 4 and 11 months,
but decreased 50 to 75 percent in the 27-month-old animals, as compared to young
004TC5/B 4-39 11-5-83
-------
adults. Senescent rats were also found to have significantly less hepatic P450
(-35 percent), NADPH cytochrome C reductase (-31 percent), and ethyl morphine
N-demethylase activity (-43 percent). Pretreatment with phenobarbital, however,
abolished these deficiencies of aging rats. These investigators suggest a reduced
metabolic capacity as a function of age, which results in a decrease in bioacti-
vation of the aliphatic halides, and hence, possible differences in toxicity in
the elderly from xenobiotics requiring bioactivation.
4.4.6 Metabolism and Cellular Toxicity
The metabolism of TCI has been suggested as a possible vector for the
hepatotoxicity and nephrotoxicity associated with excess human exposure to this
haloalkene (James, 1963; Defalque, 1961; Smith, 1966; Litt and Cohen, 1969;
Baerg and Kimberg, 1970). Experimentally, TCI has been shown to be hepatotoxic
in dogs and rats (Orth and Gillespie, 1945; Klaassen and Plaa, 1967; Cornish and
Adefuin, 1966; Carlson, 1974; Cornish et al. , 1977; Reynolds and Moslen, 1977).
Reynolds and Moslen (1977) and Henschler and Bonse (1979), on physicochemical
and biological correlative bases, have proposed that unsymmetric chlorinated
ethylenes like TCI are more hepatotoxic than symmetric ethylenes as perchloro-
ethylene and more so than vinyl chloride. The mechanism for TCI hepatotoxicity
is not definitely established, but many investigators have proposed that the
observed centrilobular cellular necrosis results from the microsomal mixed-
function oxidase system formation of chemically "reactive" metabolites which
react with and bind to tissue macromolecules and cause intracel1ular damage
(Pessayre et al., 1979; Allemand et al., 1978; Reynolds and Moslen, 1977).
Figure 4-6 indicates that these reactive metabolites may include trichloroethylene
epoxide, dichloroacetyl chloride and formyl chloride (Section 4.4.4). Chloral,
chloral hydrate and trichloroethanol, as ordinarily used hypnotics, are not
associated with significant hepatotoxicity (Goodman and Gilman, 1975).
Presently, there is little information about the degree of binding of "reac-
tive" metabolites to macromolecules that results in a toxic liver response; nor
have the critical target binding sites been identified. However, numerous factors,
such as species, age, sex, and diet, all of which modify TCI microsomal metabolism,
also markedly influence the toxic response. Drugs such as phenobarbital (Carlson,
1974) and ethanol (Cornish and Adefuin, 1966) greatly influence TCI metabolism
and hepatotoxicity. Cornish et al. (1977) found that TCI-induced liver toxicity
004TC5/B 4-40 11-5-83
-------
in rats, as measured by plasma SCOT, is potentiated by both ethanol and pheno-
barbital. Ethanol acted by competitive substrate inhibition, and also by stimu-
lation of microsomal mixed-function oxidase system; phenobarbital potentiated
toxicity by induction of mixed function oxidase system and thereby increased TCI
metabolism. Allemand et al. (1978) observed a similar potentiation of toxicity
in rats, as measured by plasma SGPT. Plasma SGPT increase correlated with the
amount of binding of 14OTCI reactive metabolites to liver microsomal protein.
Reduced glutathione had a protection effect against TCI liver toxicity. TCI
Acutely administered to rats initially decreased hepatic glutathione by 61 per-
cent, but increased glutathione levels (106 percent) 16 h later (Moslen et al.,
1977b). Glutathione added to microsomal preparations decreased binding of reac-
tive1 products of 14C-TCI, suggesting a glutathione conjugation of metabolites.
Pessayr^ et al. (1979) found that single or acute TCI administration to rats de-
creased cytochrome P450 of phenobarbital-treated rats, but not untreated animals,
and increased covalent binding of 4C-TCI metabolites to microsomal protein.
Chronic administration to normal rats simultaneously decreased Parn cytochrome
14 4bu
content and increased C-TCI metabolite binding, but also induced other micro-
somal enzyme systems. Since acute TCI exposure rarely produces functional liver
impairment in man (Smith, 1966), whereas chronic exposure does (Litt and Cohen,
1969; Baerg and Kimberg, 1970), Pessayre and co-workers (1979) suggest that the
explanation may lie in the ability of TCI to enhance its own metabolism and its
own toxicity.
Poplawski-Tabarelli and Uehleke (1982) have investigated other mechanisms
that contribute to the toxic manifestations of halogenated aliphatic hydrocarbons,
including TCI. These investigators attempted a correlation of the inhibition by
these compounds of various microsomal oxidative reactions. Of the several possible
factors influencing these mono-oxygenase activities, such as interaction with
lipid environment of microsomal cytochromes; ligand formation with reduced P450
cytochrome; covalent interactions of metabolic intermediates with cytochrome P450;
destruction of cytochromes by lipid peroxidation; and production and interaction
of CO with cytochromes, the overriding correlation appeared with the simple
physicochemical factors of high lipid solubility and vapor pressure. The
ability of chlorinated ethylene to inhibit mitochondrial respiration and hence
ATP production as a mechanism of cellular toxicity has also been investigated.
Takano and Miyazaki (1982) found that all of a series of 14 chlorinated ethanes
004TC5/B 4-41 11-5-83
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and ethylenes, including TCI, inhibited oxygen consumption of isolated rat
mitochondria; their inhibitory potency increased in the order of the number of
contained chlorines.
004TC5/B 4-42 11-5-83
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5. TOXICOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS
5.1 INTRODUCTION
Trichloroethylene (TCI) has been an important industrial chemical since
its commercial application in 1906. It has been the focus of wide research
interest and the subject of numerous case reports. The available literature
concerning the potential effects of TCI is reviewed in this chapter.
5.2 EFFECTS ON THE CENTRAL NERVOUS SYSTEM
5.2.1 Human Studies: Acute Inhalation Exposure
The first extensive study of the effects of TCI on the nervous system was
that of Stuber in 1931. Stuber reviewed a total of 283 cases of presumable
overexposure to TCI, including 26 fatalities. All cases had occurred in
European industrial operations. Stuber reported that the action of TCI
primarily involved the central nervous system (CNS), although some effects
were also observed in the gastrointestinal and circulatory systems. Adverse
effects on the kidney were rare, and no cases were reported of injury to the
liver. Among the CNS symptoms most often described were headache, dizziness,
vertigo, tremors, nausea, vomiting, sleepiness, fatigue, a feeling and appear-
ance of lightheadedness or "drunkenness" increasing to unconsciousness, and,
in some cases, to death. In addition to the general narcotic effect, specific
paralysis of the trigeminal nerve was also reported (Plessner, 1915; Persson,
1934). This observation led to the early use of TCI in the treatment of
trigeminal neuralgia.
Numerous case histories have subsequently been reported on the effects of
acute exposure to high doses of TCI (Longley and Jones, 1963; Masoero and
Lavarino, 1955; Vallee and Leclercq, 1935). The earlier studies reported on
the toxicity of TCI following anesthesia, in particular the occurrence of
trigeminal palsies (Humphrey and McClelland, 1944). Boulton and Sweet (1960)
suggested that trigeminal palsies that occurred in 24 patients resulted from
inhalation of dichloroacetylene or phosgene during closed-circuit TCI
anesthesia. These compounds are formed when exhaled TCI is passed through
soda lime to remove carbon dioxide. The formation of these impurities and
their subsequent toxicity have resulted in a decline in the use of TCI as an
anesthetic.
Kleinfeld and Tabershaw (1954) reported on five fatal cases of TCI
poisoning among industrial workers following acute overexposure. This was the
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first compilation of TCI-related fatalities. Four of the workmen, who had been
employed in a degreasing operation, continued to work despite complaints of
nausea, drowsiness, dizziness, and vomiting. All died while at work or within
a few hours after leaving work. Death was attributed to ventricular fibrilla-
tion.
St. Hill (1966) reported the case of a man who died following acute
exposure to TCI while boiling out tanks in a ship's hold after an accidental
leakage. The man was exposed for about 20 h, during which time he complained
of headache, dizziness, double vision, and paralysis of the face and neck
muscles. Two other men who had worked with the victim suffered similar
but less severe symptoms. Similar symptoms were reported by Tomasini and
Sartorelli (1971) in a patient, following chronic and acute overexposure to TCI
during operation of a dry cleaning unit. The symptoms included symmetrical
bilateral VHIth cranial nerve deafness as well as cerebral cortical
dysrhythmia and alterations in the electroencephalogram (EEC). This patient
recovered after exposure was stopped.
Loss of special senses has also been reported following TCI exposure.
Mitchell and Parsons-Smith (1969) reported the case of a worker who lost his
sense of taste after one month's exposure to high concentrations of TCI. He
subsequently developed trigeminal analgesia and cortical changes reflected in
alterations in the EEC.
Maloof (1949) reported blurring of vision and diplopia in a worker the
day after he had been exposed to hot TCI vapors. In addition to symptoms
characteristic of visual effects and effects on the CNS, the worker received
first- and second-degree burns to the skin and first-degree chemical burns to
the eyes.
Steinberg (1981) associated acute exposure to high levels of TCI with per-
manent CNS damage in a 62-year-old man. Damage persisted six years following
exposure.
Lawrence and Partyka (1981) reported an acute overexposure to TCI which
resulted in chronic involvement of the bulbar cranial nerves and esophageal
and pharyngeal motility impairment.
Many other such case histories of acute TCI exposure have been reported
with similar symptomatologies. Visual disturbances, mental confusion,
fatigue, nausea, and vomiting have all been observed. The dangers of acute
exposure to TCI may be accentuated by visual disturbances and by loss of
004TC1/F 5-2 11/8/83
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coordination. This may lead to poor manual dexterity and unsafe mechanical
operations (Patty, 1958).
Trichloroethylene has also been reported to have some abuse potential.
Cases of deliberate and repetitive inhalation have been reported by James (1963)
and by Ikeda and Imamura (1973). Abuse of TCI has been associated with hepato-
renal toxicity (Clearfield, 1970). Respiratory and cardiac failure is believed
to be the cause of death after acute inhalation exposure.
5.2.2 Human Studies: Chronic Exposure
Occupational exposure reports generally do not provide important informa-
tion that is needed to relate effects to exposure levels. Often the degree of
exposure is not well defined. Other factors often missing or given limited
coverage in such reports are (1) contamination with other noxious substances,
(2) existing pathological conditions in those exposed.
Chronic exposure to levels of TCI greatly in excess of those found or
expected in ambient air results primarily in neurological and neuropsychiatric
symptoms consisting of tremors, giddiness, increased lacrimation, reddening of
skin, decreased sensitivity of the hands, alcohol intolerance, neuroanesthetic
syndrome with anxiety states, bradycardia, and insomnia (Bardodej and Vyskocil,
1956). Other manifestations are not as well defined. The first published report
of chronic occupational overexposure to TCI in the United States summarized ten
individual cases occurring prior to 1944 (McNally, 1937; Quadland, 1944). The
symptoms in all the cases were characteristic of CNS disturbances. No quanti-
tative estimation of exposure levels was provided.
Kunz and Isenschmid (1935) reported the case of a worker who used TCI to
remove diamond powder from the rolls of a jewelry roll. It was estimated that
100 to 300 ml of the solvent evaporated directly in front of his face daily.
Blindness was preceded by increasing changes in color perception and vision.
Loss of tactile sense, decreased mobility, and the inability to grasp
objects between the thumb and fingers was noted in six women working with TCI
to remove wax remaining on optical lenses (Albahary et al., 1959).
5.2.3 Experimental Human Studies
5.2.3.1 Controlled Studies—Psychomotor function and subjective responses
to TCI have been studied in short-term, controlled, human clinical studies to
determine the effects of TCI on the CNS. Kylin et al. (1967) measured opto-
kinetic nystagmus in 12 human subjects after exposure to 1000 ppm (5380 mg/m3)
TCI for 2 h. Although a significant decrease in the fusion limit was noted,
004TC1/F 5-3 11/8/83
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this test was found not to be a sensitive indicator of the effects of TCI.
Stopps and Mclaughlin (1967) exposed one subject to four different concentra-
tions of TCI (100, 200, 300, and 500 ppm; 538, 1076, 1714, and 2690 mg/m3).
Two experiments were run at each level , with concentrations presented in ascend-
ing and descending order. Each experiment was 3 h in duration. They found no
significant effect on psychomotor performance at 100 ppm (538 mg/m3), but they
found a progressive decrease in performance as the TCI concentration was in-
creased. This study, however, was limited by the use of only one subject
and by the limited exposure-period used in the experiment, which is not com-
parable to an 8-h work day. In a larger study involving six male students
exposed to two 4-h exposures in one day at an average concentration of 110 ppm
(592 mg/m3), Salvini et al. (1971) demonstrated a statistically significant
decrease in performance ability for tests of perception, manual dexterity,
reaction time, and Wechsler memory scale.
The effects of TCI on visual motor performance, as determined by psycho-
physiological tests, were studied in 12 subjects by Vernon and Ferguson (1969).
They found a significant decrease in performance only at the highest concen-
tration tested (1000 ppm; 5380 mg/m3) after a 2-h exposure.
Stewart et al. (1970) experimentally exposed ten subjects to 100 and
200 ppm (1076 mg/m3) TCI for periods ranging from 1 h to a 5-day work week.
They reported mild fatigue and sleepiness in normal adults after 4 to 5 days'
exposure to 200 ppm (1076 mg/m3) TCI for 7 h per day. Nomiyama and Nomiyama
(1977) reported headaches in healthy male students exposed to about 100 ppm
(538 mg/m3) TCI for 4 h per day for 6 days. No effects were observed on a
flicker fusion or two-point discrimination test even at 200 ppm (1076 mg/m3).
In a pilot study to determine the effect of TCI exposure on mental capa-
city, Ettema et al. (1975) found no evident or consistent effect on task per-
formance or on physiological parameters. Fourteen adult males were exposed for
2.5 h to 0(n = 11), 75(n =11) or 300(n = 11) ppm. The tests used were described
by the authors as requiring active cooperation and exertion from the subjects;
it was cautioned that the results could not be extrapolated to vigilance situa-
tions.
The short duration of many of these exposures decreases the value of
these studies in assessing the longer-term effects of TCI on nervous system
function. They are useful primarily in measuring the threshold level for
general CNS-depressant activities of TCI.
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5.2.3.2 Epidemiological Studies—Several epidemiological studies have been
conducted in an attempt to correlate environmental concentrations and health
effects. Grandjean (1963) found that 73 workers, exposed for 5 to 15 years to
TCI concentrations of 1 to 333 ppm (5.38 to 1791 mg/m3), exhibited alterations
in autonomic nervous system function that were related to the length of time
they were exposed to TCI; these changes were also accompanied by a high frequency
of subjective symptoms. Bardodej and Vyskocil (1956) reported insomnia,
tremors, and severe neuroasthenia syndromes coupled with anxiety states and
bradycardia in 75 workers divided into four groups according to number of
years they were exposed to TCI (5 to 629 ppm; 27 to 3384 mg/m3). The groups
were exposed less than 1 year, 1 to 2 years, 2 to 9 years, and over 10 years.
They found that the symptoms increased in frequency with both longer exposures
and higher concentrations of TCI. Disturbances of the nervous system were
reported to continue for up to 1 year after final exposure. As Messite (1974)
reported, these studies did not include control groups and were based primarily
on clinical impressions, rather than statistical analyses.
In a detailed study of 104 workers exposed to TCI in metal, rubber, and
dry cleaning industries, Andersson (1957) reported that two-thirds of the
workers displayed CMS symptoms similar to those described for acute toxicity.
Follow-up studies of the workers for 3 to 7 years after exposure showed little
residual evidence of TCI intoxication. Although the average exposure level in
this study was 200 to 400 ppm (1076 to 2152 mg/m3), it should be noted that
the levels of exposure were subject to many unmeasurable fluctuations.
Lilis et al. (1969) reported a study of 70 young workers (83 percent less
than 30 years old) exposed for up to 6 years to variable concentrations of TCI.
The subjects showed symptoms of CNS and cardiac disturbances. Although the
authors estimated the TCI concentrations to be about 10 ppm (54 mg/m3), a
direct relationship between the incidence and severity of symptoms and the
duration of exposure was not established.
Konietzko et al. (1978) reported changes in EEGs that correlated with
subjective toxic effects reported by workers exposed to open basins of TCI.
Studies such as these are somewhat limited and thus provide only suggestive
information. Due to lack of background data and the potential interaction of
other chemicals, it is difficult to attribute the observed effects directly to
TCI exposure.
004TC1/F 5-5 11/8/83
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Szulc-Kuberska et al. (1976) reported occupational exposure of TCI workers
resulted in impairments of the auditory and vestibular nerves. Forty workers
were examined, ranging in age from 25 to 50. Exposure levels were not reported
nor was the test group compared to a control population. Because the indivi-
duals were reported to have had at least 40 mg TCI/1 urine, it can be concluded
that exposure levels were exceedingly high.
5.2.4 Animal Studies
5.2.4.1 Nervous System and Behavioi—There have been few in-depth studies of
the effects of TCI on the nervous system and behavior. Studies apparently have
not been performed on such behaviors as learning or extinction, accuracy or
efficiency of complex schedule-controlled behaviors, or sensory discriminations.
Most endpoints that have been evaluated were studied in only a preliminary manner.
Nervous system electrophysiology, neurochemistry, and neuropathology are areas
that warrant more attention.
Taylor (1936) found no significant serious adverse effects in rats exposed
to 500, 1000, and 2000 ppm (2690, 5380, and 10,760 mg/m3) TCI for 6 h/day, 5 days/
week, for 6 months. At 3000 ppm (16,140 mg/m3) only two of 6 mice survived.
Dogs exposed to 2000 ppm (10,760 mg/m3) TCI and guinea pigs exposed over 1100 h
to 1200 ppm (6456 mg/m3) TCI showed no adverse effects.
Baker (1958) investigated the histological changes in the CNS associated
with acute and chronic exposure of dogs to TCI. The study was designed to
produce graded exposures to TCI experimentally. Although no neuropathological
changes could be detected after a single acute exposure to 30,000 ppm
(161,400 mg/m3) TCI, repeated exposure led to brain changes at scattered loci.
Chronic exposure of dogs (2 to 8 h/day, 5 days/week) to 300 to 500 ppm (1614 to
2690 mg/m3) TCI resulted in selective destruction of the cerebellar Purkinje
cells. Similar findings were reported by Bartonicek and Brun (1970) in rabbits
chronically injected intramuscularly with a total of 53 g of TCI. The most pro-
nounced changes appeared to occur in the Purkinje cells.
Tham et al. (1979) studied the effect of TCI on the vestibular system in
rabbits by recording positional or rotational nystagmus. They found that at
blood levels greater than 30 mg/1 (following continuous i.v. infusion of 1
to 5 mg/kg/min), TCI, unlike chloral hydrate and trichloroethanol, induced
positional nystagmus. Their results suggested that TCI elicited vestibular
disturbances by stimulating the central subcortical vestibule-oculomotor
connection.
004TC1/F 5-6 11/8/83
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During the development of a potential animal model for solvent abuse,
Utesch et al. (1981) found that high levels of TCI, from 9000 to 16,000 ppm
(48,420 to 86,080 mg/m3), adversely affected CNS functioning in Wistar-Munich
rats but produced no signs of kidney or liver damage. Performance measures
used were the loss of the righting reflex and performance on a tilting plane.
Inhalation of 9000 ppm (48,420 mg/m3) for up to 15 min caused only slight
difficulty in locomotion. All of a group of 6 rats lost their righting
reflex after approximately 5 min at 14,000 ppm (75,320 mg/m3). Characteristic
signs at 14,000 ppm (75,320 mg/m3) included irritation of mucous membranes of
the eyes and respiratory tract, hyperpnea, and progressive ataxia. Performance
on the tilting plane was used to evaluate recovery. All animals appeared to be
fully recovered within 15 min after cessation of exposure to 15,000 ppm
(80,700 mg/m3). Groups of 5 or 6 rats subjected to 15,000 ppm (80,700 mg/m3)
for 5-min periods, with 15-min solvent-free intervals between subsequent expo-
sures (total = 1-5 h), did not show any elevations in SCOT or SGPT nor were
there any adverse histopathological findings. Influence of ethanol on effects
of repetitive TCI inhalation is described in Section 5.7.
Behavioral techniques have also been used to study the effect of TCI and
its metabolites in animals. Grandjean (1960) studied the effects of TCI on
food-motivated conditioned responses in three rats. Exposure was via inhala-
tion of 200 and 800 ppm (1076 and 4304 mg/m3) TCI for 3 h. He was unable to
detect obvious behavioral changes related to TCI exposure, although there
was some evidence of increased excitability or loss of inhibition. Battig and
Grandjean (1963) chronically exposed rats to 400 ppm (2152 mg/m3) TCI for
10 months (8 h/day, 5 days/ week). Although exposure to TCI did not adversely
affect their general condition, their swimming speed was reduced. They also
found a significant increase in exploratory behavior, which confirmed their
earlier studies (Grandjean, 1960). In a later study, Grandjean (1963) exposed
rats for 6 h to 400, 800, and 1600 ppm (2152, 4304, and 8608 mg/m3) TCI. Al-
though only slight decrements in swimming behavior were observed after 400 and
800 ppm (2152 and 4304 mg/m3), 1600 ppm (8608 mg/m3) produced significant de-
creases. This was also accompanied by evidence of fatigue at the higher dose.
Goldberg et al. (1964a) trained four rats in a conditioned avoidance
escape test (pole climbing). Four-hour exposures, 5 days/week for 2 weeks to
200, 500, and 1500 ppm (1076, 2690, and 8070 mg/m3) TCI did not result in
altered responses. Learning appeared to be stimulated by 200 ppm (1076 mg/m3)
004TC1/F 5-7 11/8/83
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exposure, and higher doses (4380 ppm; 23,564 mg/m3) produced a specific
inhibition of the avoidance response with no accompanying sign of motor
imbalance. The responses did not increase or decrease directly with dose
level. In a subsequent study, Goldberg et al. (1964b) evaluated further the
effects of TCI on behavioral responses in the CFE rat. Discrete avoidance
studies were performed in a 10-compartment rat-shock box. Ten rats had been
trained to 95-percent or higher efficiency in avoiding shock. Once trained, the
rats retained a stable baseline for many months. The shock-box was placed in the
exposure chamber. Each vapor study was composed of three phases: pre-exposure
control, vapor exposure, and post-exposure control. Rats stayed in the chamber
4 h a day, 5 days/week, for weeks. Exposure concentration was 128 ± 11 ppm
(689 ± 59 mg/m3). During 25 exposure days, a significant increase in the
number of shocks taken by the group was observed on 22 days. No significant
effect was observed during the first day of each of the first 3 weeks of
study. Effects were reversible during the recovery phase. Silverman and
Williams (1975), using observational methods, showed that repeated exposure (6
to 7 h/day, 5 days/week, 4 weeks) of rats to 100 ppm (538 mg/m3) TCI produced
slight behavior changes in these animals.
The neurotoxicological effects of TCI on rodents have been demonstrated
by Haglid et al. (1981). Biochemical changes and ultrastructural evidence of
neuronal cell alterations were found in various areas of the brain of adult
Mongolian gerbils exposed continuously to 60 or 320 ppm (323 or 1722 mg/m3)
for 3 months, followed by a period of 4 months free of exposure. The most
pronounced changes were in the hippocampus, the posterior part of the cerebellar
vermis, and in the brain stem. Biochemical findings indicated a glia cell
reactivity in some brain areas, which the investigations believed was caused by
a primary effect of TCI or its metabolites or secondary to damage to neuronal
cells. In affected areas, total soluble proteins were decreased, while the
S-100 fraction, a glial cytoplasmic protein, was significantly elevated, as
measured per soluble protein. In sensory-motor cortex and hippocampus,
elevations of S-100 protein per soluble protein after exposure were larger
following inhalation of 60 ppm than after 320. The investigators concluded
that observed alterations could serve as early and sensitive measurements of
toxic effects of organic solvents on the CNS. The observations on glial brain-
specific protein in discrete brain areas confirmed previous work by these in-
vestigators (Haglid et al., 1980). Previously, it had been demonstrated that
004TC1/F 5-8 U/8/83
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soluble proteins in various areas of the brain of gerbils exposed to TCI for
8 weeks (320 ppm; 1722 mg/m3) were altered differently. The authors cautioned
that the biochemical changes could reflect adaptation.
Exposure of NMRI mice to 170 ppm (915 mg/m3) TCI continuously for 30 days
did not affect brain levels of acetylcholinesterase, glutamine synthetase,
acid phosphatase and glutamate dehydrogenase activity nor cholinesterase
activity in blood. These observations suggested to the authors that nerve
cell populations and glial cells were unaffected by TCI (Kanje et al., 1981).
In contrast, plasma butyrlcholinesterase increased two-fold in exposed male
mice but was not increased in females. Liver weight in males and females
increased by factors of 1.5 and 1.9, respectively.
Kjellstrand et al. (1982) tested the possibility that acid phosphatase
could be a sensitive detector of brain damage by exposing rats, mice, and
gerbils continuously to 150 ppm (807 mg/m3) TCI for 30 days. The enzyme assay
system had a limit of detection of ± 10 to 15 percent. Acid phosphatase levels
were measured in five different areas of the brain. Small differences in
total enzyme activity and activity per mg brain tissue were seen between the
TCI and air-exposed groups in the brain stem of mice (p <0.05) and gerbils
(p <0.01). It was concluded that the alterations of acid phosphatase observed
were not prominent enough to consider acid phosphatase as a very sensitive
measure of brain damage.
5.3 EFFECTS ON THE CARDIOVASCULAR AND RESPIRATORY SYSTEMS
5.3.1 Cardiovascular Effects
The use of TCI as an anesthetic has been associated with cardiac
arrhythmias, including bradycardia, atrial and ventricular premature con-
tractions, and ventricular extrasystoles. The dose-response relationships for
these effects, however, has not been established in man or experimental animals.
High concentrations of TCI have been reported to sensitize the myocardium to
circulatory catecholamines, resulting in cardiac arrhythmias including ventri-
cular fibrillation or even cardiac arrest (Dhuner et al. , 1951, 1957; Reinhardt
et al., 1973). Metabolites of TCI, such as chloral hydrate, may also be asso-
ciated with arrhythmias involving adrenergic stimulation (DiGiovanni, 1969).
Lilis et al. (1969) observed in their study that 60 percent of the workers
exposed to inhaled TCI exhibited an increase in cardiac output, an effect
attributed to increases of epinephrine release and circulating plasma levels.
These direct cardiac effects of TCI, with sensitization to catecholamines,
004TC1/F . 5-9 11/8/83
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have been observed after accidental ingestion and have, in some cases, proved
fatal (Defalque, 1961).
Several case histories have been reported involving fatalities following
acute exposure to large concentrations of TCI. The deaths in these cases were
attributed to either cardiac arrest (James, 1963) or ventricular fibrillation
(Kleinfield and Tabershaw, 1954). Bell (1951), Bardodej and Vyskocil (1956),
Ogata et al. (1971), Andersson (1957), and others have noted that exposure to
TCI may either speed up or slow down the heart rate, depending on the degree of
exposure. The most direct evidence that TCI causes ventricular extrasystoles,
fibrillation, and cardiac arrest comes from changes that are observed in the
electrocardiograms (EKG) of subjects accidentally exposed to high concentrations
of TCI (Kledecki and Bura, 1963; Mroczek and Fedyk, 1971; Yacoub et al., 1973).
Bernstine (1954) recorded ventricular fibrillation confirmed by EKG in a worker
who sustained cardiac arrest after inhalation of TCI in analgesic concentrations.
Barnes and Ives (1944) found that 33 of 40 normal patients in deep anesthetic
planes showed signs of ventricular extrasystoles and multifocal ventricular
tachycardia.
Few systematic animal studies have been done on the effects of TCI on the
cardiovascular system. Mazza and Brancaccio (1967) studied the effects on
rabbits of chronic exposures to 2790 ppm (15,010 mg/m3) TCI, 4 h/day, 6 days/week,
for 45 days. They found that severe blood dyscrasia was produced in these ani-
mals. The characteristics of the hemochromocytometric tests and the description
of the bone marrow led the authors to conclude that chronic intoxication with
TCI has a direct effect on the bone marrow, which causes myelotoxic anemia.
Mikiskova and Mikiska (1966) studied electrophysiological responses of guinea
pigs injected intraperitoneally either with 6.7 mM/kg TCI or with 2.2 mM/kg
trichloroethanol. They recorded EKG's and EEG's and found that trichloroethanol
was at least three times as effective as TCI in inducing effects on the nervous
system and in slowing the heart, and suggested that TCI effects were due, in
part, to its metabolite, trichloroethanol.
Caffeine has been shown to potentiate the arrhythmogenicity of TCI in
rabbits upon challenge with exogenously-administered epinephrine (White and
Carlson, 1982). Rabbits were treated with a vehicle control (1 mg/kg, i.p.)
or caffeine (10 mg/kg, i.p. 30 min prior to exposure) and exposed for 1 h to
6000 ppm (32,280 mg/m3) TCI under dynamic airflow conditions. Epinephrine was
infused until arrhythmias occurred after 7.5, 15, 30, 45 and 60 min of exposure
and 15 and 30 min post-exposure. The authors reported a pronounced increase in
004TC1/F 5-10 11/8/83
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the incidence of epinephrine-induced arrhythmias in TCI-exposed rabbits, when
treated with caffeine and challenged with doses of epinephrine as low as
0.5 ug/kg.
White and Carlson (1981) also investigated the effect of ethanol on epi-
nephrine-induced cardiac arrhythmias in rabbits exposed to 6000 ppm (32,280 mg/m3)
TCI. Rabbits were treated with saline (1 ml/kg) or ethanol (1 g/kg, i.v. or p.o.)
30 min prior to exposure to 6000 ppm TCI for 1 h. Epinephrine was infused as
described above. Rabbits treated with ethanol developed epinephrine-induced
cardiac arrhythmias sooner and at lower doses of'epinephrine than control rab-
bits. More arrhythmias developed in rabbits treated with ethanol i.v. than p.o.
Ethanol administration decreased the conversion of TCI to trichloroethanol by
50 percent, and trichloroacetic acid production was completely blocked by ethanol
treatment.
Fossa et al. (1982) demonstrated that disulfiram exhibits a cardioprotec-
tive activity not related to its effects on TCI metabolism in rabbits. Disul-
firam (1.35 mmoles/kg, p.o.) was administered 24 and 6 h prior to a 1-h exposure
to 6000 ppm (32,280 mg/m3) TCI. Disulfiram or its metabolites, C$2 and diethyl-
dethiocarbamate (1.35 mmoles/kg, i.p.), were administered at similar time inter-
vals also to rabbits exposed for 1 h to 9000 ppm (48,420 mg/m3) TCI. Blood
levels of TCI and its metabolites, trichloroethanol and trichloroacetic acid,
were measured at periods ranging from 7.5 min of exposure to 30 min post-exposure.
The metabolism of TCI was inhibited in all treatment groups. When challenged
with 0.5 to 3.0 ug/kg epinephrine, disulfiram prevented the epinephrine-induced
arrhythmias resulting from exposure to 6000 ppm (32,280 mg/m3) TCI. All treat-
ment groups in the first 30 min of exposure to 9000 ppm (48,420 mg/m3) TCI
exhibited an increased percentage of animals responding with arrhythmias compared
to controls. Thirty to sixty minutes of exposure resulted in a significant
decrease in the percentage of animals responding with arrhythmias in the disul-
firam- and diethyldithiocarbamate-treated groups.
5.3.2 Respiratory Effects
In anesthetic concentrations, TCI causes little or no irritation to the
respiratory tract (Dobkin and Byles, 1963). A striking effect is observed,
however, on the rate and depth of respiration. TCI characteristically causes
increased respiratory rate (tachypnea) but decreased alveolar ventilatory
amplitude, which is associated with decreased blood oxygen tension and increased
carbon dioxide tension (Dobkin and Byles, 1963). These effects were noted in
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early animal experiments (Lehmann, 1911) and in early reports on human patients
under TCI anesthesia (Striker et al. , 1935). The incidence of tachypnea
increases as the concentration of TCI increases (Atkinson, 1960).
To investigate the mechanism responsible for the increase in respiratory
rate, Whitteridge and Bulbring (1944) studied the effects of TCI on pulmonary
afferent nerve endings in cats. They found that 0.5 to 3.5 percent (5,000 to
35,000 ppm; 26,900 to 188,300 mg/m3) TCI produced a dose-dependent increase in
the frequency of vagal discharge and an increased sensitivity of the stretch
endings. Coleridge et al. (1968) observed similar results in isolated pulmonary
receptor endings.
Stewart et al. (1970) attempted to correlate injury to rat pulmonary
alveolar parenchyma with biochemical alterations following repeated inhalation
of TCI. Mild changes in morphology were observed, including condensed mito-
chondria and vacuolation of the cells. Pulmonary surfactant secretion was
significantly decreased. The authors suggested that modification of sur-
factant secretion was a consequence of nonspecific injury following exposure
of a susceptible cell population to TCI.
5.4 HEPATIC AND RENAL TOXICITY
5.4.1 Human Studies
The similarity between TCI and chloroform and other related halocarbons
known to cause liver damage suggests that TCI might possess hepatotoxic activity.
Stuber (1931) reviewed 284 cases of industrial poisoning due to TCI and found
no evidence of liver damage. Brittain (1948) found no evidence of liver or
kidney damage in 250 neurosurgery patients who underwent prolonged TCI anes-
thesia. However, fatal acute hepatic failure has been observed following the
use of TCI as an anesthetic. Such failure has generally been in patients with
other complications such as malnutrition, toxemias and burns, or patients who
had received transfusions (Ayre, 1945; Berleb, 1953; Dodds, 1945; El am, 1949;
Heizer, 1951; and Werch et al., 1955).
Secchi et al. (1968) reported acute liver disease in three of seven cases
of poisoning following ingestion of TCI. They suggested, however, that the
results could be attributed to contamination with 1,2-dichloropropane and
1,2-dichloroethane since no liver damage was associated with ingestion of pure
TCI. Cotter (1960) described 10 patients acutely exposed to TCI in a confined
space. Four patients showed hyperglobulinemia and six exhibited hypercal-
cemia. No other liver tests were abnormal and, in all but one person, the
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clinical tests returned to normal within two months of exposure. Lachnit and
Brichta (1958) reported that of 22 workers occupationally exposed to TCI, only
three had abnormal reactions on two liver function tests (sulfobromophthalein
clearance tests and colloid stability). Albahary et al. (1959) conducted
liver function tests—measuring serum glutamic oxaloacetic transaminase (SCOT)
and serum glutamic pyruvic transaminase (SGPT)—on workers regularly exposed
to TCI. No evidence of liver disorders was found. Guyotjeannin and Van
Steenkiste (1958) found some abnormalities in cephalic flocculation, total
lipids, and plasma unsaturated fatty acids, and an increase of plasma gamma
globulins in 18 workers regularly exposed to TCI. Tolot et al. (1964) found
no evidence of liver injury, even though workers were routinely exposed to TCI
at concentrations higher than the allowable tolerance limits, that is, 0.9 to
3 mg of TCI per liter of air (167 to 558 ppm). Joron et al. (1955) reported
massive liver necrosis (particularly the left lobe) followed by death of a
chemist exposed intermittently to TCI over a 1.5-year period. Subsequent ana-
lyses of room air indicated TCI concentrations as high as 300 ppm (1614 mg/m3).
From the above reports, it is apparent that observations of liver dys-
function in workers exposed to TCI have been infrequent, and relatively little
surveillance has been conducted to ascertain latent effects. Thus, it is
unlikely that the potential magnitude of any kidney or liver abnormalities in
humans exposed for long periods to TCI is fully known. Cases of severe liver
and kidney damage resulting from ingestion of liquid TCI may provide some
evidence for latent toxicity. However, it is difficult to predict whether the
same effects would be observed if ingestion of TCI was spread over a period of
time similar to that found in inhalation exposure.
As mentioned earlier, TCI has also been reported to have some abuse
potential. Cases of deliberate and repetitive inhalation have been reported
by James (1963) and by Ikeda and Imamura (1973). Baerg and Kimberg (1970)
documented centrilobular hepatic necrosis and acute hepatic and renal damage
following repeated sniffing of cleaning fluid containing TCI and petroleum
distillates by teenagers. However, as the vapor concentration was high and
inhalation was only for a short period, the effect cannot be directly extra-
polated to inhalation of the same total dose over a long period of time, as in
occupational exposure.
Thiele et al. (1982) reported that acute overexposure to TCI, as well as
methyl chloroform, resulted in a diagnosis of hepatic cirrhosis in a 37-year-old
male industrial worker.
004TC1/F 5-13 11/8/83
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5.4.2 Animal Studies
Early experiments showed some degree of liver damage. It should be noted
that TCI test mixtures used, especially prior to 1960, likely contained contami-
nants that may have contributed to the effects observed. With two groups of dogs
exposed to 750 ppm (4035 mg/m3) TCI 8 h/day, 6 days/week, for 3 weeks, and 500 to
600 ppm (2690 to 3228 mg/m3) TCI 6 h/day, 5 days/week, for 8 weeks, Seifter
(1944) found glycogen depletion and hydropic parenchymatous liver degeneration.
Wirtschafter and Cronyn (1964) injected rats with TCI subcutaneously (0.5 g/kg);
they found elevated SCOT levels within 12 to 16 h, which were correlated with
hepatocellular damage observed in histological sections. This suggested a mild
degenerative change of liver parenchyma. Gehring (1968) observed no significant
increase in SGPT following a single exposure of mice to 5500 ppm (29,590 mg/m3).
Nowill et al. (1954) exposed one dog, three rabbits, and three rats to 0.05 to
0.1 percent (500-1000 ppm; 2690 to 5380 mg/m3) TCI for 18 h/day for 90 days.
No gross abnormality was observed in liver, renal, or blood functions. Utesch
et al. (1981) exposed rats intermittently to 15,000 ppm (80,700 mg/m3) TCI in
a manner simulating human solvent abuse. No evidence of liver or kidney damage
was observed. Pendergast et al. (1967) reported negative observations following
continuous exposure of rats, guinea pigs, dogs, rabbits, and monkeys to TCI.
Ikeda et al. (1969) injected rats every 2 days for 37 days (0.5 ml/kg and
1.5 ml/kg). No significant changes were found in liver or blood lipids.
Liver failure in experimental animals is associated with binding of TCI
metabolites to protein and nucleic acids in the liver (Bolt and Filser, 1977).
Binding of TCI metabolites and TCI hepatotoxicity are increased by compounds
that induce the liver mixed-function oxidase system and by treatment with
inhibitors of epoxide hydrase. Adams et al. (1951) and Moslen et al. (1977)
have found that TCI inhalation does not produce acute liver necrosis unless
animals are pretreated with phenobarbital, especially when fasting. In addi-
tion, in phenobarbital-treated rats, hepatic glutathione contents decreased
during TCI exposure, rebounding afterwards (Moslen et al. , 1977). Despite the
reports of hepatotoxicity in experimental animals exposed to high doses and in
humans exposed to anesthetic doses of TCI, liver damage similar to that
observed in human industrial exposure is rare (Bardodej and Vyskocil, 1956)
and difficult to produce in experimental animals with acute subanesthetic
doses (Kyi in et al., 1963).
004TC1/F 5-14 11/8/83
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Danni et al. (1981) also reported that TCI exposure of male Wistar rats
did not result in any signs of hepatic injury. Trichloroethylene had been
given orally to 5 animals, at a dose of 0.65 mmoles/100 g body weight and to 3
animals at 2.58 mmoles/100 g body weight. Relative liver weight and hepatic
triglyceride content were used as parameters in measuring liver injury. Injury
was observed only when the rats were first pretreated with phenobarbital.
In a laboratory screening study for relative hepatotoxicity in mice, Jones
et al. (1958) reported that the oral administration of TCI in olive oil (0.36 to
4.3 mg/g body weight) to 200 Swiss mice, mixed sexes, in groups of 10, resulted
in at least minimal liver injury. The minimal hepatotoxic dose reported was
0.72 mg/g. The principal change noted was fatty infiltration. The minimal
lethal dose reported was 2.92 mg/g. Histologic examination of the liver was
made 72 h after exposure.
Ramadan and Ramadan (1969) reported that 12 albino rats exposed to
anesthetic levels of TCI for 2 h did not show signs of parenchyma! liver
damage. The distribution of acid phosphatase positive granules in rat hepatic
parenchyma was, however, shown to be irregular and their number diminished,
24 h after exposure.
Dow Chemical (Stott et al. , 1981) has recently performed gavage studies
which include dosages similar to those used in the National Cancer Institute
(NCI) (1976) bioassay for carcinogenicity of TCI by gavage in B6C3F1 mice and
Osborne-Mendel rats (see Chapter 7). Male B6C3F1 mice were gavaged with 0,
250, 500, 1200, or 2400 mg TCI/kg body weight/day, 5 days a week, for 3 weeks.
Male Osborne-Mendel rats, treated similarly, received 0 or 1100 mg TCI/kg body
weight/day. Dose-related changes characteristic of hepatocellular hypertrophy
were observed at all dosage levels in mice, and ranged from slight histological
changes at the lowest dose to increased liver weights at 500 mg/kg, to in-
creased centrilobular hepatocellular swelling at 1200 mg/kg, to severe centri-
lobular hepatocyte swelling, giant cell inflammation, and mineralized cells at
the highest dose. No effect on the kidneys or on body weights was observed in
either mice or rats. Rats treated with 1100 mg TCI/kg body weight had slight-
ly increased liver weights but no histopathological changes in the livers.
The authors suggest that the greater sensitivity of B6C3F1 mice to the hepato-
toxic effects of TCI reflects their greater capacity for metabolic activation
of TCI (Section 4.6). From the results of their macromolecular binding
experiments, including DNA alkylation studies (Section 4.6), and from these
004TC1/F 5-15 11/8/83
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toxicity data, Stott and coworkers (1981) concluded that TCI induction of
hepatic tumors in B6C3F1 mice may occur via an epigenetic mechanism.
Renal failure has been an uncommon problem with TCI anesthesia (Defalque,
1961). Although depressed kidney function has been observed in experimental
animals exposed to TCI, it requires very high doses (Klassen and Plaa, 1967);
on a relative basis, TCI is much less potent than chloroform or carbon tetra-
chloride.
The acute and subchronic toxicology of TCI in weanling CD-I and ICR out-
bred albino mice has been recently investigated by Tucker et al. (1982). The
oral LD50 in female mice was found to fall in the 95 percent confidence range
of 1839 to 3779 mg/kg, compared to 2065 to 2771 mg/kg for males. In a 14-day
gavage study, the two doses selected (24 and 240 mg/kg) approximated 1/10
and 1/100 of the LD50. The only effect noted in the 14-day study was an
apparently dose-related increase in liver weight. TCI was dissolved in a
10-percent Emulphor solution. In a 5-day study to determine the effect of TCI
on hepatic microsomal activities, a group of 11 mice was given TCI according
to the following regimen: 0.73 g/kg twice on day 0; 1.46 g/kg twice on day
1, 2.91 g/kg twice on day 3, and 1.46 g/kg on days 4 and 5. Mice were sacri-
ficed on day 6. Activities found to be significantly (p <0.05) elevated in-
cluded aniline hydroxylase. Aminopyrine N-demethylase was significantly
decreased.
In the 4- and 6-month subchronic studies, 140 mice of each sex were
administered TCI in drinking water at the following levels: 0.1, 1.0, 2.5 and
5 mg/ml in 1-percent Emulphor. Weight gain was lower for each sex, compared
with vehicle group, but was not statistically significant. Decreased water con-
sumption was evident, with females at the highest dose level and with males at
the two highest doses, compared with vehicle controls.
Results of gross pathological examinations performed on mice killed at 4
and 6 months were unremarkable. A significant increase in kidney weight
occurred at the highest dose in males at 6 months and in females at both 4 and
6 months. Urinalysis indicated elevated protein and ketone levels in high-dose
females and the two highest-dose males after 6 months of exposure. Hematology
findings included a decreased erythrocyte count in the high-dose males at 4 and 6
months (13 and 16 percent, respectively); decreased leukocyte counts, particu-
larly in females at 4 months; and altered coagulation values consisting of in-
creased fibrinogen in males at both times and shortened prothrombin time in
004TC1/F 5-16 11/8/83
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females at 6 months. The observed results suggested to the authors that at
doses lower than the LD50, TCI is not a potent hepatotoxin in the mouse.
5.5 IMMUNOLOGY
The immunological status of male and female CD-I mice exposed to TCI in
drinking water for 4 and 6 months was investigated by Sanders et al. (1982).
Details of exposure and discussion of toxicological effects noted were
reported separately by Tucker et al. (1982) (see Section 5.4). In the 14-day
range-finding study, male CD-I mice had been dosed by p.o. gavage daily with
24 or 240 mg/kg TCI. Although the humoral immune response to sheep RBC was not
significantly different from control, the cell-mediated response was inhibited
in a dose-related manner. To determine the immune response over longer
periods of time, both male and female mice were exposed to TCI (0.1, 1.0, 2.5,
and 5 mg/ml) in drinking water for 4 and 6 months. Parameters evaluated
included humoral immunity, cell-mediated immunity, lymphocyte responsiveness,
bone marrow function, and macrophage function. It was observed that male mice
were relatively unaffected after both 4 and 6 months of exposure compared to
results in the 14-day study. On the other hand, females were affected, par-
ticularly in the 4-month exposure. Humoral immunity was inhibited only at the
highest TCI concentrations (2.5 and 5 mg/ml), whereas cell-mediated immunity
and bone marrow stem cell colonization were inhibited at all four concentra-
tions of TCI. The proliferation capacity of the lymphocyte was not affected
when challenged with T-cell mitogens.
5.6 DERMAL EFFECTS
Industrial use of TCI is often associated with dermatological problems
(Bauer and Rabens, 1974). These include reddening and dermatographic skin burns
on contact (Maioof, 1949), generalized dermatitis resulting from contact with
the vapor (McBirney, 1954), and possible scleroderma (Reinl, 1957). However,
irritations, burns, and rashes are usually the result of direct skin contact
with concentrated solvent and are, therefore, probably limited to effects
secondary to the solvent. Stewart and Dodd (1964), in a study of controlled
skin exposure (thumbs), showed that unless TCI is trapped against the skin,
absorption is insignificant. Absorption of TCI varies with age, skin thick-
ness and texture, as well as type of contact. No effects have been reported
with exposure to TCI in dilute aqueous solutions.
004TC1/F 5-17 11/8/83
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5.7 MODIFICATION OF TOXICITY: SYNERGISM AND ANTAGONISM
The manifestation of adverse effects of TCI appears to depend, in part,
on its metabolic products. Therefore, drugs and chemicals that enhance or
inhibit the metabolism of TCI can have a corresponding effect on the toxicity
of TCI. Reynolds (1976) and Moslen et al. (1977) have reported that the
hepatotoxic potential of TCI can be greatly enhanced by prior treatment with
drugs or chemicals that induce components of the liver mixed-function oxidase
(MFO) system. Carlson (1974) demonstrated that pretreatment of male albino
rats with phenobarbital (50 mg/kg i.p.) or 3-methylcholanthrene (40 mg/kg)
exacerbated TCI-induced liver damage. There was massive necrosis and a three-fold
increase in SCOT and SGPT levels. Groups of animals were exposed to 6900 ppm
(37,122 mg/m3) for 2 h and 10,400 ppm (55,952 mg/m3) for 2 h. This was con-
firmed in a later study of Moslen et al. (1977), who found that TCI induced
morphological changes in the liver of animals treated with only phenobarbital.
Reynolds (1976) also found acute liver injury following a 2-h exposure to
1 percent TCI in animals pretreated with other inducers of MFO, including chlo-
rinated biphenyls (Arochlor 1245). These findings suggest that a metabolite(s)
of TCI contributes to the toxicity of the compound. In this regard, Mikiskova
and Mikiska (1966) have found that trichloroethanol, a metabolite of TCI, was
three to five times more effective in inducing changes in EKG and EEG.
Masuda and Nakayama (1982) found that TCI produced moderate increases in
plasma glutamine pyruvate transminase activity in male SPF mice given an i.p.
dose of 2 ml/kg (dissolved in olive oil). Diethyldithiocarbamate trihydrate
and CS2, when given to the mice orally, 30 min before TCI, exerted a protective
effect, in that the increase in plasma glutamine-pyruvate transaminase was
lessened. Diethyldithiocarbamate was effective at each of the three dose levels
used (10, 30, and 100 mg/kg). In controls given only TCI, no marked pathological
alterations were reported upon histological examinations. CS2, at levels of
3, 10, and 30 mg/kg, acted similarly to diethyldithiocarbamate in suppressing
the TCI-associated increase in plasma glutamate-pyruvate transaminase. The
authors concluded that the protective mechanisms of diethyldithiocarbamate and
CS2 may involve their interference with metabolic activation of a variety of
hepatotoxins.
Novakova et al. (1981) reported that an open-ended i_n vitro rat liver
perfusion system could be used to demonstrate the effect of TCI and other
halogenated solvents on liver enzyme activities. TCI in such a system was
reported to increase the activities of aspartate aminotransferase, alamine
aminotransferase, alkaline phosphatase, and ornithine carbamoyltransferase.
004TC1/F 5-18 }l/8/83
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Pessayre et al. (1982) have obtained evidence that TCI is an effective
potentiator of hepatotoxic effects of CC14 i_n vivo in rats. While liver his-
tology was normal 24 hours after i.p. administration of TCI (1 ml/kg) and also
after administration of 64 pi/kg CC14, both solvents cbadministered produced
extensive centrilobular necrosis. TCI given in vitro 5 h prior to CC14
increased CC!4-induced lipid peroxidation. Similarly, coadministration marked-
ly increased serum alamine ami no transferase and decreased hepatic cytochrome
P450 concentrations. Coadministration also decreased hepatic glutathione
levels. It was concluded that the toxicologic effects produced by the con-
comitant administration of TCI and a small dose of CC14 resembled those
produced by a high dose of CC14, a finding consistent with the view that TCI
potentiated the hepatotoxicity of CC14. It was further concluded that this
potentiation may be the result of increased CC14-induced rate of lipid peroxi-
dation. Since stable metabolites of TCI failed to stimulate such peroxida-
tion, it was apparently mediated by TCI itself.
The effect of ethanol and other substances upon the manifestation of
effects of subsequent TCI exposure in animals has been studied by a number of
investigators.
One of the earliest studies was that of Cornish and Adefuin (1966). Male
Sprague-Dawley rats were orally pretreated with ethanol (50 percent in water) in
a single dose, on the basis of 5 g/kg body weight. Sixteen to eighteen hours
after pretreatment, each group (6 rats) was exposed to TCI in a dynamic exposure
chamber. Exposure levels were 100, 2000, 5000, and 10,000 ppm (538, 10,760,
26,900, and 53,800 mg/m3) TCI (3 rats in the highest exposure category died).
Animals ingesting ethanol prior to exposure to TCI showed a statistically sig-
nificant (p<0.05) increase in serum SCOT, SGPT, and serum isocitric dehydrogenase
(SICD) at 5000 ppm (26,900 mg/m3). After exposure to 5000 ppm (26,900 mg/m3) for
4 h, SGOT of animals pretreated with ethanol was about 20 times the control level,
whereas exposed rats not receiving ethanol had normal SGOT values. Animals
exposed to 2000 ppm (10,760 mg/m3) or less for 4 h showed no marked alteration of
serum enzyme levels. Neither ethanol-treated nor nontreated rats showed any
alterations of serum enzyme levels when exposed for 8 h to 100 ppm. All rats
exposed to 5000 ppm (26,900 mg/m3) TCI showed slight to widespread degenerative
lipid infiltration of the liver. Ethanol-treated rats (before TCI exposure)
exhibited early centrilobular necrosis with slight acute inflammatory cellular
reaction.
004TC1/F 5-19 11/8/83
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Muller et al. (1975) proposed that TCI and ethanol undergo similar metabolic
steps, thus allowing for a variety of interactions, depending on dose levels
and the sequence of exposure. When simultaneous exposure occurs, TCI and
ethanol may compete for both alcohol dehydrogenase and microsomal mixed-function
oxidase. Intolerance of TCI-exposed workers to alcohol has been described by
Defalque (1961). It is often manifested as red flushes on the face and upper
parts of the body and has been referred to as "degreasers flush." Stewart et
al. (1974) observed this effect in seven individuals given as little as 0.5 ml
ethanol/kg during exposure to 20,100, and 200 ppm (108, 538, 1076 mg/m3) TCI
for 1, 3, or 7.5 h. Alcohol intolerance among TCI-exposed workers has also been
described by Bardodej et al. (1952) and Bardodej and Vyskocil (1956).
Traiger and Plaa (1974) reported that isopropyl alcohol or acetone, admin-
istered to mice in an 18-h pretreatment, potentiated the hepatotoxic response to
2 doses of TCI, 11.1 mmole/kg and 16.7 mmole/kg.
Cornish et al. (1977) found that rats given i.p. doses of 0.5, 1.0, and
2.0 ml TCI/kg showed progressively increasing levels of SCOT. Pretreatment with
either phenobarbital or ethanol or both had no effect on this response. This
lack of response with ethanol is in contrast to the earlier study by Cornish and
Adefuin (1966) but may reflect different routes of exposure.
Utesch et al. (1981), in developing their animal model for solvent abuse
(see 5.4.2), found that pretreatment of Wistar-Munich rats with ethanol exacer-
bated the CNS-depressant effects of high doses of TCI. A group of rats was
given 5 ml/kg ethanol orally 1 h before initiation of the TCI inhalation regimen:
15 exposures cycles in a 5-h period; each cycle consisted of 5 min of inhalation
of 15,000 ppm TCI followed by a 15-min solvent-free interval. Ethanol was shown
to cause significant prolongation of the animals' ability to regain the righting
reflex after the second and each subsequent TCI exposure. Administration of
ethanol alone did not result in an increase in levels of SCOT or SGPT in
histopathologic alterations of the liver or kidneys. No elevations in SGPT were
seen in any of the groups that received both alcohol and TCI. Histopathologic
examination of the liver and kidneys revealed no evidence of chemically-induced
injury in any animals. Fasting for 16 h before the 5-h intermittent TCI exposure
regimen had no apparent effect on the combined CNS-depressant action of ethanol
and TCI. No histopathological effects were observed in the livers or kidneys of
these animals.
004TC1/F 5-20 11/8/83
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Moslen et al. (1977) also reported that fasting had little effect on SCOT
and SGPT levels in rats which inhaled 10,000 ppm TCI continuously for 2 h. How-
ever, pretreatment of rats with phenobarbital in combination with fasting
produced significant elevations in the levels of these serum enzymes.
Cornish et al. (1973) reported that phenobarbital pretreatment produced
no additional effect on SCOT above that produced by 1.0 and 2.0 ml TCI/kg,
given i.p. to Sprague-Dawley rats. Lower doses of TCI, 0.5 and 0.3 ml/kg, had
no effect on SCOT.
Pretreatment of male albino rats with either phenobarbital or 3-methyl
cholanthrene was shown by Carlson (1974) to potentiate the hepatotoxicity of
TCI. Rats were injected with either 50 mg phenobarbital/kg i.p. for 4 days or
3-methyl cholanthrene in corn oil, 40 mg/kg i.p. for 2 days. Animals were
exposed to TCI 24 h after the last dose of phenobarbital or 48 h after the last
dose of 3-methyl cholanthrene in a dynamic inhalation chamber. Rats were
sacrificed 22 h after exposure. Phenobarbital pretreatment increased SGOT and
SGPT three-fold above TCI controls. TCI exposure was 10,400 ppm (55,952 mg/m3)
for 2 h. Similarly, 3-methyl cholanthrene increased SGOT and SGPT; 3-methyl
cholanthrene pretreatment also increased the level of SICD about twenty-fold,
compared to TCI controls.
5.8 SUMMARY OF TOXICOLOGICAL EFFECTS
There is no reliable information concerning the toxicological effects in
humans of chronic exposure to levels of TCI below the TLV (100 ppm). Based
upon acute human overexposure information and limited animal testing, it is
unlikely that chronic exposure to TCI at levels found or expected in ambient
air would result in liver or kidney damage. Such damage has not been generally
found even when exposure greatly exceeds the TLV.
The first sign likely to be observed upon exposure to TCI is central ner-
vous system (CNS) dysfunction. In limited, acute controlled human exposure
situations, alterations in task performance have been reported only at levels
in excess of 100 ppm. There have been few in-depth studies, in rodent species,
of the effects of TCI on the nervous system and behavior. Most endpoints that
have been evaluated were studied in only a preliminary manner.
004TC1/F 5-21 11/8/83
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6. TERATOGENICITY, EMBRYOTOXIC1TY, AND REPRODUCTIVE EFFECTS
Because of its widespread use, TCI has been studied for teratogenic
potential. Teratology studies have been performed in rats, mice, and rabbits
using doses of TCI which, in some studies, produced slight signs of maternal
toxicity. Also, TCI is known to be metabolized in the maternal host by hepatic
metabolizing enzymes (and possibly also in the fetal liver) to chloral hydrate
and then to trichloroethanol and trichloroacetic acid. These metabolites,
particularly trichloroethanol, have also been shown to readily cross the human
placenta into the fetal circulation and amniotic fluid (Bernstine and Meyer,
1953; Bernstine et al., 1954) and also into the breast milk of nursing mothers
(Bernstine et al. , 1956). There is little information available on the feto-
toxic and teratogenic potential of these metabolites. Chloral hydrate and its
metabolite, trichloroethanol, have been in common medical use for a great many
years as hypnotics, including use during pregnancy, but with no reported
adverse teratogenic, fetotoxic, or reproductive effects (Goodman and Gilman,
1980). Trichloroethanol has been administered to three animal species at
various stages of pregnancy, at levels as high as 700 mg/kg/day, without
dose-related effects (Physicians Desk Reference, 1981). Other studies in
avian embryo systems (Fink, 1968; Elovaara et al., 1979) have indicated that
TCI disrupts embryogenesis in a dose-related manner. However, because admini-
stration of TCI directly into the air space of chicken embryo is not comparable
to administration of dose to animals with a placenta, it is not possible to
interpret this result in relationship to the potential of TCI to cause adverse
effects in animals or humans. The following discussion of studies subscribes
to the basic viewpoints and definitions of the terms "teratogenic" and "feto-
toxic" as summarized and stated by the U.S. Environmental Protection Agency
(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, reductions 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 continuous, and all deviations from the normal must be considered
as examples of developmental toxicity. Gross morphological terata represent
004TC3/H 6-1 11-8-83
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but one aspect of this spectrum, and while the significance of such structural
changes is more readily evaluated, such effects are not necessarily more
serious than certain effects which are ordinarily classified as fetotoxic--
fetal death being the most obvious example.
In view of the spectrum of effects at issue, the Agency suggests that it
might be useful to consider developmental toxicity in terms of three basic
subcategories. The first subcategory would be embryo or fetal lethality.
This is, of course, an irreversible effect and may occur with or without the
occurrence of gross terata. The second subcategory would be teratogenesis and
would encompass those changes (structural and/or functional) which are induced
prenatally, and which are irreversible. Teratogenesis includes structural
defects apparent in the fetus, functional deficits which may become apparent
only after birth, and any other long-term effects (such as carcinogenicity)
which are attributable to jm utero exposure. The third category would be
embryo or fetal toxicity as comprised of those effects which are potentially
reversible. This subcategory would therefore include such effects as weight
reductions, reduction in the degree of skeletal ossification, and delays in
organ maturation.
Two major problems with a definitional scheme of this nature must be
pointed out, however. The first is that the reversibility of any phenomenon is
extremely difficult to prove. An organ such as the kidney, for example, may
be delayed in development and then appear to "catch up." Unless a series of
specific kidney function tests are performed on the neonate, however, no
conclusion may be drawn concerning permanent organ function changes. This
same uncertainty as to possible long-lasting aftereffects from developmental
deviations is true for all examples of fetotoxicity. The second problem is
that the reversible nature of an embryonic/ fetal effect in one species might,
under a given agent, react in another species in a more serious and irrever-
sible manner.
6.1 ANIMAL STUDIES
6.1.1 Mouse
Schwetz and his associates (Schwetz et al., 1975; Leong et al., 1975)
investigated the potential of TCI to adversely affect fetal development in
Swiss-Webster mice (Table 6-1). Twelve mice were exposed by inhalation to TCI
(99.24-percent pure) at an air concentration of 300 ppm (1614 mg/m3) for 7 h
daily on days 6 through 15 of gestation. Day 0 of gestation was designated for
the appearance of a vaginal plug. Concurrent pregnant control mice (30) were
exposed to filtered air. The mice were observed daily throughout pregnancy,
and maternal body weights were recorded on days 6, 10, 16 and 18 of gestation
as an index of maternal toxicity. Following caesarean section on day 18, the
fetuses were weighed, measured, (crown-rump length), sexed, and then examined
for external anomalies. One-half of the fetuses were examined for soft tissue
malformation, using free-hand sectioning, and one-half of the fetuses were
004TC3/H 6-2 11-8-83
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TABLE 6-1. SUMMARY OF ANIMAL STUDIES OF FETOTOXIC AND TERATOGENIC POTENTIAL OF TCI
Fink (1968)
Elovaara
(1979)
Schwetz et al.
(1975)
Bell (1977)
Dorfmueller
et al. (1979)
Beliles et al.
(1980)
TCI
purity
Unknown
Reagent grade
Technical
99.24 % TCI
0.76 % stabi-
lizers and
impurities
(Neu-tri)
Technical
(Trichlor 132)
Technical
99 %+ TCI
0.2 % epichloro-
hydrin
(Neu-tri)
Technical
99.9 %
Conditions
(mode of administration, dosage,
Species and duration of exposure)
Chick embryo Vapor exposure, 10,000 ppm
Chick embryo Injected; 5 - 100 umole per
egg in 25 ul olive oil
S - D Rat 9 Inhalation; 300 ppm,
7h/d on 6 - 15-d gestation
S - W mouse <*
CR - SD rat 9 Inhalation; 300 ppm,
6h/d on 6 - 15-d gestation
Rat 9 Inhalation, 1800 ppm
(Long-Evans) a) Premating: 6h/d, 5d/wk
for 2 wk
b) Premating for 2 wk + first
20-d gestation, daily
c) First 20-d gestation, daily
CR - SD Rat 9 Inhalation; 500 ppm, 7h/d,
Rabbit 9 5d/wk
Premating 3 wk + first 18-d
Measures Results
Mortality Increase
anomalies Slight increase
LD50 50 - 100 umole/egg
(16 % malformations in
total survivors)
Embryo toxicity, +
teratogenicity
(maternal toxicity) (+)
Embryo toxicity, +
teratogenicity
(maternal toxicity) (+)
Embryo toxicity, +
teratogenicity, offspring
behavioral evaluation
(maternal toxicity) (+)
Embryo toxicity, +
teratogenicity
(maternal toxicity) (+)
gestation, daily (rat)
+ first 21-d gestation, daily
(rabbit)
-------
cleaned, stained, and examined for skeletal malformations. One fetus in each
litter was subjected to microscopic examination.
Exposure to TCI had no effect on either the mean, absolute, or relative
weight of the liver of mice dams when measured at the time of caesarean section.
Exposure had no effect on the average number of implantation sites per litter,
litter size, the incidence of fetal resorptions, fetal sex ratios, or fetal
body measurements. The incidence of gross anomalies observed by external
examination was not significantly greater than among control litters. TCI
exposure had no effect on the incidence of skeletal anomalies, and microscopic
examination revealed no abnormalities of organs, tissues, or cells. These
investigators concluded that 300 ppm (1614 mg/m3) TCI caused no significant
maternal, embryonal or fetal toxicity, and that TCI was not teratogenic in
Swiss-Webster mice at this dose level.
6.1.2 Rat
Schwetz et al. (1975) carried out a study in Sprague-Dawley rats. The
design of the study was similar to their protocol for mice described above.
TCI (300 ppm; 1614 mg/m3) was administered by inhalation, 7 h daily, to 18
pregnant rats during organogenesis, the period when the greatest susceptibility
to teratogenic effect is present (days 6 to 15 of gestation). Controls (30)
were provided filtered air. Day 0 of gestation was defined as the day spermatozoa
were observed in vaginal smears. The animals were killed on day 21 of gestation,
caesarean sections were performed, and fetuses in each litter were examined for
external malformations. One-half of the fetuses in each litter were then exa-
mined for soft tissue malformations, and the other half examined for skeletal
malformations. One fetus from each litter was randomly selected for serial
sectioning and histological evaluation. Schwetz et al. (1975) reported a
small but statistically significant inhibition of maternal body weight gain
when dams were exposed to 300 ppm (1614 mg/m3) TCI. However, this dose had no
effect on either the mean, absolute, or relative weight of the maternal host
livers when measured at the time of caesarean section. Exposure to TCI had no
effect on the average number of implantation sites per litter, litter size,
the incidence of fetal resorptions, fetal sex ratios, or fetal body measure-
ments. No increased incidence of soft tissue or skeletal anomalies was
observed, and histopathological examination revealed no abnormalities of
organs, tissues, or cells. Schwetz et al. (1975) concluded that 300 ppm
(1614 mg/m3) TCI caused no significant maternal, embryonal or fetal toxicity
in Sprague-Dawley rats.
004TC3/H 6-4 11-8-83
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Bell (1977) reported a similar study, conducted by Industrial Bio-Test
Lab. Inc. (Northbrook, 111.), on the teratogenic and fetal toxic effects of
TCI on Charles River rats. TCI (300 ppm; 1614 mg/m3) was administered to 17
dams by inhalation exposure, 6 h per day, from day 6 through day 15 of gestation.
Control pregnant animals (21) were killed on day 20 of gestation. Caesarean sec-
tions were performed, fetal swellings and implantation sites counted, and resorp-
tion sites and number of corpora lutea determined. Viable fetuses were counted,
and all fetuses were removed and weighed. External examination of the fetuses
was made for gross anomalies. Two-thirds of the fetuses from each litter were
examined for skeletal development, and the remaining one-third of the fetuses
were evaluated for soft tissue abnormalities. The investigators found that
300 ppm (1614 mg/m3) TCI exposure produced no evidence of maternal toxicity,
although a small but significant reduction of mean weight gain by gestation
day 15, in comparison to control dams, was recorded. There were no mortalities
or adverse behavioral reactions, gross fetal anomalies, or differences in mean
weights of fetuses exposed to TCI in comparison to unexposed controls. The
numbers of corpora lutea, implantation sites, resorption sites, and fetuses were
similar among exposed and control dams. No evidence for teratogenicity was
observed in fetuses from either exposed or unexposed dams.
Beliles et al. (1980) reported on a study of teratogenic and fetotoxic
potential of TCI in Sprague-Dawley rats conducted by Litton Bionetics, Inc.
(Kensington, MD.). The experimental design of the study included pregestational
as well as gestational exposure (Table 6-2).
TABLE 6-2. EXPERIMENTAL DESIGN OF BELILES et al. STUDY (1980)
ON SPRAGUE-DAWLEY RATS
Days of Exposure
Group
1
2
3
4
5
6
No.
21
16
20
16
20
32
TCI
ppm
0 (control)
0 (control)
500
500
500
500
(Controls exposed
Pre- gestational
None
(5d/wk) 3 wk
None
(5d/wk) 3 wk
None
(5d/wk) 3 wk
to air only)
Gestational
0-18
0-18
0-18
0-18
6-18
6-18
004TC3/H
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11-8-83
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Exposure to TCI, 500 ppm (2690 mg/m3) in air, was 7 h/d, 5 d/wk, during a
3-wk pre-gestational period, and 7 h/d each day for days 0 to 18 and days 6 to
18 of gestation. Positive identification of spermatozoa in the vaginal canal
was taken as evidence of mating and designated as day 0 of gestation. The dams
were killed on day 20 of gestation, caesarean section was performed, visceral
and thoracic organs were examined, and the numbers corpora lutea per dam deter-
mined. The maternal livers, kidneys and lungs were weighed. The numbers of
implantation sites, live and dead fetuses, and resorption sites were recorded.
The fetuses were examined for gross external anomalies and weighed, and crown-
rump length and sex were determined. One-half of the fetuses of each litter
were sectioned and examined for changes in the soft tissues; the remaining
fetuses were examined for skeletal abnormalities.
The findings reported in this study indicate that maternal toxicity was
not a factor in data interpretation; means of body weight, liver, kidneys, and
lungs of test dams were within normal expected variation of control dams. No
deaths occurred in the exposed groups. The investigators concluded that
neither the frequency nor character of the macro- or microscopic findings in
the treated groups indicated an adverse effect on fetal growth and development,
or a teratogenic potential for TCI at the dose level of 500 ppm.
Dorfmueller et al. (1979) conducted a study of the teratogenic and fetotoxic
effects of TCI in Long-Evans rats exposed to a inhalation concentration of
1800 ppm (9684 mg/m3) in air. Their experimental design was similar to that
of Beliles et al. (1980) insofar as the animals were exposed during the pre-
gestational period in addition to during gestation (Table 6-3).
The premating exposure was conducted for 6 h/d, 5 d/wk for 2 wk, and the
gestational exposure was 6 h/d each day for days 0 to 20. Day 1 of pregnancy
was considered to be the day on which sperm from mating were observed in vaginal
smears from mating. On day 21 of gestation, 15 out of 30 darns/treatment group
were sacrificed; maternal livers were removed, weighed, and frozen for later
enzyme analysis. Caesarean sections were performed and the number of live,
dead, and resorbed fetuses counted. Each fetus was weighed, the sex was record-
ed, and then examined for external anomalies. Four fetuses from each litter
were examined for soft tissue changes, and four fetuses were evaluated for ske-
letal abnormalities. Livers were removed from the remaining fetuses for measure-
ment of mixed-function oxidase activity.
004TC3/H 6-6 11-8-83
-------
TABLE 6-3. EXPERIMENTAL DESIGN OF DORFMUELLER et al. STUDY (1979)
ON LONG-EVANS RATS
Group
A
B
C
D
No.
30
30
30
30
TCI
(ppm)
1800 ± 200
1800 ± 200
1800 ± 200
none
Days of
Pre-gestational
5d/wk; 2 wk
5d/ wk; 2 wk
none
none
Exposure
Gestational
1-20
none
1-20
none
Postnatal behavioral evaluation was conducted on the offspring of the
remaining 15 dams/treatment group which were allowed to litter. At weaning, 2
male and 2 female pups from each litter were randomly selected for observation
of activity measurements at 10 days of age (Motility Meter) and 20 and 100
days of age (Activity Cage).
Dorfmueller et al. (1979) found that the female rats exposed to 1800 ppm
(9684 mg/m3) TCI before mating and/or during pregnancy did not exhibit any
signs of maternal toxicity. Weight gains throughout pregnancy were normal in
relation to filtered air controls, and the relative and absolute maternal
liver weights indicated that no significant treatment effects occurred. There
was no indication of embryotoxicity; no significant treatment effects or inter-
actions were found in numbers of corpora 1utea or implantation sites/ litter,
fetal body weights, resorbed fetuses/litter, or sex ratios. No significant
treatment effects were observed in the analysis of total soft tissue anomalies.
However, the incidence of total skeletal anomalies was increased in group C
because of an increased incidence of incomplete ossification of the sternum,
but when the frequency of this particular anomaly alone was compared in group C
versus group D, the investigators found no significant difference. Incomplete
ossification of the sternum is thought to be indicative of delays in the
general process of skeletal ossification which are not necessarily irreversible.
Thus, prenatal exposure to 1800 ppm (9684 mg/m3) TCI caused an elevation of
minor anomalies indicative of development delays in maturation, but not of
major malformations. Also, the study indicated no treatment effect on the
general activity of the offspring at 10, 20, and 100 days of age. There was,
004TC3/H
6-7
11-8-83
-------
however, a small but statistically significant depression in postnatal weight
gains of offspring in the premating exposure groups (A and B), the biological
significance of which was not apparent. The offspring are being maintained
until 18 months of age to further assess postnatal behavior and transplacental
carcinogenicity potential. This study revealed no results indicative of treat-
ment-related maternal toxicity, embryotoxicity, teratogenicity, or significant
postnatal behavioral deficits from exposure, pre-gestational and/or gestational,
to 1800 ppm (9684 mg/m3) TCI in air.
6.1.3 Rabbit
Beliles et al (1980) also studied the fetotoxicity and teratogenicity of
TCI in the female New Zealand white rabbit. Their experimental design is
presented in Table 6-4.
TABLE 6-4. EXPERIMENTAL DESIGN OF BELILES et al. STUDY (1980)
ON NEW ZEALAND WHITE RABBITS
Group
1
2
3
4
5
6
Animals/
group
21 0
18 0
23
19
23
25
TCI
ppm
(control )
(control )
500
500
500
500
Days of Exposure
Pre-gestational Gestational
None
5d/wk; 3 wk
None
5d/wk; 3 wk
None
5d/wk; 3 wk
0-21
0-21
0-21
0-21
7-21
7-21
Exposure to TCI, 500 ppm (2690 mg/m3) in air, was 7 h/d, 5 d/wk during a
3-wk pre-gestational period, and/or daily for days 0 to 21 or days 7 to 21 of
gestation. Observed copulation and/or the presence of sperm in the vaginal
canal established day 0 of gestation. On day 30 of gestation, the dams were
killed, caesarean-sectioned, and the corpora 1utea per dam determined. The
maternal livers, kidneys, and lungs were removed, weighed, and examined; the
remaining visceral and thoracic organs were also examined. The number of
uterine implantation sites, live and dead fetuses, and resorption sites were
recorded. The fetuses were examined for gross external anomalies and weighed,
and then crown-rump length and sex ratios were determined. One-half of the
fetuses of each litter were sectioned and examined for changes in the soft
tissues; the remaining fetuses were examined for skeletal abnormalities.
004TC3/H
6-8
11-8-83
-------
The number of deaths in the TCI-treated groups was not significantly greater
than among controls. Deaths occurring during the experiment account for the un-
equal numbers of animals in the groups. The mean body weights during pre-
gestational and gestational periods were determined, and there were no remarkable
differences between control and treated rabbits.
This study provided no evidence of maternal toxicity from 500 ppm
(2690 mg/m3) exposure to TCI. Means of body weight, liver, and lungs were within
normal expected variation of control dams. A small increase in means of kidney
weights for groups 2, 4, and 6 (compared to group 1) was found, but these
increases were judged to be unrelated to treatment. No difference was observed
in the mean weight of fetuses exposed to TCI in comparison to those of unexposed
controls. The numbers of corpora lutea, implantation sites, resorption sites,
fetuses and sex ratio were similar among exposed and control dams.
Gross examination of fetuses, with regard to external morphology and
internal abnormalities, revealed no unusual changes except for hydrocephalus in
4 fetuses of 2 litters of group 3. This anomaly occurred only in the one treat-
ment group 3 and the malformation was not observed in the litters of rabbits
that were exposed to 500 ppm (2690 mg/m3) TCI during a 3-week pre-gestational
period plus the same gestational period (Group 4). The authors confirmed the
anomaly as an external hydrocephalus, which, in their experience, does not
occur in untreated control rabbits. They, therefore, felt that, although not
statistically significant, this finding could not be discounted entirely as
occurring by chance. Unless other factors can be identified which are respon-
sible for increasing the susceptibility of the rabbits during the gestational
period, it would be expected that the malformation would occur in animals ex-
posed to the same dose of TCI for a longer period.
Examination of the fetuses for soft tissue anomalies showed no changes
(except external hydrocephalus discussed above) that were not frequently observed
in 30-day-old rabbit fetuses of the strain and source used in the study. Ske-
letal examination revealed no unusual features, except for an increased incidence
of changes related to retarded bone ossification, which were not considered mal-
formations as such. Neither frequency nor the character of these changes in the
treated groups indicated an adverse effect on fetal growth and development or of
a teratogenic potential. In summary, this study in rabbits exposed to 500 ppm
(2690 mg/m3) TCI revealed no evidence of maternal toxicity or embryotoxicity; the
004TC3/H 6-9 11-8-83
-------
authors did report the occurrence of hydrocephalus in a few fetuses of one of
the study groups, but no definitive conclusion was made regarding this occur-
rence.
6.2 FETOTOXICITY IN HUMANS
There are no epidemiologic studies in the literature specifically investi-
gating fetotoxicity or overt birth anomalies that may occur from TCI exposure in
the workplace.
Corbett and his associates (1972; 1974) reported an increased incidence
of miscarriages in operating room nurses exposed to various general anesthetics
(volatile halogenated aliphatic hydrocarbons). TCI was only one of several anes-
thetics in use, and, therefore, it is impossible to make a specific association
of TCI with the miscarriages. In addition, the study and its methodology have
been severely criticized by Cote (1975), particularly with respect to clinical
definition of anomalies and to methods of clinical evaluation.
Aviado et al. (1976), in his review, concluded that TCI exposure may produce
respiratory difficulties in neonates. However, respiratory effects on neonates
have not been established, and TCI has been in use for many years in Europe as a
self-administered inhalant analgesic and anesthetic during labor without
reported effects.
6.3 SUMMARY
Wei 1-designed inhalation exposure studies in the mouse (300 ppm), and rat
(300,500 and 1800 ppm; 1614, 2690, and 9684 mg/m3) by different investigators
demonstrate no significant maternal toxicity, embryo toxicity, teratogenicity,
or postnatal behavioral effects. In rabbits, 500 ppm (2690 mg/m3) inhalation
exposure provides no evidence of maternal toxicity or embryo toxicity, but an
assessment of teratogenic potential was made based on only a few hydrocephalic
fetuses observed in one group (of four) exposed to TCI.
At present, the available studies do not indicate that TCI is toxic to
the fetus at levels below the maternally toxic level. Also, no definitive
clinical evidence of fetotoxicity or teratogenicity from TCI exposure has been
reported. Therefore, the available information does not indicate that the con-
ceptus is uniquely susceptible to the effects of TCI. It should be noted,
however, that additional studies of appropriate rodent species are needed to
more fully examine the teratogenic potential of T,CI.
004TC3/H 6-10 11-8-83
-------
6.4 REFERENCES
Aviado, D., S. Zakhari, J. Simaar, and A. Ulsamer. Methyl Chloroform and TCE in
the Environment. CRC Press, Cleveland, OH, 1976.
Beliles, R. P., D. J. Brusick, and F. J. Mecler. Teratogenic-Mutagenic Risk of
Workplace Contaminants: Trichloroethylene, Perchloroethylene and Carbon
Disulfide. U.S. Department of Health, Education and Welfare (Contract No.
210-77-0047), 1980.
Bell, Z. Written Communication with contractor reports of: (a) Dominant Lethal
Study with Trichloroethylene in Albino Rats Exposed via Inhalation, February
9, 1977, and (b) Teratogenic Study via Inhalation with Trichlor 132,.
Trichloroethylene in Albino Rats, March 8, 1977.
Bernstine, J. B. and A. E. Meyer. Passage of metabolites of chloral hydrate
into amniotic fluid. Proc. Soc. Exp. Biol. Med. 84:456-457, 1953.
Bernstine, J. B., A. E. Meyer and R. L. Bernstine. Maternal blood and breast
milk estimation following the administration of chloral hydrate during
the puerperium. J. Obst. Gyn. 63:228-231, 1956.
Bernstine, J. B. , A. E. Meyer and H. B. Hayman. Maternal and foetal blood
estimation following the administration of chloral hydrate during labour.
J. Obst. Gyn. 61:683-685, 1954.
Corbett, T. H. Anesthetics as a cause of abortion. Fert. Steril. 23:866-869,
1972.
Corbett, T. H., R. G. Cornell, J. L. Endres, and K. Leiding. Birth defects among
children of nurse anesthetists. Anesthesiology 41.: 341-344, 1974.
Corbett, T. , G. Hamilton, M. Yoon, and J. Endres. Occupational exposure of
operating room personnel to trichloroethylene. Can. Anesth. Soc. J. 20:657-678,
1973.
Cote, C. J. Birth defects among infants of nurse anesthetists. Anesthesiology
42:514-515, 1975.
Dorfmueller, M. A., S. P. Henne, R. G. York, R. L. Bornschein, and J. M. Manson.
Evaluation of teratogenicity and behavioral toxicity with inhalation exposure
of maternal rats to trichloroethylene. Toxicol. 14:153-166, 1979.
Elovaara, E., K. Hemminki, and H. Vainio. Effects of methylene chloride, trichloro-
ethane, trichloroethylene, tetrachloroethylene and toluene on the development
of chick embryos. Toxicology 12:111-119, 1979.
Fink, R. Toxicity of Anesthetics. Williams and Wilkins Co., Baltimore, MD, 1968.
pp. 270.
Goodman, L. S. , and Gilman, A. G. The Pharmacological Basis of Therapeutics.
Ed. A. G. Gilman, L. S. Goodman and A. Gilman, 6th ed. New York: McMillan
Pub. Co. , 1980, pp. 361-363.
004TC3/H 6-11 11-8-83
-------
PDR, Physician's Desk Reference, Grade!1, N.Y.: Medical Economics Co., 1981,
p. 1258.
Schwetz, B. A., B. K. J. Leong, and P. J. Gehring. The effect of maternally
inhaled trichloroethylene, perchloroethylene, methyl chloroform, and
methylene chloride on embryonal and fetal development in mice and rats.
Toxicol. Appl. Pharmacol. 32:84-96, 1975.
U.S. Environmental Protection Agency. Determination not to initiate a rebuttable
presumption against registration (RPAR) of pesticide products containing
carbaryl; Availability of decision document. Fed. Regist. 45: 81869-
81876, December 12, 1980.
004TC3/H 6-12 11-8-83
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7. MUTAGENICITY
Trichloroethylene (TCI) has been tested for its ability to cause gene
mutations in bacteria, yeast, higher plants, insects, and rodents. It has also
been evaluated for its ability to cause chromosome aberrations in insects, rodents,
occupationally exposed workers and in cultured cells, and for its ability to
cause other effects which are indicative of DNA damage (gene conversion, mitotic
recombination, sister chromatid exchange and unscheduled DNA synthesis and DNA
alkylation). The results of these tests will be discussed below and summarized
in Tables 7-1 to 7-9. Consideration will also be given to studies bearing on
the potential of TCI to reach the germinal tissue of mammals.
7.1 GENE MUTATION STUDIES
7.1.1 Prokaryotic Test Systems (Bacteria)
Eight reports were evaluated concerning testing TCI for its mutagenic
potential in bacteria. Seven used Saljnpne11 a ty ph imuriurn (Henschler et al.
1977, Simmon et al. 1977, Margard 1978, Waskell 1978, Baden et al. 1979, Bartsch
et al. 1979, Kline et al. 1982), and one employed Escherichia coli (Greim et
al. 1975).
Because of the possibility of mutagenic contaminants in TCI samples,
Henschler et al. (1977) chemically analyzed and subsequently tested samples of
TCI for mutagenicity. They determined that a sample of technical grade TCI used in
the National Cancer Institute carcinogenicity bioassay (NCI 1976) contained the
following impurities (%w/w) by gas chromatography-mass spectrometry analysis:
epichlorohydrin, 0.22; 1,2-epoxybutane, 0.20; carbon tetrachloride, 0.05;
chloroform, 0.01; 1,1,1-trichloroethane, 0.035; diisobutylene, 0.02; ethylacetate,
0.052; pentanol-2, 0.015; butanol, 0.051. These impurities, were tested for
7-1
-------
mutagenicity with and without S9 mix from RGB-induced Wistar rat livers. In
Salmonella typhimurium, two of them, epichlorohydrin and 1,2-epoxybutane, were
found to be mutagenic in tests conducted without activation. Increases of
25-fold and 8-fold over the spontaneous revertant rates were observed for the
two compounds, respectively (extrapolated from Figure 1 of the paper). Negative
responses were observed when the tests were repeated using S9 mix. Negative
responses were also obtained with carbon tetrachloride, chloroform, and 1,1,1-
trichloroethane both with and without activation, but no data were presented.
Similarly, no increased mutation frequency was obtained with "purified" TCI
(source and purity not given). Roughly 140 TA100 revertants were observed at
each point tested over the entire dose range (from 1.12 x 10~^M to 1.12 M)
both with and without metabolic activation; the spontaneous revertant count
was 138 4- 8 (without activation) and 242 +_ 10 (with activation) colonies per
plate. In their testing, Henschler et al. (1977) added the chemical substances
directly to the top agar. No precautions were reported to have been taken to
prevent excessive evaporation of the test compound, and thus, the significance
of the negative response obtained with TCI is weakened by the possibility that
the material may have evaporated before adequately exposing the indicator
cells. This aspect of the study design could not be evaluated because no
toxicity data were presented. The positive response after exposure to other
volatiles like 1,2-epoxybutane may reflect the potency of these materials
compared to TCI. Based on their results, the authors concluded that the carcinogenic
activity of technical grade TCI in the NCI bioassay experiment was probably
predominantly, if not exclusively, due to the epoxide contaminants. This
study is inadequate for reaching these conclusions for a number of reasons
including flaws in the study design such as those mentioned above (i.e., the
7-2
-------
lack of a positive control requiring metabolic activation and not taking precautions
to prevent the escape of TCI).
Margard (1978) also conducted studies addressing the hypothesis that the
mutagenic activity of technical grade samples of TCI is due to mutagenic stabilizers
and not TCI itself by comparing the mutagenic properties of technical and
purified grades of TCI in the Salmonella typhimuriurn/mammalian microsome plate
incorporation assay. He used tester strains TA1535, TA1537, TA1538, TA98, and
TA100 in the presence and absence of a metabolic activation system (S9 mix)
from adult Sprague-Dawley rats induced with Aroclor-1254. Prior to plating,
two aliquots of cells were taken. One was diluted and grown on complete media
for determining cell survival and the other was grown on histidine deficient
plates for selection of his+ revertants. Cells were then incubated for 48
hours at 37°C and colony counts were made. The technical grade contained
0.08% (w/w) epichlorohydrin, 0.02% N-methylpyrrole, 0.13% N-propyl alcohol,
0.05% nitromethane, 0.10% 1,1,1-trichloroethane, and 0.45% butylene oxide.
The purified grade contained no detectable epoxides nor other stabilizing
ingredients (written communication with L. Schlossberg, Detrex Chemical Industries,
Inc.). A concentration-related increase in the number of revertants was observed,
as described in Table 7-1, in strain TA1535 both with and without metabolic
activation after exposure to technical grade TCI. This sample was not mutagenic
in strains TA98, TA100, TA1537, and TA1538. Purified TCI was not mutagenic in
any strains. TCI levels >^ 0.1 ml/plate were toxic as indicated by reduced
cell viability (49-86% killing). It has been reported that precautions were
taken to inhibit evaporation of the test samples during treatment of the cells
(Schlossberg, personal communication), but it was not specified in writing
what those precautions were. The toxicity data suggest that the cells were
exposed to the test material, but it is not possible to determine toxicity
7-3
-------
TABLE 7-1. MUTAGENICTY OF TECHNICAL GRADE TRICHLOROETHYLENE IN SALMONELLA/MAMMALIAN MICROSOME ASSAY
(adapted from Margard 1978)
Activation
Toxicity
Tester
Strain
TA1535
TA100
TA1535
TA100
Cone.
ml /pi ate
1.0
0.5
0.1
0.05
0.01
0
1.0
0.5
0.1
0.05
0.01
0
0.1
0.5
0.1
0.05
0.01
0
1.0
0.5
0.1
0.05
0.01
0
Plate
Count
27
142
185
196
53
207
255
283
MUTAGEN
97
172
22
246
175
175
225
317
Rel.
Via.*
0.14
0.72
0.94
—
0.19
0.73
0.90
—
ICITY OF
0.39
0.70
0.90
—
0.55
0.55
0.71
" '- i" . .T~
Mutagenicity
No.
Rev.t
58
40
17
24
184
263
123
167
PURIFIED GRADE
17
19
21
22
70
142
157
154
'. ' •"•"< -k ', "
Nonactivation
Toxicity
Plate
Count
22
122
149
157
66
167
232
234
Rel.
Via.*
0.14
0.78
0.95
—
0.28
0.71
1.0
--
Mutagenicity
Rol
Rev.t
58
42
21
23
173
181
136
160
TRICHLOROETHYLENE
89
121
156
175
166
162
354
305
0.51
0.69
0.89
--
0.54
0.53
0.82
--
15
20
20
19
114
158
145
158
I \ \_ I VI V I V V~ T I U l«r I I I \*J V^l w X- ****** wwnif^ui w-w w-w
tNumber of revertants observed (average).
-------
adequately in a plate incorporation test. To do this a liquid suspension
assay must be done. Appropriate positive controls were reported and demonstrated
the S9 had metabolic activity. From the data presented, it is not clear how
many plates were employed for each dose nor is it clear whether or not replicate
testing was performed. Under the conditions of test "purified" TCI was not
mutagenic.
To overcome the concern that the volatility of certain halogenated
hydrocarbons may prevent them from being adequately tested in standard plate
incorporation tests, Waskell (1978) investigated the mutagenic properties of
commonly used volatile anesthetics, including TCI, and several of their known
metabolites in testing conducted in sealed chambers. Salmonella typhimurium
strains TA100 and TA98 were used in tests for gene reversion. Anesthetic-grade
TCI from Ayerst was tested in closed containers at doses up to 10% in the
atmosphere. The purity of the sample tested was not reported, but it would
have been high with an approximate minimum purity of 99.5% and stabilization
by 0.1% thymol (personal communication with Ayerst). Holes were punched in
the top of the Petri dishes to allow the test vapor access to the bacterial
strains. Tests were conducted in the presence and absence of phenobarbital- and
PCB-induced Sprague-Dawley rat liver microsomes. No toxicity testing was
conducted nor were the actual concentrations of TCI measured in the exposure
chamber so it is not known whether the bacteria received a sufficiently high
dose. After a 48-hour exposure TCI did not produce a mutagenic effect in
strains TA100 and TA98 with or without metabolic activation. However, no data
were presented to support this conclusion. With respect to the metabolites of
TCI, the non-volatile compound 1,1,1-trichloroacetic acid (_> 99.0% pure, obtained
from Matheson, Coleman, and Bell, personal communication with Matheson,
Coleman, and Bell), chloral hydrate (recrystallized from chloroform six times
7-5
-------
to achieve a purity of >^ 99.0%), and trichloroethanol (purity not determined,
personal communication with L. Waskell, V.A. Hospital, San Francisco, CA) were
tested at maximum nontoxic quantities in plate incorporation tests. Doses of
trichloroethanol at 7.5 mg/plate and trichloroacetic acid at 0.45 mg/plate
were not mutagenic in strains TA98 and TA100 chloral hydrate at 10 mg/plate
increased the number of TA100 revertants above control (200) by 121 with metabolic
activation and 89 without metabolic activation. The data are inconclusive but
suggestive of a weak positive response. These strains were responsive to the
positive control chemicals diethyl sulfate and 2-aminofluorene.
Further testing was done to evaluate the dose-response of chloral hydrate-
induced mutagenesis in strain TA100. The number of revertants per plate in the
control groups were 190 and 210 with and without metabolic activation, respectively.
Doses of chloral hydrate from 0.5 mg/plate to 10.0 mg/plate did not result in
a mutation frequency greater than twofold above the spontaneous level. However,
increases in the number of revertants per plate above control values were
observed both with and without metabolic activation: 255 to 260 at 0.5 mg; 285
to 290 at 1.0 mg; 300 to 320 at 5.0 mg; and 290 to 310 at 10.0 mg. Although the
author reported these increases to be statistically significant by the t test
(P < 0.01), the increases are, at best, merely suggestive of a positive response.
The negative response for the induction of gene mutations in bacteria induced
by exposure to TCI reported by Waskell (1978) is consistent with the results
of Henschler et al. (1977) and Margard (1978). However, equivocal weak positive
responses suggestive of a positive effect have been reported by other investigators
using air tight exposure chambers and metabolic activation.
In tests conducted employing Salmonella tester strain TA100 both with and
without an exogenous S9 metabolic activation, Bartsch et al. (1979) assessed
7-6
-------
the mutagenic potential of over 20 chemical substances including a TCI sample
obtained from Merck, Darmstadt, Federal Republic of Germany. The sample was
reported to be 99.5% pure and contained no detectable amounts of epichlorohydrin
or 1,2-epoxybutane at levels _> 1 ppm. Experiments were conducted using a
vapor exposure assay in which Petri dishes containing a lawn of TA100 with or
without S9 mix were placed, uncovered, in 9-liter desiccators for various
lengths of time at 37°C in the dark. Upon completion of the exposure period
the compound was removed, the plates were then covered, inverted, and placed
in an incubator for an additional 48 hours at 37°C and scored. TCI was not
found to be mutagenic under the conditions used (see below).
Concentration Revertants/plate
in Air (5 v/v) Time (h) +S9 -S9
Trichloroethylene
0
8
20
Vinyl Bromide
2
20
16
16
16
16
75
85
55 (toxic)
240
715
70
72
50 (toxic)
190
695
When the test conditions were modified such that plates containing a liver S9
fraction from phenobarbital-treated OF-1 mice, an NADPH-generating system, TA100,
and 0.1 mM EDTA were preincubated at 37°C for 4 hours and then exposed to 5%
TCI vapor in air for 2 hours, a nearly twofold increase over the spontaneous
mutation frequency was observed (see below).
Concentration Revertants/plate
in Air (% v/v) Time (h) +S9 -S9
0 75 70
5* ( 2 100 80
5* 2* 135 70
*0.l mM EDTA added to soft agaT!
tPreincubated with S9, TA100, and 0.1 mM EDTA for 4 hours then exposed
to TCI vapors.
7-7
-------
The results of only one test were presented, but it was stated that near twofold
increases over the spontaneous mutation frequency were observed consistently.
Baden et al. (1979) evaluated the mutagenic properties of TCI in Salmonella
typhimurium strains TA100 and TA1535 both with and without S9 metabolic activation
from PCB-induced male Sprague-Dawley rats. The sample was > 99.5% pure, contained
no detectable epoxides, and was stabilized with 0.1% (w/w) thymol (personal
communication with M. Kelly, V.A. Hospital, Palo Alto, CA). Liquid TCI was
directly added to bacterial cell cultures for incubation at 37°C for 2 hours,
and concentrations in the gas phase above the incubator, which were the same
as those in the desiccator experiment described below, were monitored for the
first 2 hours of exposure showing a variation of _< 10%. These cultures were
plated and incubated for 2 days at 37°C before counting revertant colonies.
TCI was not found to be mutagenic in this experiment. However., no monitoring
was conducted after the initial 2 hours, and it is not known whether the desired
exposure levels were maintained throughout the 2-day incubation period. Toxicity
data were not reported and therefore it is not known whether the target cells
were actually exposed to a sufficiently high concentration of the test material.
In another experiment, reported in the same article, cultures were exposed to
vapor concentrations ranging from 0.1 to 10.0% (_+ 10% tolerance) in a desiccator
for 8 hours at 37°C with further incubation without treatment for 40 hours
before counting of colonies. The experiment was repeated, and each test concentration
was evaluated with triplicate assays. TCI vapor produced a less than twofold
increase in the number of revertant colonies to approximately 30% above background
(149 +_ 12, n=15) at concentrations of 1% (196 +_ 13, n=15) and 3% (194 +_ 12,
n=15) in TA100 with metabolic activation. No thymol control was reported, and
the response was not dose-related. At best this study provides only suggestive,
supportive evidence for mutagenic activity caused by TCI.
7-8
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Simmon et al. (1977) also reported that reagent grade TCI, with no detectable
epoxides, gave less than a twofold increase in revertant counts in Salmonella
typhimurium strain TA100. The sample was 99.85% pure, and contaminants included
0.09% chloroform, 0.02% tetrachloroethylene, 0.01% pentachlorobutadiene, and
0.03% hexachlorobutene; epoxides were not detected (personal communication with
R. Spanggord, SRI International). Plates containing TA100 with and without
metabolic activation were exposed to TCI vapor evaporated from a known amount
of liquid in a desiccator for 7 hours at 37°C before counting. TCI was detected
as mutagenic only with metabolic activation. The number of revertants per
plate was increased above the spontaneous control value of about 140 both with
and without activation by TCI treatment as follows: 180, 230, 240, and 200
revertants at 0.5, 1.0, 1.5, and 2.0ml TCI/9-liter desiccator, respectively,
in the presence of S9 mix from B6C3F1 male mice; and 160, 210, 200, and 170
revertants at 0.5, 1.0, 1.5, and 2.0ml TCI/9-liter desiccator, respectively,
in the presence of S9 mix derived from PCB-induced male Sprague-Dawley rats.
The revertant counts were extrapolated from Figure 22 of the paper. The results
of these assays were reproduced several times (personal communication with V.
Simmon, Genex Corp.), but no estimation of the variance of the results (stated
to be low) was presented and no data are presented to allow its calculation.
Greim et al. (1975) exposed Escherichia coli strain K12 to 3.3 mM
analytical grade TCI during 2 hours of incubation at 37°C in the presence of
metabolic activation from phenobarbital-induced male NMRI mouse liver microsomes.
A chemical analysis of the test sample was not done by the investigators (written
communication with D. Henschler, University of Wurzburg); however, the approximate
composition of the TCI product was probably: trichloroethylene,
>99.9%; either <, 100 ppm diisopropylamine or _< 120 ppm of a combination of
diisopropylamine, N-methylpyrrole, and 4-tertbutylphenyl as stabilizers; < 500 ppm
of other chlorinated hydrocarbons combined (chloroform, carbon tetrachloride,
7-9
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1,2-dichloroethane, 1,1,2-dichloroethylene [sic] , and tetrachloroethylene);
no epoxides present at a detection level of 1 ppm (written communication with
Dr. Fries and Dr. Koppe, E. Merck Company, the producer of the sample used in
this study). Several loci in E. coli K12 were used to test for mutagenicity:
back mutation at three loci, gal+, arg+, and nad+, and the forward mutation
MTR system which yields resistance to 5-methyl-DL-tryptophan. Survival of
bacteria on complete medium at the TCI concentration used (3.3 mM) was 76 _+
4%. A weak twofold mutagenic effect of TCE, expressed as a percentage of the
spontaneous mutation rate, was observed for arg+, 232 _+ 36, but the significance
of this response cannot be ascertained because no increase was observed for
the other markers; gal + , 123 _+ 23; MTR, 114 _+ 18; nad+, 100. The reporting
of the conduct of testing and the observed results were inadequate. For instance,
information concerning spontaneous mutation frequencies, number of replications,
revertant count data, etc. for each test was not reported. Only one dose was
used so it was not possible to obtain dose-response data. Thus, the significance
of the results cannot be adequately assessed.
None of the tests described previously clearly showed TCI to be mutagenic.
However, the weak, less than twofold, increases in revertant counts observed in
several studies, using several samples of TCI and different strains and
species of bacteria suggest that commercially available TCI may be weakly
mutagenic to bacteria after metabolic activation. The data for concluding
that TCI itself is mutagenic are best suggestive but the fact that weak but
less than twofold responses were obtained with such samples only after metabolic
activation argues against the possibility of the responses being caused by the
presence of epoxide stabilizers because these agents are direct-acting mutagens.
It is important to note that only very weak positive responses (< 2-fold above
the spontaneous level) were obtained with "purified" samples of TCI. In these
7-10
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studies the responses were not always dose-related and were observed only at
very high doses (i.e. vapors ranging from 1 to 5% v/v). The data are not
typical of mutagenic agents in Salmonella, but it is not possible to unequivocally
rule out that TCI causing a slight increase in mutant yield. The studies
described below discuss the observations from tests with other organisms.
7.1.2 Eukaryotic Test Systems
7.1.2.1 Plants--
7.1.2.1.1 Yeast—Employing the yeast Schizosaccharomyces pombe in a host-mediated
assay with B6C3F1 mice, Loprieno et al. (1979) reported findings similar to those
obtained in bacteria by Henschler et al. (1977). In this study the mutagenic
potential of technical grade TCI (composition, % (w/w): trichloroethylene,
99; 1,2-epoxybutane, 0.19; epichlorohydrin, 0.09; ethylacetate, 0.04; n-methyl
pyrrole, 0.02; diisobutylene, 0.03) was compared with that of a purified grade
of TCI (composition w/w: trichloroethylene, 99.98%; chloroform, 10 ppm;
carbon tetrachloride, 17 ppm; 1,1,2-trichloroethane, 21 ppm; 1,1,2,2-tetrachloro-
ethane, 14 ppm; other contaminants, 16 ppm). One and one-half milliliters
(2 x 109 cells/ml) of yeast cell suspension (SP ade 6-60/rad 10-198h-) was
injected into the peritoneal cavity of mice untreated (controls) or treated
with a total volume of 0.5 ml doses of TCI (ranging from 0 to 2000 mg/kg)
administered by gavage; cells were removed from the peritoneum and plated
for counting of mutant colonies 6 or 16 hours after treatment. Each assay was
performed in at least three animals. The report states that results of this
test (summarized in Table 7-2) indicate that the technical grade material was
weakly positive showing a dose-related increase in the induction of mutants.
These results are judged to be equivocal for the following reasons. The linear
regression analysis presented in the report was statistically incorrect, because
it was calculated using mean values rather than the individual data points.
7-11
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TABLE 7-2. MUTAGENICITY OF TRICHLOROETHYLENE AND EPICHLOROHYDRIN IN S. POMBE
BY MEANS OF THE HOST-MEDIATED ASSAY IN B6C3F1 MICE
(16 hours of incubation)
(Loprieno et al. 1979)
Treatment Dose Mutants
mg/kg M.F. x 10-4 (M 4. S.E>)
Trichloroethylene None (control) 0.94^0.08*
technical grade 500 1.33 T 0.28
1,000 1.32 TO.29
2,000 1.61 +_ 0.45
Methyl methanesulfonate 50 4.16 +_ 0.54
Trichloroethylene None (control) 1.45 +_ 0.06
purified grade 1,000 1.59 7 0.22
2,000 1.36^0.21
Dimethylnitrosamine 100 16.73 + 3.24
Epichlorohydrin None (control) 1.66 +_ 0.03
2 1.50 + 1.20
10 2.05 +_ 0.51
50 3.71 ^ 1.05
*Lower than the other negative control values.
7-12
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The correlation coefficient was reported to be statistically significant (r =
0.86); recalculating using their data r was found to equal 0.35 which is not
statistically significant. Furthermore, the number of mutants in the negative
controls for the technical product was much less, by 44% and 35%, respectively,
than those in the controls for the "purified" TCI and epichlorohydrin. The
experimental values for the "purified" samples were in the same range as those
for the technical material and they showed no increase in mutation frequency
compared to the concurrent negative controls. The mutagenic response to
epichlorohydrin tested by itself was, however, positive (3.71 _+ 1.05 x 10-4
mutants at 50 mg/kg) as was the response of the positive control dimethylnitrosamine
(16.73 +_ 3.24 x lO-4 mutants at 100 mg/kg) indicating that for these chemicals
the assay was working properly. No data were presented bearing on the ability
of the administered TCI to reach the indicator organisms (e.g., chemical binding).
The negative response in this host mediated assay obtained for the pure sample
and the equivocal response for the technical sample in this study may be due to a
number of reasons including the possibility that TCI (or its metabolites)
did not reach the indicator organisms in the peritoneum under the test conditions
employed.
Subsequent testing by the same laboratory (Rossi et al. 1983) was conducted
to further investigate the mutagenic potential of pure and technical samples of
TCI, and two stabilizers contained in technical grade sample: epichlorohydrin
and 1,2-epoxybutane. Schizosaccharomyces pombe strain PI was used in in vitro
testing and in intraperitoneal and intrasanguineous host mediated assays. S9
mix from phenobarbital-or B-napthoflavone-induced mice or rat livers was used
in the in vitro studies where TCI was tested at doses up to 100 mM. No increases
in the frequency of forward mutations over negative controls (1.38 _+ 0.67
revertants) was found. By contrast epichlorohydrin and 1,2-epoxybutane caused
7-13
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dose-related increases in mutant frequency up to about a 10-fold increase
compared to negative controls at 6.4 mM and 12.8 mM, respectively.
Negative responses were obtained when 2 g/kg doses of TCI were used in
intraperitoneal and intrasanguineous host-mediated assays with CD 1 x C57BL
mice (Rossi et al. 1983). Negative responses were also obtained when
epichlorohydrin and 1,2-epoxybutane were used in intraperitoneal host-mediated
assays at doses up to 2g/kg. These data suggest that the intraperitoneal host
mediated assay in mice with Schizosaccharomyces pombe may not be appropriate
for testing halocarbons and their stabilizers.
Mondino (1979) also reported negative results for purified TCI in a yeast
host-mediated assay. He used Schizosaccharomyces pombe and adult male B6C3F1
mice. The components of the TCI sample were identified as follows: trichloroethylene,
99.9%; water, evaporation residue, chloroform, 1,2-dichloroethane, carbon
tetrachloride, 1,1,2-trichlorethane, other impurities, all j( 20 ppm; diisopropylamine,
198 ppm; diisobutylene, 104 ppm; butylhydroxytoluene, 12 ppm. Yeast cell
suspension was injected into the peritoneum of the mice, who had received 500,
1000, or 2000 mg/kg dosages of trichloroethylene by gavage. Three animals
comprised each treatment group as well as the untreated control and positive
control (100 mg/kg of dimethylnitrosamine) groups. After a 16-hour incubation
period the animals were sacrificed for removal of yeast cells from the peritoneum
for plating and counting. Plates were incubated at 32°C for 4 to 5 days prior
to mutant colony counting. Mean numbers (+_ S.D.) x 10~4 of mutant colonies
were as follows: control, 1.80jt 0.48; low dose, 1.18 +_ 1.18; mid-dose, 1.03
+^ 0.91; high-dose, 1.84 +_ 0.28; positive control (dimethylnitrosamine 100 mg/kg
31.1 +_ 3.31. As was the case for the studies above the negative result obtained
by Mondino (1979) may result from TCI not being mutagenic, or it may be that
the compound or its metabolites did not reach the test organisms, in the peritoneum
7-14
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of the test mice.
The mutagenic properties of technical grade TCI in the XV 185-14C haploid
strain of Saccharomyces cerevisiae were assessed j_n_ vitro by Shahin and Von
Borstel (1977). The ability of TCI to induce mutations at the lys 1-1, his
1-7, and horn 3-10 loci was assessed in this study. Stock cultures were incubated
in liquid YPG medium at 30°C until a stationary growth phase was achieved.
Cells were harvested, washed, and adjusted to a population of 1 x 108 cells/ml.
In order to estimate its mutagenic effect in the absence of metabolic activation,
cells treated with 1.11, 5.56, 111.1, or 222.2 mM of TCI during incubation at
30°C were withdrawn at 2, 6, 24, and 48 hours for plating and counting of
colonies. No precautions were reported to have been taken to prevent excessive
evaporation of the compound and ensure exposure to the test organism, but the
reported toxicity indicates this was not a problem and that the cells were
adequately exposed. Treated cells were compared with cells from similarly
maintained untreated control and positive control experiments (222.2 mM of
2-acetylaminofluorene for tests without metabolic activation and 8 ug/ml of
ethyl methanesulfonate for tests with and without metabolic activation).
Plates were maintained at 30°C for 5 days to estimate survival, and the assays
were performed in triplicate.
In the study described above, concentrations of 111.1 mM and 222.2 mM TCI
in the presence of metabolic activation increased the reversion frequencies at
the lys 1-1, horn 1-10, and his 1-7 loci (Table 7-3) but only at levels that were
also highly toxic (> 99% cell killing). The revertants/107 survivors for lys
1-1, his 1-7, and horn 3-10, corresponding to 1 hour treatments of 111 and 222 mM,
were 2552 and > 23,200, 3553 and > 29,000, and 5313 and > 25,000, respectively
compared to negative control values of 15.3, 19.5, and 8.6. The significance
of the reported increased incidences of mutations in tests conducted with
7-15
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TABLE 7-3. MUTAGENICITY TECHINICAL GRADE OF TRICHLOROETHYLENE AT THE HIS 1-7
LOCUS OF SACCHAROMYCES CEREVISIAE
(adapted from Shahin and von Borstel 1977)
Dose (mM)
0
1.11
5.56
111.1
222.2
Positive Control
-S9 (6-hr
Revertants
14.3
16.7
22
0
0
83*
incubation)
%Survival
100
90
72
<0.13
<0.013
61
+S9 (4-hr
Revertants
19.2
15.9
16.8
22,000
>29,000
6300"!"
incubation)
%Survival
99
72
65
<0.1
<0.1
0.23
*2-acetylaminofluorene at 20 ug/ml
tEMS at 8 ul /ml.
7-16
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metabolic activation is uncertain because the increases were noted only when
there was greater than 99% killing. The positive control in the tests with
metabolic activation (EMS) was inappropriate because it is a direct-acting
mutagen. TCI was ineffective as a mutagen without metabolic activation at all
doses tested, but increased numbers of revertants were noted with activation.
With respect to the 48-hour treatment without activation, the maximum number
of revertants per 107 survivors for the three loci individually was 10 to 15
for untreated controls (98% survival), 12.8 to 21.4 for 1.11 mM (73% survival),
11.4 to 17.1 for 5.56 mM (64% survival), and 0 for 11.1 mM and 222.2 mM (< 0.1%
survival) of TCI. The lack of revertants with the two highest concentrations
appears to correspond to failure of the cells to survive after plating. The
largest number of revertants per 107 survivors produced by 2-acetylaminofluorene
(222.2 mM) was 102 (lys 1-1) after a 6-hour treatment, and 673 (his 1-7) and
488 (horn 3-10) after a 24-hour treatment. It may be that there is a "window"
of effective concentrations where TCI is mutagenic but not excessively toxic.
Such a concentration effect cannot be ruled out because levels between 5.56 mM
(ineffective) and 111.1 mM (suggestive response but highly toxic) were not
tested and it was in this dose range that the weak positive responses discussed
below were obtained by Bronzetti et al. (1978) and Callen et al. (1980) in
strain 07. It is important to note that the composition of the TCI sample
used in the study by Shahin and Von Borstel (1977) was not determined, and
the possible contribution of contaminants to the response cannot be ascertained
or ruled out (personal communication with R.C. Von Borstel, University of
Alberta). These results, do not allow a conclusive evaluation to be made.
An increase in point mutations and gene conversion by a certified ACS
reagent grade of TCI in Saccharomyces cerevisiae strains 04 and 07 was observed
by Bronzetti et al. (1978) in cell suspension and in host-mediated assays.
7-17
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The authors did not report a detailed chemical analysis of the sample; however,
a description of the product obtained by personal communication with Fisher
Scientific Company, the producer of the test sample indicates a purity of > 99.5%
and stabilization with either 25 ppm diisopropylamine or 20 ppm triethylamine.
In the cell suspension experiment, strain D7 cells, which can detect mitotic
gene conversion at the trp locus, point (reverse) mutation at the ilv locus,
and mitotic recombination at the ade locus, were incubated in the presence of
TCI with or without S9 mix derived from livers of uninduced male CD-I mice for
4 hours at 37°C before plating on selective media (for counting of ilv revertants,
acte recombinants, and trp convertants) and on complete medium (for survivor
counts). The results for ilv revertants are presented in Table 7-4. The other
results will be discussed later in the section on tests indicating DNA damage.
In the host-mediated assay yeast cell culture was instilled into the retroorbital
sinus of male adult CD-I mice. Two experiments were included in this assay:
an acute experiment in which a single gavage dose of 400 mg/kg was given immediately
after introduction of strain D4 or D7 cells on the day of the assay. Mice
were sacrificed 4 hours following introduction of the yeast cells and liver,
lungs, and kidneys were removed for homogenization and subsequent plating of
yeast cells as described for the suspension assay. Assays in this study were
done in triplicate or quadruplicate, and the data for gene revertants are
presented in Table 7-4 as mean _+ S.E.
The results of Bronzetti et al. (1978) were as follows. In the absence
of metabolic activation, 10 mM and 40 mM concentrations of TCI effected a
dose-related reduction in cell viability in the cell suspension system but did
not induce a mutagenic response. Survival was reduced to approximately 87%
with the 10 mM dose and to 65% with the 40 mM dose compared to 100% for the
controls. TCI did produce concentration-dependent increases in point mutation
7-18
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TABLE 7-4. MUTAGENICITY OF REAGENT GRADE TRICHLOROETHYLENE AT THE ILV LOCUS IN
SACCHAROMYCES CEREVISIAE
(adapted from Bronzetti et al. 1978)
Suspension Tests Strain D7
Concentration (mM)
Revertants/106
x + S.E.
% Survival
Without Metabolic Activation
0
10
40
With Metabolic Activation
0
10
20
30
40
Host-Mediated Assay in CD-I
Strain Organ
Acute Exposure (400 mg/kg)
D7 Liver
Lungs
Kidneys
0.75 +_ 0.12
0.85 _+ 0.15
0.87 _+ 0.06
0.85 +_ 0.06
1.16 i 0.16
1.77 _+ 0.12
2.24 +_ 0.17
3.23 jf 0.32
Mice
No. of
Treatment Mice
Control 3
TCE 4
Control 2
TCE 3
Control 2
TCE 4
100
87.5 jf 1.0
64.1 _+ 2.1
100
81.6 +_ 2.9
67.1 +_ 2.8
55.5 jh 1.7
50.3 + 1.4
Revertants/106
x + S.E.
0.08 + 0.04
0.56 +_ 0.52
0.08 + 0.002
0.40 Jh 0.19
0.21 + 0.02
0.79 + 0.37
7-19
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frequencies with metabolic activation at doses of 10, 20, 30, and 40 mM with
metabolic activation. Positive responses were also observed in the host-mediated
assays. Fourfold to eightfold increases were obtained in the number of revertants
compared to the negative controls. The highest response was observed in yeast
recovered from the kidney where 0.79 +_ 0.37 x 10-6 revertants were found in
the treated animals vs. 0.21 +_ 0.02 x 10"6 in the negative controls.
Like Bronzetti et al. (1978), Callen et al. (1980) also employed a TCI
sample obtained from the Fisher Scientific Company. They used Saccharomyces
cerevisiae strains D4 and 07 to assess TCI's genetic activity and strain D5
intact yeast cells or microsomal suspensions to study its metabolism indirectly.
Gene conversion at the trp 5 locus, mitotic recombination at the ade 2 locus,
and gene reversion of the ilv 1 locus (Table 7-5) of strain D7 were assessed by
exposures of the cells to 0, 15, and 22 mM solutions in suspension at 37°C for
1 hour followed by plating on appropriate media, allowing for expression of
genetic activity and, finally, scoring. A twofold increase in reverse mutations
at the ilv 1 locus over the spontaneous control values was observed at 15 mM (7.4
vs. 3.6 revertants x 10~6 survivors, respectively) without an exogenously supplied
metabolic activation system (67% cell survival). A severely toxic effect was
observed at 22 mM. A positive response without activation was observed by
Callen et al. (1980) but not by Bronzetti et al. (1978). The reason for this
is not known, but it may be due to the stages of growth the yeast cells were in
when tested. Callen et al. (1980) found TCI to be mutagenic to cells from log
phase cultures but not to cells from stationary cultures of D7. Bronzetti et
al. (1978) did not report the growth phase of the cells used in their test, but
if they employed stationary phase cells a negative response without activation
would be expected.
Callen et al. (1980) provided additional suggestive evidence that Saccharomyces
7-20
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TABLE 7-5. MUTAGENICITY OF TRICHLOROETHYLENE AT THE ILV LOCUS IN
SACCHAROMYCES CEREVISIAE D7
(adapted from Callen et al. 1980)
Survival
ilv 1
Concentration
(mM)
Total %
Colonies
Total
Revertants
Revertants
106 Survivors
0
15
22
1201
802
3
100
67
0.3
43
59
3.6
7.4
7-21
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can metabolize TCI. They analyzed whole cell suspensions of strain 05 in the
log phase of growth in a spectrophotometer after exposure to unspecified amounts
of TCI. Peaks with maximum of 450 and 505 mM were observed. The peak at 450
nm appeared to reach a maximum after roughly five minutes and after this period
an additional peak at 423 nm was observed. This spectrum is similar to that
observed when TCI is added to mammalian microsomes. These results indicate that
trichloroethylene enters exposed yeast cells and suggest it is metabolized by a
yeast cytochrome P-450-independent monooxygenase system.
The yeast studies show that commercially available TCI is mutagenic in
Saccharomyces cerevisiae. The data are consistent with TCI metabolites being
mutagenic, because the samples tested are represented as being free from epoxide
stabilizers (which are direct-acting mutagens), and metabolic activation appears
to be required for the genetic activity of TCI. The doses required to elicit
twofold to fourfold increases in gene reversion (i.e., 10-40 mM in suspension
tests and 400 mg/kg in the host-mediated assay) show the commercial samples of
TCI to be weakly active. The negative responses obtained with the Schizosaccharomyces
pombe host-mediated assays are not considered to diminish the positive results
in Saccharomyces because of the inherent insensitivity of the j>. pombe test (forward
mutation assay with high spontaneous control levels and no selection scheme).
7.1.2.1.2 Angiosperms. Testing for somatic cell mutations in the stamen
hairs of Tradescantia clone 4430, an interspecfic hybrid (T. subacaulis x T.
hirsutiflora), was done by Schairer et al. (1978) to assess the mutagenic
potential of TCI. A group of unrooted fresh cuttings containing young inflorescences
possessing flower buds in a range of developmental stages was exposed to 0.5
ppm TCI for 6 hours. Following treatment, the cuttings were grown in aerated
Hoagland's nutrient solution and analyzed each day as they bloomed for 3 weeks
after treatment. One hundred forty-eight mutations were observed out of 44,000
7-22
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stamen hairs scored. The mutation frequency (+_ S.E./100 cells), after the
negative control values (not reported) were subtracted, was reported to be
positive (0.112 _+ 0.036; P < 0.01). Based on the data given, however, this
value cannot be verified. The mutation frequency (+_ S.E./100 cells) for 6
hour exposures to 5 ppm EMS and 75 ppm vinyl chloride were 1.012 +_ 0.133 and
0.112 +_ 0.046, respectively. Because the purity and source of the sample were
not provided, it is not possible to ascribe with certainty the observed positive
effects to the genotoxic action of TCI per se.
7.1.2.2 Animals--
7.1.2.2.1 Insects (Drosophila). Abrahamson and Valencia (1980) fed 1000
ppm TCI in 0.2% sucrose for 72 hours to male Canton-S (wild type) Drosophila
melanogaster. and subsequently mated 102 of these flies with female FM6 (a
marked and balanced X-chromosome) flies and screened the F£ descendents for
mutagenicity using the sex-linked recessive lethal test. Ninety-six of the
treated males were fertile. Administration of the test compound was performed
in a glass shell vial containing filter material saturated with the feeding
solution which was renewed twice daily. However, because of the volatility of
TCI the actual dose given to the flies might have been much less than expected
in the feeding study. Treated and untreated control males were mated individually
with three females each and then transferred without anesthetization to fresh
females at 2- to 3-day intervals until a total of four broods of progeny were
produced. The fertile females were left in the vials for 1 week. In a simultaneous
experiment, 0.3 ul of TCI was injected into the abdomen of the male flies
before mating. A technical grade sample from Allied Chemical Co. was used,
and the purity and composition of the sample was not ascertained (personal
communications with L. Valcovic, Food and Drug Administration, sponsor of the
study; and Allied Chemical Co.).
7-23
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TCI was not mutagenic in the above test. The frequency of lethals (number
of lethals/number of chromosomes tested) was 0.12% (19/9248) In the feeding
experiment, 0.17% (4/2344) in the injection experiment, 0.31% (31/10,009) in
concurrent controls, and 0.24% (230/94,491) in the historical controls used in
the testing laboratory for 2 years prior to completion of this study. The
negative result obtained in this test indicates that either the test substance
did not reach the target site (in the testes) or that TCI is not mutagenic.
Beliles et al. (1980) also tested TCI in Drosophila for its ability to
cause sex-linked recessive lethals and, in addition, for its ability to cause
X or Y chromosome loss. Three-hundred one-day old B$Y y+ac+, (K^l+2)"/X.Y^,
INEN2,f males were placed in each of three screen cages and subsequently exposed
to 0, 100, and 500 ppm TCI vapor from North Strong (estimated to be 99.9% pure by
infrared spectroscopy) for 7 hours. An unspecified number of males serving as
positive controls were fed 0.025 M EMS in 1% sucrose for 8 hours. Two-hundred
males per treatment group were individually mated to sequential groups of 3
In(l)sc8, sc8 1 B/In(l)dl-49 BM1, y ac v BM1 virgin females. The brooding
scheme consisted of a 2-3-3-2 day mating sequence enabling specific sperm cell
stages to be tested (i.e., spermatozoa, spermatid, spermatocyte, and spermatogonia),
Losses of the entire X or Y chromosome and the short or long arm of the Y
chromosome were detected by scoring for specific phenotypic classes in the Fj
generation. F^ males of the genotype y ac v B^/In(l)EN2,f were mated to
virgin Y^/X.Y^-, In(l)dl-49, v f B females and the occurrence of recessive
lethal mutations on the In(l)EN2, f chromosome were detected by the lack of
males with forked bristles in the F2 generation. The frequencies of X or Y
chromosome loss were 0.07% and 0.17% for the low and high doses, respectively,
compared to 0.06% for the concurrent negative control. These values are not
signficantly different; however, the 0.17% was close to a significant P value.
7-24
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The positive control EMS did not induce sex chromosome loss under these test
conditions; thus it is not possible to determine whether the test was conducted
properly. The frequencies of losses of the long arm of the Y chromosome,
0.03% and 0.05% for the low and high doses, respectively, and losses of the
short arm of the Y chromosome, 0.02% for both dose levels, were not significantly
different from the 0.01% value of the concurrent negative control for losses
of both arms. Like the study by Abrahamson and Valencia (1980), Beliles et
al. (1980) found TCI to be ineffective in causing an increase in the incidence
of sex-linked recessive lethal mutations. The frequencies were 0.03% and
0.16% for the low and high doses, respectively, compared to 0% for the concurrent
negative control and 0.11% of the historical negative control. The frequency
of sex-linked recessive lethal mutations for the positive control (EMS) was 21.56%
showing a significant positive response. No significant variations were observed
among the four germ cell (brood) stages employed in any of the assays.
7.1.2.2.2 Rodents. TCI was tested in a mouse spot test by Fahrig (1977).
Virgin females of the inbred C57BL/6J Han strain (a/a, wild type) were mated
with males of the Han-rotation bred T-stock (a/a=non-agouti, a/a=brown, ccn
p/ccn p=chinchilla and pink-eyed dilution; d se/d se=dilute and short ear;
s/s=piebald spotting) to yield embryos of the genotype a/a, b/+, cc*1p/+ +, d
se/+ +, s/+ (black coat, dark eyes). Each male was mated with two females.
On day 11 of gestation, a single intraperitoneal (i.p.) injection of TCI (99.5%
pure) was made into 50 and 26 females at doses of 140 and 350 mg/kg, respectively.
Impurities detected in this test sample were 60 to 150 ppm ethanolamine, 0.0003%
chloride (Cl2), and 0.0001% free chloride (Cl-1) (personal communication
with R. Fahrig, University Tubingen, West Germany). Litters were born on day
21 of gestation, and pups were examined for color spots, the mutagenic end-point,
twice weekly between 2 and 5 weeks of age.
7-25
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The number of offspring (number of litters) surviving for examination was
144 (23) in the untreated control group, 145 (38) in the low-dose group, and 51
(13) in the high-dose group. The number of coat color (brown or gray) spots
found were: none in concurrent control pups, 2/794 historical control pups
(0.25%), two low-dose pups (1.3%), and two high-dose pups (3.92%). Known
mutagens including ethyl methanesulfonate were positive (26% at 0.9 mM EMS).
Results of this study indicate that the TCI sample tested or one or more of
its metabolites is able to produce mutations in somatic tissue of an intact
mammal.
Based on the weak positive responses obtained in Saccharomyces, Tradescantia,
and (57B46J) mice, commercial samples of TCI are judged to be capable of causing
gene mutations after metabolic activation. Because of the requirement for activation,
it is not likely that epoxide stabilizers present in the commercial samples account
for the effect. However, because high doses of TCI were required to elicit a
positive effect it must be considered to be only weakly mutagenic. The data for
purified samples of TCI do not show it to be positive. However, weak, less
than twofold, increases are reported with metabolic activation at high doses.
7.2 CHROMOSOME ABERRATION STUDIES
TCI has been examined for its ability to cause changes in chromosome number
(chromosomal loss tests with Drosophila, micronucleus test in mice, and hypodiploid
cells in humans), and structure (in studies employing mice, rats, and humans).
The results of the chromosome loss test were described in the previous section
(Beliles et al. 1980). The rest of the studies are presented below.
7.2.1 Experimental Animals
7.2.1.1 Bone Marrow--Beli1es et al. (1980) tested TCI for its ability to
cause chromosome aberrations in rat bone marrow cells. Adult male and female
rats [CRL: COBS CD (SD) BR] were exposed to 0, 100, or 500 ppm TCI by inhalation
7-26
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both acutely and subchronically. The positive control was TEM at 0.3 mg/kg
given i.p. in 0.85% saline. Acute exposure consisted of a single 7-hour session
in the exposure chamber. Subchronic exposures consisted of 5 daily exposures
of 7 hours. Bone marrow cells were then collected and slides prepared, coded,
and 50 cells/animal scored. There was no increase in the number of aberrations
in either study. The percentage of cells with aberrations were 0.4-2.4 in the
negative control group compared to 0-2.7 in the TCI treated group. The positive
control TEM was clastogenic causing 7.2-8.5% aberrant cells. TCI failed to
cause the induction of aberrations or aneuploidy under the conditions of this
study; however, it is not considered to be an adequate test of the clastogenic
potential of TCI. The positive control was not appropriate because it was
given by i.p. injection while TCI was administered via inhalation. In addition,
the doses used may have been too low to detect weak activity. The lowest
concentration in air reported to cause death in the rat is 8000 ppm/4 hrs
(American Industrial Hygiene Association Journal 30:470, 1969). It would have
been appropriate to have tested higher doses of TCI and to have incorporated a
volatile and preferably a chemically related substance as the positive control.
7.2.1.2 Dominant Lethal Test—The precise nature of damage causing dominant
lethal effects is not known, but there is a good correlation between chromosome
breakage in germ cells and dominant lethal effects (Matter and Jaeger 1975).
When dominant lethal effects are observed it can be concluded that the test
substance reaches the gonads and likely causes genetic damage.
Slacik-Erben et al. (1980) assessed the ability of 0, 50, 202, and 450 ppm
TCI in air to cause dominant lethal mutations in NMRI-Han/BGA mice. The sample
contained 99.5% trichloroethylene stabilized with 100 mg/1 triethanolamine.
Analysis of a liquid sample also identified 5 ppm 1,2-dichloroethylene, 9 ppm
cis-l,2-dichloroethylene, 80 ppm 1,1,1-trichloroethylene, 120 ppm carbon
7-27
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tetrachloride, 10 ppm 1,1,2-trichloroethane, and 815 ppm tetrachloroethylene.
Fifty males per dose were exposed for 24 hours in a 1000 liter volume glass
chamber equipped with a ventilator. The temperature was kept between 22-24°C
and the humidity between 70-80%. For rapid equilibration the calculated volume
of liquid TCI was placed in the chamber, and mixtures of TCI and air were streamed
through the chamber at a rate of 200 (50 and 202 ppm) and 300 (450 ppm) I/hour.
After the 24-hour exposure period, the male mice were mated individually with
one untreated NMRI-Han female for days. At this time, the mated females were
replaced by fresh virgin females who were kept for 4 days and then replaced,
(and this was repeated, etc.) until each male had been mated singly to 12
females for 4 days each (up to 48 days after treatment). By this mating regimen
all stages of spermatogenesis were sampled from mature sperm to gonial cells.
Each pregnant female was sacrificed by cervial dislocation, dissected, and
examined for dead and living implants. The results of the testing are presented
in Table 7-6. Under conditions of this test, TCI was not detected as mutagenic,
but there are several important points to consider with respect to this finding.
The males exhibited no visible effects during the exposure, and doses up to
450 ppm did not alter the fertilization rate. Thus, the doses selected for
test may not have approached the maximum tolerated dose. Even if the maximum
tolerated dose was approached, the dominant lethal test is not recognized to
be a sensitive test for detecting mutagens (Russell and Matter 1980) due to
the high spontaneous level of dominant lethal events.
7.2.1.3 Micronucleus Test--Duprat and Gradiski (1980) used TCI in a micronucleus
test. CD-I mice were divided into nine groups, six of which were given up to
3000 mg/kg TCI (analytical grade 99.5% purity; unspecified source) in gum
arabic orally in two dosings separated by 24 hours. The animals were sacrificed
16 hours later, bone marrow smears were made, stained, and polychromatic
7-28
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TABLE 7-6. MUTAGENICITY OF TRICHLOROETHYLENE IN THE DOMINANT LETHAL ASSAY
USING NMRI MICE
(adapted from Slacik-Erben et al. 1980)
Dose
in ppm
(rate)
Female Total
Mated % w/Implants Implants
Total Total Live % Dominant
Dead Live Implants/ Lethal
Implants Implants Female Factors*
(200 1/h)
0
50
202
(300 1/h)
498
482
493
89.6
90.1
91
5578
5360
5703
443
479
531
5135
4881
5172
*Dominant lethal factors
FL% = 1 - (live implants per female of test group
(live implants per female of the contro
11.5
11.2
11.5
2.6
0
0
450
503
488
93.4
92.4
6142
5927
502
533
5640
5394
12.0
11.97
0.25
x 100
group)
7-29
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erythocytes (PCEs) and mature erythrocytes (MEs) were examined for the presence
of micronuclei. The incidence of micronuclei in PCEs was higher in the vehicle
control group (i.e., treated with 10% gum arabic solution) than in the untreated
control (1.0 +_ 0.5 vs. 0.5 +_ 0.4%). Also, the incidence of what were considered
micronuclei in PCEs and MEs from treated animals was greater than the vehicle
control levels at all doses and showed a dose-response effect. At the highest
dose (3000 mg/kg) the percentage of PCEs with "micronuclei" was 15.8 +_ 5.7
compared to the vehicle and untreated control values presented above. The
corresponding values for "micronuclei" in MEs were 3.4 _+ 1.8, 0.2 +_ 0.3, and
0.3 +_ 0.3, respectively (P < 0.01). Because of their occurrence in MC the
micronuclei observed by Duprat and Gradiski (1980) are likely artifactual (See
discussion by Schmid 1975).
7.2.2 Humans
Konietzko et al. (1978) studied the incidence of chromosome aberrations
(structural and numerical) in peripheral lymphomycytes of 28 chemical workers
engaged in the manufacture of TCI. The workers ranged in age between 23 and
67 years with an average age of 42.5. Their exposures ranged in duration from
1 to 21 years; the average length of exposure was approximately 6 years.
Since many of the workers had been exposed over a period of many years, it was
not known what other chemical substances they may have been exposed to. Thus,
the study could only address the long-term effects associated with working in
a factory that manufactured TCI. A control group of 10 healthy young men
working in the Institute of Human Genetics where the study was conducted and
ranging in age from 15 to 45 years (average age 28.3 years) was selected for
comparison. However, this group was not a matched control group. The exposed
group had a frequency of 10.96 _+ 4.4% (X _+ SO) hypodiploid cells which was
significantly elevated compared to the control group, 6.5 _+ 3.2% hypodiploid
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cells. Concerning chromosome breaks/100 cells the frequencies were 3.1 +_ 3.6
and 0.6 _+ 0.69, respectively. Because the control group was not a matched
control group and since the mean frequency of chromosome breaks observed in
the exposed workers was within the normal range, the increased incidence of
chromosome breaks was not considered significant by the authors, although they
did consider the increase in hypodiploid cells significant. Because of this
it was thought appropriate to further investigate factors which may have contributed
to the increased incidence of hypodiploid cells. Based on the frequency of
hypodiploid cells in the controls, it was calculated that individuals possessing
> 13% hypodiploid cells were abnormal. Workers were divided according to
whether or not they possessed > 13% hypodiploid cells and these groups of
workers were subsquently further characterized (Table 7-7). Nine individuals
had greater than 13% hypodiploid cells. These individuals worked under
higher daily peak concentrations of TCI (as measured on three occasions over a
2-week period for 8 hours each time) than did their fellow workers. The average
daily concentrations for these two groups of workers were 75 +_ 14 ppm vs. 61 _+
23 ppm, respectively. These exposure levels were used as estimates of previous
years of exposure. This was thought to be a good approximation of the actual
exposures received because no technical changes had been made in the plants.
This report may provide suggestive evidence that the exposures resulting from
the manufacture of technical grade TCI may cause chromosomal loss or nondisjunction
in humans. However, the lack of an appropriate matched control group and the
possibility that the incidence of hypodiploid cells was due to preparation of
the chromosomes rather than exposure to TCI renders this study inconclusive.
7.3 OTHER STUDIES INDICATIVE OF MUTAGENIC ACTIVITY
Additional studies have been conducted bearing on the genotoxicity of TCI.
These studies do not measure mutagenic events per se in that they do not
7-31
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TABLE 7-7. NUMERICAL CHROMOSOME ABERRATIONS IN TRICHLOROETHYLENE WORKERS
(adapted from Konietzko et al. 1978)
Parameter
> 13% Hypodiploid Cells
(n* = 9)
Mean Value Standard Error
(X) (Sx)
< 13% Hypodipoloid Cells
(n* = 18)
Mean Value Standard Error
(X) (Sx)
Daily average
concentration
(ppm)
Average maximum
concentration
75.0
17.4
61.4
23.1
(ppm)
Exposure (years)
Total dose
(ppm x day)
Age (years)
Alcohol (g/day)
Nicotine
(cigarettes/day)
205.8
5.9
0.55 x 106
38
18.9
12.8
96.3
4.8
0.72 x 106
9.4
29.3
12.7
115.7
5.9
0.31 x 106
40.4
33.6
10.1
57.6
5.1
0.39 x 106
9.7
37.5
10.2
*n =
n = Number of individuals studied.
7-32
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demonstrate the induction of heritable genetic alterations, but positive results
in these test systems show that DMA has been affected. Such test systems provide
supporting evidence useful for qualitatively assessing genetic risk.
7.3.1 Gene Conversion in Yeast
An increase in the frequency of mitotic gene conversion has been observed
after exposure to chemical mutagens as a consequence of genetic repair (Fabre
and Roman 1977). Bronzetti et al. (1978) tested TCI for its ability to induce
this endpoint at the ade and trp loci in strains D4 and D7 of Saccharomyces
cerevisiae. Strain D7 cells were incubated with TCI (ranging up to 40 mM)
with or without CD-I mouse liver S9 mix for 4 hours at 37°C in a liquid suspension
assay. A dose-related increase in trp+ convertants was observed in the tests
conducted with metabolic activation (41.0 +_ 2.6 x 10~6 convertants at 40 mM
compared to 16.3^ 0.42 x lO'6 convertants for the negative control). No
increase was noted in the test conducted without activation. Toxicity was
observed in both tests with a 40-50% reduction in cell survival at the highest
dose (40 mM). Twofold to fivefold increases in the incidences of gene conversion
were noted in these tests and also in the host-mediated assays where strains
D4 and D7 were instilled into the retroorbital sinus of adult male CD-I mice.
TCI was given by gavage in an acute exposure experiment (single dose of 400
mg/kg) or a subacute exposure experiment (22 pretreatment dosages of 150 mg/kg
5 days/week followed by a single 400 mg/kg dose). The largest increases (fourfold
over spontaneous values) were found in yeast isolated from the liver and kidney
(target organs for TCI toxicity), and gene conversion frequencies were higher
with subacute dosing compared to acute dosing.
Callen et al. (1980) treated D7 cells to 0, 15, and 22 mM solutions of TCI
in liquid suspension tests at 37°C for 1 hour followed by plating on selective
media. Cell survival was reduced by 30% at 15 mM and by > 99% at 22 mM TCI. A
7-33
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fivefold increase in trp convertants was detected at 15 mM compared to the
negative controls (76 x 10~6 vs. 14 x 10~6, respectively). There was also a
fourfold increase in mitotic recombination at the ade 2 locus at 15 mM TCI
compared to the negative controls (12 x 10~3 vs. 3 x 10~3, respectively).
These endpoints were not reported for the cultures exposed to 22 mM TCI because
of the extreme toxicity at this dose. The positive responses for gene conversion
and mitotic recombination in Saccharomyces strain D7 indicate TCI is capable of
interacting with DNA in yeast.
7.3.2 Sister Chromatid Exchange (SCE) Formation
White et al. (1979) evaluated the ability of anesthetic grade TCI (source
and purity not reported) to cause sister chromatid exchanges (SCE) in Chinese
hamster ovary cells. The material was administered as a gas to exponentially
growing cells at 0.17% of the atmosphere in the presence of 19% oxygen, 5%
carbon dioxide and 75.83% nitrogen for 1 hour with S9 mix prepared from Aroclor
1254-treated male rats. After exposure, the concentration of TCI gas in the
flask was measured by gas chromotography and found to be 59% of that delivered.
The cells were allowed to go two rounds of cell division in the presence of
bromodeoxyuridine (BrdU). Subsequently, chromosome preparations were made and
stained with Giemsa. One-hundred control and 100 treated metaphases were
scored for SCEs by a single observer. The number of SCEs/chromosome were
*? 0.536 +_ 0.018 and 0.514 +_ 0.018, respectively. Under the conditions of this
test, anesthetic grade TCI did not yield an increased incidence of SCEs.
However, it should be noted that the conditions of the test may not have provided
an adequate assessment of TCI's ability to cause sister chromatid exchanges.
In the first place, only one dose was administered and it may have been too
low. There was no indication of toxicity in the treated cells further suggesting
that they did not receive an adequate dose. In the second place, no concurrent
7-34
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positive controls were employed to ensure that the test system was working
properly. These deficiencies reduce the weight of the negative response reported
by White et al. (1979).
Gu et al. (1981) collected blood from six individuals occupationally exposed
to TCI, cultured their peripheral lymphocytes, and scored the chromosomes for
SCEs. Blood was also collected from nine additional persons who served as the
control group and as blood donors for testing trichloroethanol and chloral
hydrate, metabolites of TCI, in vitro for the induction of SCEs. A
significantly higher number of SCEs/cell was reported for the exposed group
compared to the controls (9.1 _+ 0.4 vs. 7.9 jf 0.2). There was no information
about the levels of TCI the subjects were exposed to in the workplace, but
there was a good correlation between the average number of SCEs/cell per person
and the levels of trichloroacetic acid and trichloroethanol measured in the
blood. Those with the highest mean value for SCEs also had the highest level
of these metabolites of TCI in their blood. Cultured lymphocytes were exposed
to trichloroethanol and chloral hydrate at 178 and 54.1 mg/1, respectively for
68-72 hours, and increases in SCEs were observed compared to the untreated
cultures. The results are tabulated as follows:
Group No. of Karyotypes X + SD Between from t Test *
1.
2.
3.
4.
5.
Control
Trichloroethanol (1)
Trichloroethanol (2)
Chloral Hydrate
TCI in vivo
212
24
72
52
194
7.9 +_ 3.0
9.8 _+ 3.2
9.2 4- 3.3
10.7 4. 4.0
9.1 +• 4.9
182
183
184
185
<0.05
<0.01
<0.001
<0.01
Calculated by the authors.
The results obtained by Gu et al. (1981) are suggestive of a positive response.
7-35
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The chemical substances to which the TCI workers in this study (Group 5) were
exposed and the extent to which such exposures occurred are unknown. It is
likely that they were exposed to other substances which have been shown to be
mutagenic (e.g., 1,2-epoxybutane and epichlorohydrin) and these may be responsible
for the weak effects. However, the positive responses obtained by Gu et al. (1981)
in in vitro studies with trichloroethanol (> 99.5% pure, Fluka Groups 283)
and crystalline chloral hydrate (Group 4) show that these metabolites of TCI could be
responsible for the induction of SCEs in TCI workers.
7.3.3 Unscheduled DMA Synthesis (UPS)
Four tests have been performed to assess the ability of TCI to cause
unscheduled DNA synthesis (Beliles et al. 1980, Perocco and Prodi 1981, Williams
and Shimada 1983, and Williams 1983). Although demonstration that a chemical
causes unscheduled DNA synthesis does not provide a measurement of its mutagenicity
it does indicate that the material interacts with DNA.
Beliles et al. (1980) exposed human WI-38 cells to TCI at doses ranging
from 0.1 to 5.0 ul/ml to evaluate its ability to cause UDS. The positive
controls N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benz(a)pyrene (BaP)
were used at concentration ranges betweem 1.25 to 10 ug/ml and 2.5 to 20 ug/ml,
respectively. Both agents were positive; however, BaP produced a non-linear
dose-response effect. All studies were conducted at the same time on a single
batch of WI-38 cells. TCI yielded a weak increase at the two lowest doses
(1.5 to 1.8-fold increase) both with and without activation (Table 7-8). The
response is only suggestive of an effect, but the testing done with S9 may not
have provided an optimal test of its gentoxic potential because the positive
control (BaP) was only effective at one dose (2.5 ug/ml); higher doses resulted
in a negative response although this may have due to cell killing.
Perocco and Prodi (1981) collected blood samples from healthy humans,
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TABLE 7-8. UNSCHEDULED DNA SYNTHESIS IN WI-38 CELLS
(adapted from Bellies et al. 1980)
Test
Nonactivation
Solvent Control, DMSO
MNNG -
T1 Trichloroethylene
Activation
Solvent Control, DMSO
BaP
Trichloroethylene
Compound
Concentration
(ul/ml)
0
5 ug/ml
0.1
0.5
1.0
5.0
0
2.5 ug/ml
0.1
0.5
1.0
5.0
DNA
(ug)
23.76
6.27
24.75
23.76
26.07
13.20
24.75
29.70
28.38
21.78
21.78
DPM
988.7
472.9
1561.3
1616.1
1221.3
150.6
834.5
1500.3
1622.1
860.6
1006.8
DPM/
(ug DNA)
41.6
75.4
63.1
68.0
46.8
11.4
33.7
50.5
57.2
39.5
46.2
Percent of
Control Response
100
181 +
152 +
164 +
113
27
100
170 +
100
170 +
117
137
-------
separated the lymphocytes, and cultured 5 x 105 of them in 0.2 ml medium for 4
hours at 37°C in the presence or absence of TCI (Carlo Erban, Milan, Italy or Merck-
Schuchardt, Darmstadt, FRG, 97-99% pure). The tests were conducted both in the
presence and in the absence of PCB-induced rat liver S9 mix. A comparison was
made between treated and untreated cells for scheduled DNA synthesis (i.e., DNA
replication) and unscheduled DNA synthesis. A difference was noted between the
groups with respect to scheduled DNA synthesis measured as dpm of [3H]
deoxythymidylic acid (TdR) after 4 hours of culture (2661 +_ 57 dpm in untreated
cells compared to 1261 +_ 36 dpm in cells treated with 5 ul/ml TCI). Subsequently,
2.5, 5, and 10 ul/ml (0.028, 0.056, and 0.11 umole/ml) TCI was added to cells
which were cultured in 10 mM hydroxyurea to suppress scheduled DNA synthesis.
The amount of unscheduled DNA synthesis was estimated by measuring dpm from
incorporated [3H]TdR 4 hours later. At 10 ul/ml TCI 392 +_ 22 and 439 +_ 23 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 (1320^ 57 at 5 ul/ml CMME versus 612 _+ 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.
TCI was evaluated by the authors as positive in the test, but they did not
state their criteria for classifying a chemical as positive. It is important
to note that none of the experimental values from cells treated with TCI without
metabolic activation had higher dpm values than the controls. Furthermore,
only one out of three experimental values was greater than the controls with
metabolic activation (668 4- 34 at 5 ul/ml compared to control value of 612 +_
7-38
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26). The increase was not statistically significant. Thus, the positive finding
reported in this work is judged to be inconclusive.
In unpublished papers, Williams and Shimada (1983) and Williams (1983)
tested samples of TCI for their ability to cause unscheduled DNA synthesis in
rat and mouse primary hepatocyte cultures (HPC DNA repair assay). In the
Williams and Shimada (1983) report two samples were tested using rat primary
hepatocytes. One was fully stabilized, and the other, referred to as
"non-stabilized," contained 99.9% TCI. No information was provided about the
remaining components. Two types of tests were conducted. One was a conventional
HPC DNA repair assay in that the hepatocytes were exposed to TCI added to the
culture medium. The other was a modified assay, and the cells were placed in
small glass dishes in a sealed incubator and exposed to TCI vapor. Tritiated
thymidine (^H-TdR) was added to the culture medium in both types of tests.
If TCI caused DNA damage repaired by the excision repair pathway, ^H-TdR
would be incorporated into the repaired DNA. To detect such incorporation,
the cells were fixed on glass slides and subsequently coated with radiotrack
emulsion. After an incubation period the slides were developed and examined
microscopically to count developed silver grains in the radiotrack emulsion
over the cell nuclei.
Williams and Shimada (1983) used 2-acetylaminof1uorene and vinyl chloride
as positive controls for the conventional and modified exposure assays,
respectively. 2-acetylaminof1uorene was highly active (170 grains/nucleus at
1Q-5M) and by comparison vinyl chloride was weakly active at 2.5% (5 grains/nucleus)
and 5% (11 grains/nucleus) in increasing unscheduled DNA synthesis compared to
the negative controls (3 grains/nucleus).
In the modified HPC DNA repair assay, cells were exposed to dosages up to
2.5% TCI in the air for 3 or 18 hours. No increase in the grain count was
7-39
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observed (the number of grains/nucleus ranged from 0.2 +_ 0.2 to 3.2 _+ 2.5 in
treated cells compared to 2.75 JH 2.76 in the untreated controls). Similarly,
negative results were reported in the conventional assay where the cells were
exposed to concentrations as high as 1% TCI in the medium.
There are several factors to consider in the analysis of this assay.
First, the system is designed to detect the occurrence of a specific type
of repair (i.e., "long-patch" excision repair). Xenobiotics which cause damage
that is not repaired or that is repaired by another mechanism will not be
detected as possessing genotoxic activity in the system.
Second, the endpoint measured may not actually reflect repair synthesis of
chromosomal DNA in the nucleus (Lonati-Galligani et al. 1983). Enhanced
incorporation of ^H-TdR into mitochondria! DNA or suppressed -^H-TdR incorporation
into mitochondria! DNA without a concomitant increased or decreased incorporation
into nuclear DNA can lead to false negative or positive results, respectively.
Thus, both nuclear incorporation and cytoplasmic incorporation should be measured
and the dose-responses plotted for each to serve as a basis for deciding whether
or not the compound has induced UDS.
Third, nuclear grain counts vary with nuclear size (Lonati-Galligani et al.
1983); thus, it may be appropriate to express grain counts as a percentage of
the measured nuclear area. In the studies by Williams and Shimada (1983) the
values were transformed by subtracting cytoplasmic grain counts from total
nuclear grains counts to give a net grain count; as a result the data are difficult
to evaluate, and the way they are presented may preclude the detection of weakly
active agents. Thus, although the results of this study are negative they do
not provide convincing evidence that TCI does not induce UDS. This is a
relevant point because vinyl chloride, a structurally related compound and known
mutagen, was only weakly active in this test and then only at doses as high or higher
7-40
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than the highest dose tested for TCI.
In the Williams (1983) study, TCI (source and purity not reported) was
assayed in the HPC DMA repair test using a conventional liquid exposure protocol.
Primary hepatocytes from B6C3F1 mice and Osborne-Mendel rats were tested in two
separate test series. A positive dose-related response was obtained in the
mouse cells between 10~4M and 10~2M (18.24 _+ 3.95 grains/nucleus at 10~2M vs.
0.78 _+ 1.18 grains/nucleus in the concurrent negative control). TCI was reported
to be nontoxic between 10~6M and 1Q-2M. A negative response was obtained in
testing conducted in rat cells. Positive and negative controls were employed
for both sets of experiments and responded appropriately, but the concerns
noted above regarding the Williams and Shimada (1983) study apply here as
well. In spite of this, the positive response in the mouse cells shows that a
commercially available sample of TCI is genotoxic in B6C3F1 mouse liver cells.
The positive responses for gene conversion and mitotic recombination in
yeast, and UDS in mouse cells provide evidence that commercially available TCI
reaches and is weakly active in damaging DNA.
7.4 EVIDENCE THAT TCI REACHES THE 60NADS
Although not bearing on the ability of TCI to cause mutations, two studies
have been conducted to assess its ability to induce sperm morphological abnormalities
in mice (Land et al. 1979 and Beliles et al. 1980). A statistically significant
increase was observed in both studies strongly suggesting that the compound or
an active metabolite reaches the gonads. Land et al. (1979) exposed groups of
(C57BL/C3H)Fj male mice (13 weeks old) to air (negative control), 0.02% and
0.2% anesthetic grade TCI vapor 4 hours/day for 5 consecutive days. Twenty-eight
days after the first day of exposure the animals were sacrificed. Both cauda
epididymides were removed, minced, strained, and stained in 1% Eosin Y. Slides
were prepared, mounted, coded, and evaluated by scoring 1000 sperm/slide at
7-41
-------
400 X magnification. Data were recorded as percent abnormal sperm. The means
were calculated for each group and compared with controls using the sample
t-test. As noted below, TCI exposure resulted in statistically significant
elevations in the percentage of morphologically abnormal sperm.
Number of % Abnormal Sperm
Agent Concentration % Animals Studied + S.E.
Air
Trichloroethylene
0.02
0.2
15
5
5
1.42 +_ 0.08
1.68 + 0.17
2.58 + 0.29*
*Statistically significant difference compared to controls (P < 0.001).
This study provides suggestive evidence that exposure to a high concentration
of TCI, during the period of meiotic division damages early spermatocytes.
Beliles et al. (1980) also observed a significant increase in sperm head
abnormalities in male CD-I mice but not albino rats [CRL: COBS CD(SD) BR].
Groups of 12 animals were dosed 7 hours/day for 5 days to 0, 100, or 500 ppm TCI.
After dosing, groups of four animals were killed at the end of 1 week, 4 weeks, and
10 weeks. Sperm was collected from the caudae epididymides and suspended in
0.85% saline, stained, and scored for abnormalities. At least. 2000 sperm were
analyzed/group (500/animal). The negative responses obtained in the rat study
are not considered adequate for assessing the ability of TCI to reach the testes
because the positive controls gave a negative response. Increased incidences
of abnormal sperm were observed in the mice of the 500 ppm group compared to
the negative controls for weeks 1 and 4 (i.e., 18.9% vs. 6.8% and 23.5% vs.
8.1%, respectively). A significant increase was also noted in the 100 ppm
exposure group compared to the negative control for week 4 (14.8% vs. 8.1%).
7.5 MUTAGENICITY OF METABOLITES
Trichloroethylene is metabolized to trichloroethanol, trichloroethanol
7-42
-------
glucuronide, and trichloroacetic acid. There are several intermediates leading
to the formation of these end products of metabolism, including some which have
been identified in man (e.g., chloral hydrate) and some of which have not been
identified but which are thought to occur (e.g., trichloroethylene oxide).
Some mutagenicity studies bearing on a few of TCI's metabolites are discussed
below. See the chapter on metabolism for a more detailed discussion.
Kline et al. (1982) tested the mutagenic potential of TCI oxide in Salmonella
strain TA1535 and £. coli strain WP2 uvrA. They observed no increases in
revertant colonies at doses ranging from 0.25 to 5 mM. Preincubation tests
were conducted and toxicity was observed at the highest doses demonstrating
that the bacteria were exposed to sufficient concentrations of the test material.
In the same paper epoxide metabolites of other chloroalkenes were found to be
mutagenic under the same conditions of test (i.e., cis- and trans-1-chloropropene
oxide, and cis- and trans-l,3-dichloropropene oxide). The genotoxicity of
these compounds was also assessed by differential growth inhibition of the DMA
polymerase-deficient JE. coli mutant polA~ compared to its polymerase proficient
polA+ parent. TCI oxide was positive in this assay yielding a dose-related
decrease in the survival of po!A~ cells (up to 40%) compared to the polA+
cells at doses ranging from 0.006 to 0.11 mM/ml.
Both Waskell (1978) and Gu et al. (1981) assessed trichloroethanol for its
ability to cause genetic effects. As discussed in the section on gene mutation
studies in Salmonella (using TA98 and TAIOO), Waskell (1978) tested trichloroethanol
at doses up to 7.5 mg/plate and found no increases in the revertants compared
to the negative controls.
Gu et al. (1981), on the other hand, found suggestive evidence that
trichloroethanol (at 178 mg/1) may be an inducer of SCEs in primary cultures
of human lymphocytes. In two experiments (of 24 and 72 karyotypes analyzed
7-43
-------
respectively), Gu et al. (1981) found 9.8 +_ 3.2 and 9.2 _+ 3.3 SCEs/nucleus
compared to the control value of 7.9 +_ 3.0.
Trichloroacetic acid and chloral hydrate were also tested by Waskell (1978)
in the study mentioned above, Trichloroacetic acid at 0.45 mg/plate was not
mutagenic to either TA98 and TA100. Chloral hydrate, however, was weakly active
yielding consistent but always less than twofold increases in the number of
TA100 revertants compared to the spontaneous level over a dose range from 0.5
to 10 mg both with and without metabolic activation. These increases suggest
chloral hydrate is mutagenic to Salmonella strain TA100.
In their testing of chloral hydrate Gu et al. (1981) found that chloral
hydrate at 54.1 mg/1 caused increases in the number SCEs in cultured human
lymphocytes compared to untreated control cells (i.e., 10.7 +_ 4.0 vs. 7.9 +_ 3.0).
Again the response was suggestive of an effect.
The weak increases obtained with trichloroethylene oxide, trichloroethanol,
and chloral hydrate are consistent with the hypothesis that they may be responsible
for the mutagenic effects observed with TCI after metabolic activation.
7.6 BINDING TO DNA
Stott et al. (1982) studied the pharmacokinetics and macromolecular inter-
actions of TCI in B6C3F1 male mice and male Osborne-Mendel rats. As part of
their study, mice were given 1200 mg/kg [14C] TCI (> 500 uCi/animal, specific
activity = 2.4 mCi/mmol) in corn oil. Five hours later, the animals were
killed, their livers were removed and frozen, and DNA was extracted two days
later. After enzymatic digestion, fractions corresponding to nucleosides or
purine and pyrimidine bases were separated and identified by high-pressure
liquid chromatography. Aliquots were placed in scintillation fluid and the ^C
content was determined by liquid scintillation counting. Anaylsis of the data
indicated that the ability of TCI to alkylate DNA was low. A maximum estimate
7-44
-------
of the average DNA alkylation level was 0.62 _+ 0.42 alkylations/106 nucleotides.
These data suggest TCI or its metabolites are capable of binding to DNA, but
only to a limited extent, and are consistent with the suggestive mutagenic
responses reported for TCI and certain of its metabolites.
7.7 SUMMARY AND CONCLUSIONS
Commercially available samples of TCI have been shown to cause responses
suggestive of a positive effect in gene mutation studies using bacteria, fungi,
higher plants, and somatic cells of mice in vivo. These responses occurred with
metabolic activation only, suggesting the involvement of one or more metabolites
of TCI. This requirement for metabolic activation argues against the possibility
that epoxide stabilizers found in many commercial samples are responsible. The
marginally increased incidences of revertant counts were only observed at high
doses. Thus, commercially available TCI is only weakly mutagenic at most.
TCI was not shown to cause structural chromosomal aberrations in the one test
conducted to assess this endpoint.
Other tests provide evidence that commercially available TCI damages DNA.
Suggestive and weak-positive responses have been observed in yeast (gene conver-
sion and mitotic recombination), mice (UDS), and humans (SCE and UDS). Metabolic
activation was required to obtain the positive responses. Certain metabolites
of TCI have been tested for their mutagenic potential, and suggestive positive
effects have been shown. TCI or a metabolite(s) may be capable of binding to
DNA, but only minimally.
TCI causes weak increases in morphologically abnormal sperm providing
evidence that its reaches the gonads. A synopsis of the results of these
studies is presented in Table 7-9.
The available data provide suggestive evidence that commercial-grade TCI
is a weakly active indirect mutagen, causing effects in a number of different
7-45
-------
TABLE 7-9. SUMMARY OF TESTS FOR MUTAGENICITY OF TCI
Test
Category Organism Type of Test
I. Gene Salmonella Reverse mutations
Mutations typhimurium in vitro
Plate
incorporation
tests
Vapor
exposure
Escherichia Forward and
col i reverse mutations
Schi zosaccharomyces Forward mutations
pombe (Host-mediated
assays)
Purity
of TCI Results
Technical-
grade
Technical- +
grade
Purified
Anesthetic-
grade
Purified3 *
Purified3 *
Reagent3 *
grade
Analytical- *
grade
Technical- *
grade
Comments
No control to test
effectiveness of
S9 mix. No
precautions to
prevent evaporation.
Two-fold increase
1.8-fold increase
1.3-fold increase
1.7-fold increase
Positive for reverse
mutations only at
arg locus (two-fold
increase).
1.7-fold increase
Reference
Henschler et
al., 1977
Margard, 1978
Margard, 1978
Waskell, 1978
Bartsch et
al., 1979
Baden et
al. 1979
Simmon et
al., 1977
Greim et
al . 1975
Loprieno et
al., 1979
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = inconclusive
-------
TABLE 7-9. (continued)
.e-
—i
Test
Category Organism
Purity
Type of Test of TCI Results Comments
Purified
Reference
Loprieno et
al., 1979 !
Saccharomyces
cerevisiae
Tradescantia
Drpsophila
melanogasTer
Mouse
Purified
Purified3
Reverse mutations Technical- x
(in vitro) grade
ACS regeant- +
grade
Technical- +
grade
Forward mutations Unknown *
Sex-linked Technical
recessive lethals grade
Spot test
Technical'
grade
Technical
grade
Epichlorohydrin
and epoxybutane
were also negative.
High toxicity
Four-fold increase
both host-mediated
assay and liquid
suspension test.
Two-fold increase"
Six-fold increase
Rossi et al.,
1983
Mondino,
1979
Shahin and
Von Borstel,
1977
Bronzetti et
al., 1978
Call en et
al., 1980
Schairer
et al., 1978
Abrahamson
and Valencia,
1980
Bellies et
al., 1980
Fahrig et
al., 1977
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
TABLE 7-9. (continued)
-c-
oo
Test
Category Organism
II. Chromosomal Drosophila
Aberrations melanogaster
Rat
Mouse
Human
III. Other Saccharomyces
Studies cerevisiae
Indicative
of Mutagenic
Damage
Purity
Type of Test of TCI Results
Chromosome Technical -
loss grade
Bone marrow Technical- x
grade
Dominant Purified3
lethal
Micronucleus Analytical- x
grade
Breaks Occupational
exposure
Hypodiploid cells Occupational x
exposure
Gene conversion ACS reagent- +
grade
Comments
Positive control
given by different
route of exposure.
Doses of TCI may
have been too low.
Positive response
reported by authors
may be due to arti-
facts in mature
erythrocytes.
Unmatched control
group. Hypodiploid
cells can be caused
by preparation of
chromosomes.
Two-fold increase
with metabolic
activation.
Reference
Beliles et
al., 1980
Beliles et
al., 1980
Slacik-
Erben et
al., 1980
Duprat and
Gradiski,
1980
Konietzko et
al., 1978
Konietzko et
al., 1978
Bronzetti
et al., 1978
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
TABLE 7-9. (continued)
Test
Category Organism
Mouse
Human
Type of Test
Mitotic
recombination
Sister chromatid
exchange
Sister chromatid
exchange
Purity
of TCI
Technical-
grade
Technical-
grade
Anesthetic-
grade
Occupational
exposure
Results Comments
+ Five-fold increase
+ Four-fold increase
No positive
controls. No
evidence of
toxicity.
* Increases correlated
with presence of TCI
Reference
Call en et
a.1., 1980
Call en et
al., 1980
White et
al., 1979
Gu et al. ,
1981
I
.p-
Rat
Unscheduled DNA
synthesis
HPC DNA
repair
assay
Technical- *
grade
Technical- x
grade
Stabilized
Unstabi li zed
Technical-
grade
metabolites tri-
chloroethanol and
trichloroacetic acid
in the blood.
1.5 to 1.8-fold
increases
Vinyl chloride only
weakly active.
Beliles et !
al., 1980 ;
Perocco and.
Prodi, 1981
Williams and
Shimada,
1983
Williams and
Shimada,
1983
Wi11i ams,
1983
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
TABLE 7-9. (continued)
Test
Category Organism Type of Test
Mouse HPC DNA
repair
assay
IV. Evidence Mouse Morphological
TCI Reaches sperm
the Gonads abnormalities
Purity
of TCI Results Comments Reference
Technical- +
grade
Anesthetic- *
grade
Technical- +
grade
8- to 20-fold Williams,
increases 1983
1.8-fold increase Land et
al., 1979
Three-fold iqcrease BeTiles et
al., 1980
V. Mutagenicity of Metabolites
A. TCI-oxide Salmonella
typhimurium
Reverse mutations
Kline et
al., 1982
Escherichia
coTi
Reverse mutations
Kline et
al., 1982
B. Trichloroethanol
Salmonella
typhimurium
Human
lymphocytes
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
Differential
killing of repair
deficient bacteria
Reverse mutations
Sister chromatid
exchange
40 % decreases in
survival of Pol-
vs. Pol+ cells
Kline et
al . , 1982
Waskell,
1978
Gu et al.,
1981
-------
TABLE 7-9. (continued)
Test
Category
Organism
Type of Test
Purity
of TCI
Results
Comments
Reference
vn
C. Trichloroacetic Acid
Salmonella
typhimurium
D. Chloral Hydrate
Salmonella
typhimurium
Human
lymphocytes
Reverse mutations
Reverse mutations
Sister chromatid
exchange
1.6-fold increase
Waskell,
1978
Waskell,
1978
Gu et al.,
1981
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
test systems representing a wide evolutionary range of organisms, including
humans. Based on this, commercial TCI may have the potential to cause weak or
borderline increases above the spontaneous level of mutagenic effects in exposed
human tissue. The observations that TCI causes adverse effects in the testes
of mice suggest TCI may cause adverse testicular effects in man, too, provided
that the pharmacokinetics of TCI in humans also results in its distribution to
the gonads. The data on pure TCI do not allow a conclusion to be drawn about
its mutagenic potential. However, mutagenic potential cannot be ruled out. If
it is mutagenic, the available data suggest TCI would be a very weak, indirect
mutagen.
7-52
-------
7.8 REFERENCES
Abrahamson, S., and R. Valencia. 1980. Evaluation of substances of interest
for genetic damage using Drospphila melanogaster. Final sex-linked recessive
lethal test report to FDA on 13 compounds. FDA Contract No. 233-77-2119.
Baden, J.M., M. Kelley, R.I. Mazze, and V.F. Simmon. 1979. Mutagenicity of
inhalation anesthetics: Trichloroethylene, divinyl ether, nitrous oxide and
cyclopropane. Br. J. Anaesth. 51:417-421.
Bartsch, H., C. Malaveille, A. Barbin, and G. Planche. 1979. Mutagenic and
alkylating metabolites of halo-ethylenes, chlorobutadienes and dichlorobutenes
produced by rodent or human liver tissues. Arch. Toxicol. 44: 249-277.
Beliles, R.P., D.J. Brusick, and F.J. Heeler. 1980. Teratogenic-mutagenic
risk of workplace contaminants: Trichloroethylene, perchloroethylene, and
carbon disulfide, contract report, contract No. 210-77-0047. U.S. Department
of HHS, NIOSH, Cincinnati, OH 45226.
Bronzetti, G., E. Zeiger, and D. Frezza. 1978. Genetic activity of
trichloroethylene in yeast. J. Environ. Pathol. and Toxicol. 1:411-418.
Callen, O.F., C.R. Wolf, and R.M. Philpot. 1980. Cytochrome P-450 mediated
genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons
in Saccharomyces cerevisiae. Mutat. Res. 77: 55-63.
Duprat, P., and D. Gradiski. 1980. Cytogenetic effect of trichloroethylene in
the mouse as evaluated by the micronucleus test. IRCS Medical Science Libr.
Compendium 8:182.
Fabre, F., and H. Roman. 1977. Genetic evidence for inducibility of recombination
competence in yeast. Proc. Natl. Acad. Sci. (U.S.A.) 74(4): 1667-1671.
Fahrig, R. 1977. The mammalian spot test (Fellfleckentest) with mice. Arch.
Toxicol. 38:87-98.
Greim, H., G. Bonse, Z. Radwan, D. Reichert, and D. Henshcler. 1975.
Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as
a function of metabolic oxirane formation. Biochem. Pharmacol. 24:2012-2017.
Gu, Z.W., B. Sele, P. Jalbert, M. Vincent, C. Marka, D. Charma, and J. Faure.
1981. Induction d'echanges entre les chromatides soeurs (SCE) par le
trichloroethylene et ses metabolites. Toxicological European Research 3:63-67.
Henschler, D., E. Eder, T. Neudecker, and M. Metzler. 1977. Carcinogenicity
of trichloroethylene: Fact or artifact? Arch. Toxicol. 37:233-236.
Kline, S.A., E.C. McCoy, H.S. Rosenkranz, and B.L. Van Duuren. 1982. Mutagenicity
of chloroalkene epoxides in bacterial systems. Mutat. Res. 101:115-125.
Konietzko, H.W., W. Haberlandt, H. Heilbronner, G. Reill, and H. Weichardt.
1978. Cytogenetische Untersuchungen an Trichlorathylen-Arbeitern. Arch.
Toxicol. 40:201-206.
7-53
-------
Land, P.E., E. L. Owen, and H.W. Linde. 1979, Mouse sperm morphology following
exposure to anesthetics during early spermatogenesis. Anesthesiology 51(3):
S259.
Lonati-Galligani, M., P.H.M. Lohman, and F. Berends. 1983. The validity of the
autoradvographic method for detecting DNA repair synthesis in rat hepatocytes
in primary culture. Mutat. Res. 113:145-160.
Loprieno, N., R. Barale, A.M. Rossi, S. Fumero, G. Meriggi, A. Mondino., and
S. Silvestri. 1979. In vivo mutagenicity studies with trichloroethylene and
other solvents (prelimTn~ary results). Unpublished.
Margard, W. 1978. Summary report on in vivo bioassay of chlorinated hydrocarbon
solvents. Unpublished.
Matter, B.E., and I. Jaeger. 1975. The cytogenetic basis of dominant lethal
mutations in mice. Studies wth TEM, EMS, and 6-mercaptopurine. Mutat. Res.
33:251-260.
McCann, J., and B.N. Ames. 1977. The Salmonella/microsome mutagenicity test:
Predictive value for animal carcinogenicity in H.H. Hiatt, J.D. Watson, and J.A.
Vlinsten eds. Origins of Human Cancer, Cold Spring Harbor Laboratory, New York,
pp. 1431-1450.
Mondino, A. 1979. Examination of the live mutant activity of the trichloroethylene
compound with "Schizosaccharomyces pombe." Unpublished.
National Cancer Insititute (NCI). 1976. Carcinogenesis bioassay of
trichloroethylene. CAS no. 79-01-6. NCI-CG-TR-2.
Ogli, K. 1977. Reversible growth suppressing effects of various anesthetics
in Euglena gracilis. Med. Jour, of Osaka Univer. 28(1):15-21.
Okuda, C., and K. Ogli. 1976. The effects of anesthetics on microtubule
protein. Jap. J. Anesthesiology 25:785-791.
Perocco, P., and G. Prodi. 1981. DNA damage by haloalkenes in human lymphocytes
cultured in vitro. Cancer Letters 13:213-218.
Russell, L.B., and B.E. Matter. 1980. Whole-mammal mutagenicity tests.
Evaluation of five methods. Mutation Research 75:279-302.
Schairer, L.A., J. Van't Hof, C.G. Hayes, R.M. Burton, and F.J. deSerres.
1978. Measurement of biological activity of ambient air mixtures using a mobile
laboratory for in situ exposures. Preliminary results from the Tradescantia
plant test sytem. ~EPA~ Pub. 600/9-78-027:421-440.
Schmid. W. 1975. The micronucleus test. Mutat. Res. 31:9-15.
Shahin, M.M., and R.C. Von Borstel. 1977. Mutagenic and lethal effects of
-benzene hexachloride, dibutyl phthalate and trichloroethylene in Saccharomyces
cerevisiae. Mutat. Res. 48:173-180.
7-54
-------
Simmon, V.F., K. Kauhanon, and R.G. Tardiff. 1977. Mutagenic activity of
chemicals identified in drinking water. Pages 249-258 in D. Scott,
B.A. Bridges, and F. H. Solves, eds. Progress in genetic toxicology.
Elsevier/North Holland Biomedical Press.
Slacik-Erben, R., R. Roll, 6. Franke, and H. Uehleke. 1980. Trichloroethylene
vapours do not produce dominant lethal mutations in male mice. Arch. Toxicol.
45: 37-44.
Sturrock, J.E., and J.F. Nunn. 1975. Mitosis in mammalian cells during exposure
to anesthetics. Anesthesiology 43(l):21-33.
Waskell, L. 1978. A study of the mutagenicity of anesthetics and their
metabolites. Mutat. Res. 57:141-153.
White, A.S., S. Takehisa, E.I. Eger II, S. Wolff, and W.C. Stevens. 1979.
Sister-chromatid exchanges induced by inhaled anesthetics. Anesthesiology 50:
426-430.
Williams, G.M. 1983. Draft Final Report TR-507-18. DNA repair test of 11
chlorinated hydrocarbon analogs. For ICAIR Life Systems, Inc. and U.S.
Environmental Protection Agency. Dr. Harry Milman, Project Officer, 401 M St.,
SW, Washington, DC 20460. Unpublished.
Williams, 6.M., and T. Shimada. 1983. Evaluation of several halogenated ethane
and ethylene compounds for genotoxicity. Final report for PPG Industries,
Inc., Dr. A.P. Leber, PPG Industries, Inc., One Gateway Center, Pittsburgh, PA
15222. Unpublished.
7-55
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8. CARCINOGENICITY
8.1 ANIMAL STUDIES AND CELL TRANSFORMATION STUDIES
There have been a number of laboratory investigations of the carcinogenic
potential of trichloroethylene (TCI) in experimental animals. These studies
have been done using rats, mice, and hamsters, with TCI administered by inhalation,
gavage, subcutaneous injection, and topical application. These results are
summarized in Table 8-1. Of the studies done, the evidence for the carci nogenicity
of TCI consists of statistically significant increases of hepatocellular carcino-
mas in male and female B6C3F1 mice [National Toxicology Program (NTP) 1982,
National Cancer Institute (NCI) 1976, Bell et al. 1978], malignant lymphomas in
female NMRI mice (Henschler et al. 1980), and renal adenocarci nomas, by life
table and incidental tumor tests, in male Fischer 344 rats (NTP 1982). However,
inadequacies in the NTP study in rats and the IBT study in mice and limitations
in the interpretation of the data in the Henschler et al. study in mice are
evident, as discussed herein.
8.1.1 Oral Administration (Gavage): Rat
8.1.1.1 National Toxicology Program (NTP 1982)--A carcinogenicity bioassay on
TCI in Fischer 344 rats was recently completed for the National Toxicology
Program, and a 1982 draft report of the study has been reviewed by its Board of
Scientific Counselors.
The TCI test sample was described as a high-purity "Hi-Tri" product obtained
in two lots, one being Dow Chemical Company Lot No. TB05-206AA, used for a
preliminary dose-finding study and for the first 7 months of the bioassay, and
the other being Missouri Solvents Lot No. TB08-039AA, used for the duration of the
bioassay. Samples of TCI were chemically analyzed by gas-liquid chromatography,
8-1
-------
TABLE 8-1. TCI CARCINOGEN 1C ITY BIOASSAYS IN ANIMALS
TCI chemical
Study purity
NTP 1982 Purified
SCI 1976 Technical grade
Bel 1 et al . Technical grade
(MCA) 1978
Maltonl 1979 Purified
Henschler et al . Purified
1980
Species
Mice, B6C3F1
Males
Females
Rats, Fischer
344
Males
Females
Mice, B6C3F1
Males
Females
Rats, Osborne-
Mendel
Males
Females
Mice, B6C3F1
Males
Females
Rats, Charles
River
Males
Females
Rats, Sprague-
Dawley
Males
Females
Mice, Han:NMRI
Males
Females
Rats. HanrWist
Males
Females
Hamsters, Syrian
Males
Females
Dose levels,
route
1000 ing/ kg/ day
gavage, 103 wk
500, 1000 mg/kg/day
gavage, 103 wk
1119, 2339 rog/kg/day
869. 1739 mg/kg/day
gavage, 78 wk
549, 1097 ing/kg
gavage, 88 wk
100, 300, 600 ppm
inhalation, 24 mo
100, 300. 6UO ppm
inhalation, 24 mo
250, 50 mg/kg
gavage, 62 wk
100, 500 ppm
inhalation, 78 wk
100, 500 ppm
inhalation, 78 wk
100, 500 ppm
inhalation, 78 wk
...I -.,..— — --..— .— .,-.. .
Results
Treatment -related
hepatocellular carcinomas
In males and females
Renal adenocard nomas
in treated males
Treatment-related
hepatocellular carcinomas
in males and females
Negative
Increased incidence of
hepatocellular carcinomas
In males and females
with dose
Negat 1 ve
Negative
Increased incidence of
malignant lymphomas
in females
Negat 1 ve
Negative
8-2
-------
TABLE 8-1. (continued)
Study
Van Duuren et al .
1979, 1983
TCI chemical
purity
Purified
Species
Swiss mice
ICR/Ha
Female
Dose levels,
route
1 mg, 3x/wk, 581 d
topical
Results
Negative
Purified TCI
epoxlde
NTP 1982
Ma) ton) 1979
Henschler et al,
19BU
Purified
Purified
Purified and
stabilized
Female
Female
Female
Male
Female
Female
Female
Rats, Osborne-
Mendel
Marshall 540,
August 28807,
AC I
Mice, B6C3F1
Swiss albino
Rats, Sprague-
Oawley
Swiss mice
ICR/Ha
1 mg, 3x/wk, 14 d
2.5 ug phorbol
myrlstate acetate,
topical 452 d
0.5 mg sc/wk, 622d
0.5 mg, once wk
gavage, 622 d
1 mg TCI epoxlde,
3x/wk, 2.5 ug
phorbol myrlstate
acetate, topical,
452 d
2.5 mg TCI epoxlde
3x/wk, topical for
526 d
0.5 my TCI epoxlde,
once wk, sc, 547 d
Gavage, 104 wk
Inhalation, 78 wk
Inhalation, 104 wk
Gavage, 78 wk
Negative
Negative
Negative
Negative
Negative
Negative
In progress
In progress
In progress
8-3
-------
mass spectroscopy, infrared spectrophotometry, and nuclear magnetic resonance
spectroscopy. Analytical results showed a TCI purity of greater than 99.9%.
Epichlorohydrin was not found by gas chromatography-mass spectroscopy at a
detection level of 0.001% (v/v). The TCI product contained 8 ppm of
diisopropylamine as a stabilizer. Refrigeration at 4°C created no analytically
detectable decrease in purity over the course of product usage.
Stock solutions of TCI in corn oil were made once weekly for the first 16
weeks and once monthly for the remainder of the bioassay. Analysis by gas
chromatography showed that these solutions were stable for 7 days at room
temperature and for 4 weeks at 4°C. Stock solution levels of TCI were within
10% of nominal concentrations.
Based on survival, body weights, and pathology in a preliminary 13-week
dose-finding study, estimated maximally tolerated and one-half maximally
tolerated doses were selected for the bioassay. For the bioassay, 50 males
and 50 females per group were given nothing (untreated controls), corn oil
alone (vehicle-controls), 500 mg/kg/day of TCI in corn oil (low dose), or 1,000
mg/kg/day of TCI in corn oil (high dose). Rats were dosed daily, 5 days per
week, for 103 weeks. The volume of administered corn oil was 1 ml.
Rats were randomly assigned to dose groups when 8 weeks old at the start
of the study. The animals were caged in groups of 5, and decedents were
replaced during the first 1.5 weeks. Temperature and humidity in the animal
quarters were 22 to 24°C and 40 to 60%, respectively, and there were 10 to 15
room air exchanges every hour.
The animals were observed daily. Body weights were recorded weekly
for the first 12 weeks and monthly thereafter. Each animal was necropsied,
and tissues and organs, as well as all lesions and tumors, were evaluated
histopathologically.
8-4
-------
Terminal sacrifice of survivors occurred at 111 to 115 weeks.
Body weight trends in Figure 8-1 show reduced mean weight gains in high-dose
males and in both treatment groups of females. A dose-related effect on mean
body weights in females became evident after 60 weeks, and mean weight gain
between vehicle-control and low-dose males was similar (mean body weights at the
start of the study were 161 and 141 g for vehicle-control and low-dose males,.
respectively).
A dose-related reduction in survival of treated male rats and a smaller
decrease in survival of high-dose females were evident through survival pro-
babilities estimated by the Kaplan and Meier (1958) technique (Figure 8-2).
Differences in survival among female groups were not significant (P < I).05);
however, survival was significantly (P < 0.05) lower in low-dose males
(P = 0.005) and high-dose males (P = 0.001) compared to vehicle-control males.
The following numbers of rats which were killed by "gavage error" were censored
from the survival curves in Figure 8-1: 1 vehicle-control male, 3 low-dose
males, 10 high-dose males, 2 vehicle-control females, 5 low-dose females, and 5
high-dose females. The numbers of rats alive at terminal sacrifice were as
follows: 35/50 vehicle-control males, 20/50 low-dose males, 16/50 high-dose
males, 36/50 vehicle-control females, 33/50 low-dose females, and 26/50 high-
dose females.
It is indicated in the NTP (1982) report that, because of reduced survival
in treated male rats, the statistical methods used which adjust for intercurrent
mortality (life table analysis, incidental tumor test) would provide more mean-
ingful results, statistically, than unadjusted statistical methods (Fisher
Exact Test, Cochran-Armitage Trend Test). Kidney tumor data in Table 8-2 show
a significant (P < 0.05) increase in kidney tubular adenocarcinoma incidence in
high-dose males (3/16) compared to vehicle-control males (0/33) at terminal
8-5
-------
a
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Figure 8-1
MALE RATS
D VEHICLE CONTROL
O LOW DOSE
A HIGH DOSE
I 1 I I I I
20 30 40 50 60 70
TIME ON STUDY (WEEKS)
80
I
30
100
1 10
a a
a a
a °
2 A
a a
a a
a
o u
A A
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a ° A 6
P o © e
FEMALE RATS
D VEHICLE CONTROL
O LOW DOSE
A HIGH DOSE
I
10
1
20
I
30
I
40
I
50
I
60
I
70
I
80
90 100 110
TIME ON STUDY (WEEKS)
Growth curves for rats administered trichloroethylene in corn
oil by gavage. (nTP 1982)
8-6
-------
PROBABILITY OF SURVIVAL
PROBABILITY OF SURVIVAL
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-------
TABLE 8-2. ANALYSIS OF PRIMARY TUMORS IN MALE
(NTP 1982)
KIDNEY: TUBULAR-CELL ADENOCARCINOMA
Tumor rates
Vehicle-
control
Low
dose
High
dose
Overallb
Adjusted0
Terminal d
Statistical tests6
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
0/48 (0%)
0.0%
0/33 (0%)
P = 0.009
P = 0.009
P = 0.038
0/49 (0%)
0.0%
0/20 (0%)
(f)
(f)
(f)
3/49 (6%)
18.8%
3/16 (19%)
P = 0.028
P = 0.028
P = 0.125
KIDNEY: TUBULAR-CELL ADENOMA OR ADENOCARCINOMA
Tumor rates
Vehicle-
control
Low
dose
High
dose
Overall13
Adjusted0
Terminald
Statistical tests6
Life table
Incidental tumor test
Cochran-Armitage Trend
Fisher Exact Tests
0/48 (0%)
0.0%
0/33 (0%)
P = 0.019
P = 0.030
P = 0.084
2/49 (4%)
5.6%
0/20 (0%)
P = 0.194
P = 0.327
P = 0.253
3/49 (6%)
18.8%
3/16 (19%)
P = 0.028
P = 0.028
P = 0.125
aDosed groups received doses of 500 or 1,000 mg/kg of trichlorethylene by
gavage.
^Number of tumor-bearing animals/number of animals examined at the site.
cKaplan-Meier estimated lifetime tumor incidence after adjusting for
intercurrent mortality.
"^Observed tumor incidence at terminal kill.
eBeneath the control incidence are the P-values associated with the trend test.
Beneath the dosed group incidence are the P-values corresponding to pairwise
comparisons between that dosed group and the controls. The life table analysis
regards tumors in animals dying prior to terminal kill as being (directly or
indirectly) the cause of death. The incidental tumor test regards these
lesions as non-fatal. The Cochran-Armitage Test and the Fisher Exact Test
compare the overall incidence rates directly.
configuration of tumor incidences precludes the use of this statistic.
8-8
-------
sacrifice by life table (P = 0.028) and incidental tumor (P = 0.028) tests.
Each test for linear trend was significant (P < 0.05), as shown in Table 8-2.
Historical corn oil control data for male Fischer 344 rats evaluated in the NTP
bioassay program as of June 15, 1981 for studies of at least 104 weeks showed a
renal tumor incidence of 3/748 (0.4%) adenocarcinomas; thus, this tumor type is
rare in this strain of rats. However, the comparison for kidney adenocarclnomas
between high-dose and vehicle-control male rats was not significant (P < 0.05) by
the Fisher Exact Test, and the statistical significance achieved with the other
tests above reflects the significantly (P <_ 0.005) higher mortality in high-dose
males as compared to vehicle-control males. Other observed kidney tumors in rats
include a transitional cell carcinoma of the renal pelvis in a low-dose male,
tubular adenomas in two low-dose males, a carcinoma (NOS) of the renal pelvis in
a high-dose male, a transitional cell papilloma of the renal pelvis in an
untreated control male, and a tubular adenocarcinoma in a high-dose female.
Renal tubular adenocarcinomas had a general histopathologic appearance of
solid sheets of cells, often atypical, and small areas of necrosis. These
tumors commonly invaded surrounding normal tissue. Small renal tubular adenomas
consisted of solid collections of tubular epithelial cells filling contiguous
tubules. In larger adenomas there was greater evidence of cellular atypia, and
there was no sharp demarcation between larger adenomas and adenocarcinomas.
Renal adenocarclnomas were distinguished from renal adenomas by basement membrane
invasion.
Toxic nephrosis characterized as cytomegaly was found in 98 treated male
rats and 100 treated female rats, but not in control rats. This lesion was
found in early deaths, and it progressed in severity during the course of the
study. Enlargement of tubular cells was detected early and progressed to the
extent that some tubules were dilated and hard to identify. With longer sur-
8-9
-------
vival the number of dilated cells decreased, corresponding tubules became dilated,
and the basement membrane had a striped appearance. The most advanced stage of
cytomegaly extended into the renal cortex. Cytomegaly was graded as: 1 - slight,
a subtle change in a limited area of the kidney; 4 - severe, an obvious lesion
which involved a substantial part of the kidney, and which could alter its
function; 2 - moderate; 3 - well marked. Average grades for each group of rats
were: 0 for male and female controls, 2.8 for low-dose males, 3.1 for high-dose
males, 1.9 for low-dose females, and 2.7 for high-dose females. In contrast,
renal nephropathy, a common spontaneous lesion in aging rats, was of lower
incidence in treated males (88% in untreated controls, 85% in vehicle controls,
59% in low-dose, and 37% in high-dose) which could relate to the higher mortality
in treated males.
Increases of tumor incidences at other sites in treated rats were not
apparent. Malignant mesothelioma incidence was higher in low-dose males [vehicle-
controls, 1/50; low-dose, 5/50 (P = 0.042, life table test); high-dose, 0/48],
but high-dose animals did not develop this tumor type, and the low-dose incidence
was not significantly (P < 0.05) increased according to the other statistical
tests used in this study.
The TCI doses used in this study produced statistically significant decreases
in the survival of male rats, and a decreased survival in high-dose female rats
compared to matched vehicle-controls was apparent, although this difference was not
found to be statistically significant. By definition (NCI 1976) the maximum
tolerated dose in cardnogenicity bioassays is the highest dose that can be
administered during the chronic study which will not be expected to alter the
animals' survival rate from effects other than carci nogenicity. Thus, TCI doses
beyond that maximally tolerated, particularly in male rats, appear to have been
used in this study. The toxicity of TCI in the kidney tubule region, as
8-10
-------
cytotnegaly, approximated the "well-marked" grade on the average in both groups
of treated male rats and the high-dose group of female rats, and it has been
noted that the severity of this lesion increased with longer survival. Although
a small but statistically significant excess of renal tubular adenocarcinomas in
high-dose males compared to vehicle-control nales was calculated by statistical
methods which adjusted for mortality, the NTP and its Board of Scientific
Counselors have concluded that this study in Fischer 344 rats is inadequate for
a judgment on the carcinogenicity of TCI. The following is quoted from the
latest version (received May 24, 1983) of the abstract on this study from the
NTP:
Under the conditions of these studies, epichlorohydrin-free
trichloroethylene caused renal tubular-cell tumors in male K344/TI
rats, produced nephropathy in both sexes, shortened the survival
times of males and did not produce a carcinogenic response among
females. This experiment was considered to be inadequate to
evaluate the presence or absence of a carcinogenic response to
trichloroethylene in male F344/N rats.
Use of additional lower doses in this study co'uld have provided a broader
evaluation of dose-response for the observed toxic effects of TCI.
8.1.1.2 National Cancer Institute (NCI 1976)—A carcinogenicity study of TCI
in Osborne-Mendel rats was reported by the National Cancer Institute (NCI)
in 1976. The composition of the TCI sample used, determined by vapor-phase
chromatography-mass spectrometry, was as follows: TCI, _> 99.0% (w/to);
1,2-epoxybutane, 0.19%; ethyl acetate, 0.04%; N-methylpyrrole, 0.02%;
diisobutylene, 0.03%; epichlorohydrin, 0.09%. The rats were 48 days old when
the study began. Treatment groups included 50 animals/sex/dose, and 20 animals/
sex comprised matched vehicle-control (corn oil) groups. Ninety-nine male and
98 female rats used as vehicle "colony controls" were also compared with treat-
-------
merit groups in this study. Vehicle "colony controls" included matched controls
for TCI and other chemicals being tested concurrently.
Maximum and one-half maximum tolerated doses for the carci nogenici ty study
were estimated from survival, body weight, and necropsy results from a preliminary
8-week subchronic test. In the carcinogenicity study, treated male and female
rats were given time-weighted average doses, by gavage, of 549 and 1,097 mg/kg/
treatment of TCI 5 days/week for 78 weeks. Doses were presented as time-weighted
averages due to the dose level changes made during the study, as shown in Table 8-3,
on the basis of apparent toxicity from treatment with TCI.
Rats were allowed to survive until terminal sacrifice at 32 weeks post-
treatment. Animals were observed daily for mortality and toxic signs, and
body weights and food consumption were recorded weekly for the first 10 weeks
and monthly thereafter. Survivors and decedents were necropsied, and tissues
and organs, including tumors and lesions, were examined histopathologically.
At the beginning of the study, rats were randomly assigned to treatment
and control groups according to body weight. Body weight trends in Tables 8-4
and 8-5 show decreased body weight gain in males and females in both treatment
groups compared to matched vehicle-controls. Food consumption trends in Tables
8-4 and 8-5 indicate decreased food intake by both groups of treated female rats
as compared to matched vehicle-controls, whereas food intake by treated male
rats appeared comparable to that of matched vehicle-controls. Although decreased
body weights in treated animals could reflect decreased food consumption, it
would appear that decreases in food consumption and body weights ultimately
were a result of toxicity from treatment with TCI. For example, food con-
sumption between matched control and low-dose males was approximately equal,
whereas body weight gain was less in the latter group.
8-12
-------
TABLE 8-3. DOSAGE AND OBSERVATION SCHEDULE - TRICHLOROETHYLENE CHRONIC STUDY
ON OSBORNE-MENDEL RATS
(NTP 1982)
Dosage
group
Low-dose
males and
females
High-dose
males and
females
Dose (mg
TCI Ag
body wt)
650
750
500
500
no treatment
1300
1500
1000
1000
no treatment
Percent of
TCI in
corn oil
60.0
60.0
60.0
60.0
60.0
60.0
60.0
60.0
60.0
Age at
dosing3
(weeks)
7
14
23
37
85
7
14
23
37
85
Treatment Time-weighted
period av. dose° (mg
(weeks) TCI /kg body wt)
7C
9c
I4c
48d
32 549
7C
9c
14C
48d
32 1097
Matched controls received doses of corn oil by gavage calculated on the basis
of the factor for the high dose animals.
aAge at initial dose or dose change.
bTime-weighted average dose = £ (dose in mgAg x no. of days at that dose)/ £
(no. of days receiving any dose). In calculating the time-weighted average
dose, only the days an animal received a dose are considered.
GDosing 5 days per week each week.
^Dosing 5 days per week, cycle of 1 week of no treatment followed by 4
weeks of treatment. (Animals were treated for 38 weeks of the 48-week
period. )
Survival patterns in Figures 8-3 and 8-4 indicate a rather poor survival
of animals in this study, particularly in treated animals where a decline in
survival was evident early in the study. A dose-related decrease in survival
of treated male rats was observed; however, survival of low-dose females was
less than that of high-dose females early in the study. Statistical analysis
of survival patterns in the NCI (1976) report showed a significant (P < 0.05)
decrease in the survival of high-dose males (P = 0.001) vs. matched control
8-13
-------
oo
I
TABLE 8-4. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - MALE RATS
(NCI 1976)
Vehicle-control
Time
interval
(weeks )
0
14
26
54
78
110
Body
Mean
(g)
193
504
570
616
559
382
wei ghta
Std. dev.
(g)
15.0
39.4
43.3
50.2
76.7
26.9
Foodb
(g)
0
153
156
149
153
91
No. of
animals
wei ghed
20
20
20
20
16
2
Body
Mean
(g)
193
474
533
573
523
383
Low dose
weight
Std. dev.
(g)
15.8
40.4
47.1
47.6
67.3
101.5
Food
(g)
0
150
158
147
158
114
No. of
animals
weighed
50
50
47
40
31
8
Body
Mean
(g)
194
446
500
513
462
423
High dose
weight
Std. dev.
(g)
16.7
45.3
47.2
48.3
47.2
45.3
Food
(g)
0
140
150
134
148
255
No. of
animals
weighed
50
45
43
30
12
3
aCalculated using individual animal weight.
bAverage weight per animal per week.
-------
CO
TABLE 8-5. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - FEMALE RATS
(NCI 1976)
Vehicle-control
Time
interval
(weeks )
0
14
26
54
78
110
Body
Mean
(g)
146
302
351
388
373
326
wei ghta
Std. dev.
(g)
11.4
33.8
37.6
51.3
58.2
80.1
Foodb
(g)
0
107
124
128
146
119
No. of
animals
wei ghed
20
20
19
17
16
8
Body
Mean
(g)
144
271
293
315
317
311
Low
wei ght
Std. dev.
(g)
11.0
24.4
28.8
29.8
39.9
86.0
dose
Food
(g)
0
98
110
120
145
114
No. of
animal s
wei ghed
50
44
34
28
20
13
Body
Mean
(g)
144
262
286
307
317
311
High
weight
Std. dev.
(g)
9.5
26.3
30.8
38.6
43.5
67.7
dose
Food
(g)
0
104
106
113
135
136
No. of
animals
weighed
50
47
45
37
23
13
Calculated using individual animal weight.
^Average weight per animal per week.
-------
PROBABILITY OF SURVIVAL
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males and of both high-dose females (P = 0.028) and low-dose females (P = 0.049)
vs. matched control females. Survival at terminal sacrifice was as follows:
3/20 matched control males, 8/50 low-dose males, 3/50 high-dose males, 8/20
matched control females, 15/48 low-dose females (2 missing females were excluded
from the denominator), and 13/50 high-dose females.
A carcinogenic effect of TCI was not discerned in this study. Total tumor
incidences expressed as the number of rats with tumors were as follows: 5/20
matched control males, 7/50 low-dose males, 5/50 high-dose males, 7/20 matched
control females, 2/48 low-dose males, and 12/50 high-dose females. No statis-
tically significant (P < 0.05) differences in tumor incidence between matched
control and treatment groups were found.
Chronic nephropathy was common in treated animals of both sexes and both
doses. This nephropathy was described as slight to moderate degenerative and
regenerative changes in tubular epithelium. This nephropathy was stated to be
unlike the chronic nephropathy encountered as a commonly occurring lesion in
aging rats and recognized frequently in treated and control rats in this study.
The occurrence of unilateral malignant mixed tumors in kidneys of two matched
control males, a hamartoma in the kidney of one low-dose male and one matched
control male, and an adenocarcinoma in the kidney of one low-dose male were
noted. High incidences of chronic respiratory disease were found in each
treated group as well as in matched controls.
Under the conditions of this study, TCI was not carcinogenic in Osborne-
Mendel rats. However, animal survival was rather low, particularly in treated
animals, and greater survival during the course of the study might have per-
mitted a stronger evaluation for carcinogenicity. Reduction in survival and
body weight gain indicate that the TCI doses used were toxic to the animals,
and a broader evaluation of dose response could have been possible if addi-
8-18
-------
tional lower doses had been tested and if dose levels had not been adjusted
during the study. Rats treated with TCI, and their controls, were housed in a
room with other rats treated with dibromochloropropane, ethylene dichloride,
1,1-dichloroethane, or carbon disulfide. There were 10 to 15 air exchanges
each hour in the room, but ambient air was not tested for the presence of
volatile materials.
8.1.1.3 Maltoni (1979)--Ma1toni (1979) summarized the results of a study done
to estimate the carcinogenic potential of TCI in Sprague-Dawley rats. Thirty
males and 3U females, 13 weeks old, were assigned to each group. Two treatment
groups received 50 or 250 mg/kg of TCI by gavage daily, 4 to 5 days weekly for
52 weeks, and a vehicle-control group was given olive oil. The TCI sample was
at least 99.9% pure and was contaminated with <_ 50 ppm each of 1,2-dichloroethy-
lene, chloroform, carbon tetrachloride, and 1,1,2-trichloroethane. No detectable
epoxides were found in this test material, and 10 ppm di isopropylamine was
added as a stabilizer. Surviving animals were sacrificed at 140 weeks. All
animals were given a necropsy examination, and tissues and organs were evaluated
microscopically.
A carcinogenic effect was not demonstrated in this study. However, although
the experiment lasted 140 weeks, the animals were dosed for 52 weeks, which is a
duration below potential lifetime exposures. Tumor findings were presented in the
study report; however, mortality patterns and toxic signs, if any, were not
described to indicate the sensitivity of the animals to the TCI treatment used.
Use of animals younger than 13 weeks of age at the beginning of the study could
have given a broader assessment of the carcinogenic potential of TCI through an
evaluation of treatment with TCI during the early growth and development of the
rats.
8-19
-------
8.1.2 Oral Administration (Gavage): Mouse
8.1.2.1 National Toxicology Program (NTP 1982)—A carcinogenicity bioassay on
TCI in B6C3F1 mice was recently completed for the National Toxicology Program,
and a 1982 draft report of the study has been reviewed by the NTP's Board of
Scientific Counselors.
The TCI test sample and the experimental design were those used in the
NTP (1982) carcinogenicity bioassay in Fischer 344 rats previously described
in this document. Evaluation of mortality, body weights, and pathology in a
13-week preliminary dose-finding study led to the selection of 1,000 mgAg/day,
5 days per week, for 103 weeks as the single dose level used in the bioassay.
Fifty males and 50 females per group were assigned to untreated control groups,
corn oil vehicle-control groups, and treatment groups, when 8 weeks old.
Vehicle-control and treated animals received 0.5 ml corn oil with each gavage
administration. Mice were caged in groups of 10 for the first 8 months of the
study, and in groups of 5 for the rest of the study. The animals were evaluated
for mortality, body weight, and pathology until terminal sacrifice at 112 to
115 weeks.
Results of this study were analyzed by the statistical methods used for
the NTP (1982) carcinogenicity bioassay in rats. Mean body weights of treated
males were lower than those of vehicle control males, whereas mean body weights
between vehicle control and treated females were comparable (Figure 8-5). Sur-
vival was significantly (P = 0.004) lower in treated males compared to vehicle-
controls, and although survival in treated females was lower after 95 weeks,
the overall difference in survival between vehicle-control and treated females
was not significant (P < 0.05) (Figure 8-6). Deaths of two vehicle-control males,
three treated males, one vehicle-control female, and three treated females were
attributed to "gavage error," and these animals were censored from the survival
8-20
-------
50
40
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10 20 30 40 50 60 70
TIME ON STUDY (WEEKS)
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90 100 110
Figure 8-5. Growth curves for mice administered trichloroethylene in corn oil
by gavage. (NTP 1982)
8-21
-------
oo
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patterns shown in Figure 8-6. At terminal sacrifice, 33/50 vehicle-control
males, 16/50 treated males, 32/50 vehicle-control females, and 23/50 treated
females were alive.
A carcinogenic effect of TCI was not apparent in this study except for a
significant (P j< 0.002) increase in the incidence of hepatocellular carcinomas
in treated male and female mice compared to corresponding vehicle-controls
(Tables 8-6 and 8-7). Hepatocellular carcinomas metastasized to the lung in five
treated males and one vehicle-control male. Hepatocellular adenoma incidence was
significantly (P < 0.05) increased in treated female mice by each statistical
method indicated in Table 8-7, but in treated male mice only by life table analysis
(Table 8-6). Spontaneous hepatocellular tumor formation is a characteristic of
the B6C3F1 mouse strain, and incidences of hepatocellular carcinoma in historical
corn oil control B6C3F1 mice as of June 15, 1981 for carcinogenicity studies of
104 weeks duration in the NTP bioassay program were 18% (120/256) in males and
2.9% (22/751) in females.
Hepatocellular carcinomas had a general histopathologic appearance of
markedly abnormal cytology and architecture, with diffuse patterns of tumor
tissue infiltrating normal tissue, whereas hepatocellular adenomas consisted of
circumscribed areas of distinct parenchyma cells. A treatment-related effect
on nonneoplastic pathology in liver was not evident in the study report.
Cytomegaly in the kidney was found in 90% of treated male and 98% of
treated female mice, but not in corresponding vehicle-controls. The average
grades of severity for cytomegaly, according to the scheme used for grading
cytomegaly in kidneys of treated Fischer 344 rats in the previously described
NTP (1982) study, were 1.5 in dosed males and 1.8 in dosed females. Comparison
with the average grades for male and female Fischer 344 rats given 1,000 mgAg/day
of TCI in the NTP (1982) bioassay indicates that toxic nephrosis of the kidney
8-23
-------
TABLE 8-6. ANALYSIS OF PRIMARY TUMORS IN MALE MICE3
(NTP 1982)
LIVER: ADENOMA
Tumor rates
Overal 1&
Adjusted0
Terminal d
Vehicle-
control
3/48 (6%)
9.1*
3/33 (9%)
Dosed
8/50 (16%)
27.5%
1/16 (6%)
Statistical tests6
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisner Exact Tests
P = 0.022
P = 0.191
P = 0.113
LIVER: CARCINOMA
Tumor rates
Overal lb
Adjusted0
Terminal d
Vehicle-
control
8/48 (17%)
22.1%
6/33 (18%)
Dosed
30/50 (60%)
92.5%
14/16 (18%)
Statistical tests6
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
P < 0.001
P < 0.001
P < 0.001
LIVER: ADENOMA OR CARCINOMA
Tumor rates
Overal 1&
Adjusted0
Terminal d
Vehicle-
control
11/48 (23%)
30.7%
9/33 (27%)
Dosed
38/50 (76%)
97.1%
15/16 (94%)
Statistical tests5^
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
P < 0.001
P < 0.001
P < 0.001
aThe dosed group received doses of 1,000 mg/kg of trichlorethylene by gavage.
^Number of tumor-bearing animals/number of animals examined at the site.
cKaplan-Meier estimated lifetime tumor incidence after adjusting for intercurrent
mortality.
^Observed tumor incidence at terminal kill.
eBeneath the dosed group incidence are the P-values corresponding to pairwise
comparisons between that dosed group and the controls. The life table analysis
regards tumors in animals dying prior to terminal kill as being (directly or
indirectly) the cause of death. Tne incidental tumor test regards these
lesions as non-fatal. The Fisher Exact Test compares the overall incidence
rates di rectly.
8-24
-------
TABLE 8-7. ANALYSIS OF PRIMARY TUMORS IN FEMALE MlCEa
(NTP 1982)
LIVER: ADENOMA
Tumor rates
Vehicle-
control
Dosed
Overal lb
Adjusted0
Terminal d
Statistical tests8
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
2/48 (4%)
6.2%
2/32 (6%)
8/49 (16%)
30.2%
6/23 (26%)
P = 0.016
P = 0.023
P = 0.049
LIVER: CARCINOMA
Tumor rates
Overall6
Adjusted0
Termi nal^
Statistical tests6
Vehicle-
control
2/48 (4%)
6.2%
2/32 (6%)
Dose
13/49 (27%)
43.9%
8/23 (35%)
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
P < 0.001
P = 0.002
P = 0.002
LIVER: ADENOMA OR CARCINOMA
Tumor rates
Overall6
Adjusted0
Terminal
Statistical tests6
Vehicle-
control
4/48 (8%)
12.5%
4/32 (13%)
Oosed
19/49 (39%)
63.8%
13/23 (57%)
Life taole
Incidental tumor test
Cochran-Armitage Trend,
Fisner Exact Tests
P < 0.001
P < 0.001
P < 0.001
aThe dosed group received doses of 1,000 mg/kg of tnchlorethylene by gavage.
^Number of tumor-bearing animals/number of animals examined at the site.
cKaplan-Meier estimated lifetime tumor incidence after adjusting for intercurrent
mortal i ty.
^Observed tumor incidence at terminal kill.
eBeneath the dosed group incidence are the P-values corresponding to pairwise
comparisons between that dosed arouo and the controls. The lifa i-abi» ane^is
regards tumors in animals dying prior to terminal kill as oeing (directly or
"indirectly) the cause of death. The incidental tumor test regards these
lesions as non-fatal. The Fisher Exact Test compares the overall incidence
rates directly.
8-25
-------
was less severe in the mice in this study at an equivalent dose. Only one dosed
mouse had a cytomegaly grade above 2. One vehicle-control male and one treated
male each had a renal tubular cell adenocarcinoma.
Under the conditions of this bioassay, TCI treatment produced statistically
significant increases of hepatocel lular carcinomas in male and female B6C3F1
mice. These results repeated those of the NCI (1976) carcinogenicity study
discussed herein, showing statistically significant increases in the incidence
of hepatocellular carcinomas in male and female B6C3F1 mice given TCI stabilized
with epoxides. In effect, the repeat study excludes the epoxides as necessary
factors in the increased incidence of hepatocel lular carcinomas in male and
female B6C3F1 mice treated with TCI under the conditions of the NTP (1982) and
NCI (1976) bioassays. Use of additional doses in the NTP (1982) study could
have provided an evaluation of dose response for hepatocellular carcinomas in
mice.
8.1.2.2 National Cancer Institute (NCI 1976)—A carcinogenicity study of TCI
in B6C3F1 mice was reported by the National Cancer Institute (NCI) in 1976.
The TCI sample was that used in the NCI (1976) study in Osborne-Mendel rats
described herein. Treatment groups included 50 animals/sex/dose, and 20
animals/sex comprised matched vehicle-control (corn oil) groups. Seventy-seven
male and 80 female mice used as vehicle "colony controls" and 70 male and 76
female mice serving as untreated "colony controls" were also compared with the
treatment groups in this study; "colony controls" included matched controls for
TCI and other chemicals being tested concurrently. Mice were 35 days old when
the study began.
Maximum and one-half maximum tolerated doses for the carcinogenicity study
were estimated from survival, body weight, and necropsy results from a preliminary
8-week subchronic test. In the carcinogenicity study, time-weighted average doses
8-26
-------
of TCI were given by gavage, 5 days/week, for 78 weeks, to the mice at levels
of 1,169 and 2,339 mg/kg/treatment in males and 869 and 1,739 mg/kg/treatment
in females. Doses were presented as time-weighted averages due to the dose
level changes made during the study, as shown in Table 8-8, in an attempt to
maintain maximum and one-half maximum tolerated doses.
TABLE 8-8. DOSAGE AND OBSERVATION SCHEDULE - TRICHLOROETHYLENE
CHRONIC STUDY ON B6C3F1 MICE
(NCI 1976)
Dosage
group
Low-dose
males
High-dose
males
Low-dose
females
High-dose
females
Dose (mg Percent of
TCI/kg TCI in
body wt) corn oi 1
1000
1000
1200
no treatment
2000
2000
2400
no treatment
700
900
no treatment
1400
1400
1800
no treatment
15.0
10.0
24.0
15.0
20.0
24.0
10.0
18.0
10.0
20.0
18.0
Age at
dosing3
(weeks)
5
11
17
83
5
11
17
83
5
17
83
b
11
17
83
Treatment Time-weighted
period Av. doseb (mg
(weeks) TCI/kg body wt)'
6^
faC
66C
12
6C
6C
66C
12
12C
66C
12
6C
6C
66C
12
1169
2339
869
1739
Matched controls received doses of corn oil by gavage calculated on the basis
of the factor for the high dose animals.
aAge at initial dose or dose change.
^Time-weighted average dose = £ (dose in mg/kg x no. of days at that dose)/£
(no. of days receiving any dose). In calculating the time-weighted average
dose, only the days an animal received a dose are considered.
cDosing 5 days per week each week.
8-27
-------
Mice were permitted to survive until terminal sacrifice at 12 weeks
following treatment. Animals were observed daily for mortality and toxic
signs, and body weights and food consumption were recorded weekly for the first
10 weeks and monthly thereafter. Survivors and decedents were necropsied, and
tissues and organs, including tumors and lesions, were examined histopatho-
logically.
At the beginning of the study, mice were randomly assigned to treatment
and control groups according to body weight. Body weight trends in Tables 8-9
and 8-10 show comparable weight gain between matched vehicle-controls and both
treatment groups for both males and females. Food consumption trends in Tables
8-9 and 8-10 indicate similar consumption between the female groups and slightly
higher consumption in treated males as compared to matched controls. Therefore,
it is possible that, if food consumption between matched control and treated
groups of males had been equivalent, body weight gain in treated males might
have been less than that in matched control males.
Survival patterns are presented in Figures 8-7 and 8-8. Differences in
survival reported as statistically significant (P < 0.05) were those between
high-dose and low-dose males (P = 0.001), matched control males and low-dose
males (P = 0.004), and both treatment groups of females and matched control
females (P = 0.035). Survival at terminal sacrifice was as follows: 8/20
matched control males, 36/50 low-dose males, 22/50 high-dose males, 20/20
matched control females, 42/50 low-dose females, and 42/47 high-dose females (3
high-dose females were missing and not included in the denominator).
Prior to terminal sacrifice, approximately 50% of the treated males and "a
few" treated females exhibited bloating or abdominal distension. Results of
necropsy and histopathologic examinations revealed a statistically significant
(P < 0.05) increase in the incidence of hepatocellular carcinomas in male and
8-28
-------
OO
I
CO
V.O
TABLE 8-9. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - MALE MICE
(NCI 1976)
Vehi cle-control s
Time
interval
(weeks )
0
14
26
54
78
90
Body
Mean
(g)
17
28
31
32
34
34
wei ghta
Std. dev.
(9)
U.5
0.1
0.2
0.4
0.6
0.7
Foodb
(g)
0
23
24
21
24
34
No. of
animals
weighed
20
20
20
18
8
8
Body
Mean
(g)
17
28
31
33
34
33
Low dose
weight
Std. dev.
(g)
2.0
0.8
0.9
1.0
0.8
0.7
Food
(g)
0
27
28
26
32
32
No. of
animal s
weighed
50
50
48
45
40
35
Body
Mean
(g)
17
29
32
34
35
34
High dose
weight
Std. dev.
(g)
1.1
0.4
0.7
0.4
1.0
1.3
Food
(g)
0
26
27
30
39
38
No. of
animals
weighed
50
49
42
33
24
20
Calculated using individual animal weight.
^Average weight per animal per week.
-------
TABLE 8-10. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - FEMALE MICE
(NCI 1976)
Vehicle-controls
Time
interval
(weeks)
oo
i
° 0
14
26
54
78
90
Body
Mean
(g)
14
23
25
28
29
28
wei ghta
Std. dev.
(g)
0.0
0.4
0.2
1.1
0.6
0.6
Foodb
(g)
0
27
22
19
23
28
No. of
animals
wei ghed
20
20
19
18
18
17
Body
Mean
(g)
14
23
26
27
29
30
Low dose
wei ght
Std. dev.
(g)
0.6
0.3
0.7
0.5
0.7
0.6
Food
(g)
0
23
23
21
25
27
No. of
animals
wei ghed
50
49
49
45
41
40
Body
Mean
(g)
14
24
25
26
28
28
High dose
weight
Std. dev.
(g)
0.7
0.5
0.5
0.3
0.4
0.7
Food
(g)
0
24
24
22
26
26
No. of
animals
weighed
50
49
47
41
40
39
Calculated using individual animal weight.
^Average weight per animal per week.
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female mice treated with TCI (Table 8-11). A dose-related response in females is
evident. Metastasis of hepatocellular carcinoma to the lung was observed in four
low-dose males and in three high-dose males. The time to observation of the first
hepatocellular carcinoma in each group was as follows: 72 weeks in matched
control males, 81 weeks in low-dose males, 27 weeks in high-dose males, and 90
weeks in females. With regard to "colony control" mice, hepatocellular carcinomas
were diagnosed in 5/77 (6.5%) vehicle-control males, 5/70 (7.1%) untreated
control males, 1/80 (1.3%) vehicle-control females, and 2/76 (2.6%) untreated
control females. Hepatocellular carcinoma incidences in "colony control" mice
were comparable to those in matched vehicle-control males (1/20 or 5%) and
females (0/20 or 0%). Statistically significant (P < 0.05) differences in the
incidence of other tumor types between control and treatment groups were not
found.
The general pathology of hepatocellular carcinomas consisted of various
characteristics, including an orderly cord-like arrangement of neoplastic cells,
a pseudoglandular pattern resembling adenocarci noma, or sheets of highly
anaplastic cells with minimal cord- or gland-like arrangement.
Under the conditions of this study, TCI induced a statistically signi-
ficant (P < 0.05) increase in hepatocellular carcinoma incidence in male and
female B6C3F1 mice. Although the number of matched vehicle controls was low,
the use of pooled colony controls gives additional support for treatment-related
effects.
A more precise estimate of dose response perhaps could have been
obtained if additional lower doses had been used and if constant doses rather
than time-weighted averages had been used. Treated animals were housed in the
8-33
-------
TABLE 8-11. INCIDENCE OF HEPATOCELLULAR CARCINOMAS IN B6C3F1 MICE RECEIVING TRICHLOROETHYLENE BY ORAL GAVAGE
(NCI 1976)
CO
1
-p-
Dose 0 mgAg
Number with
tumors /number
of animals 1/20
Incidence 0.05
P-val ue
Number with
metastases 0/20
Males
1169 mgAg 2339 mgAg 0 mgAg
26/50 31/48 0/20
0.52 0.646 0
a a
1.39 x 10~4 3.44 x 10-6
4/50 3/48 0/20
Females
869 mgAg 1739 mgAg
4/50 11/47
0.08 0.234
1.75 x 10-1 1.35 x ID'2
0/50 0/47
Statistically significant (P < 0.05) increase compared to matched vehicle-controls (0 mg/kg) by Fisher Exact
Test.
-------
same room as mice treated with other volatile compounds*; however, since
1) controls were in the same room as treated animals, 2) oral TCI doses probably
would have been much higher than ambient levels of other volatiles, 3) the cages
had filters to limit the amount of chemical released into the ambient air, 4)
the total room air was exchanged 10 to 15 times per hour, and 5) dosing was done
in another room under a large hood, the likelihood that the other volatile com-
pounds were responsible for the observed results is considered low. It should
be noted that ambient levels of volatiles in the animal quarters were not
measured. Further evidence that the induction of hepatocellular carcinomas in
B6C3F1 mice was due to treatment with TCI in this study is the repeat study
(NTP 1982) with a similar experimental design in which the mice were not housed
with mice treated with other volatile chemicals. Additionally, the repeat NTP
(1982) study of purified TCI in B6C3F1 mice devoid of detectable levels of
epoxides indicates that the presence of epoxides was not necessary for the
induction of hepatocel lular carcinomas in B6C3F1 mice in the NCI (1976) study.
8.1.2.3 Van Duuren et al. (1979)t--A carcinogenicity study of purified TCI in
ICR/Ha Swiss mice was described by Van Duuren et al. (1979). Experimental
details provided below include those obtained through written communication
with B.L. Van Duuren, New York University. The TCI sample was > 99% pure, and,
although contaminants were not identified, epoxides were considered not to be
present in the test material. It was stated that although range-finding studies
*1,1,2,2-tetrachloroethane, 3-chloropropene, chloropicrin, 1,1-dichloroethane,
chloroform, sulfolene, iodoform, ethylene dichloride, methyl chloroform, 1,1,2-
trichloroethane, tetrachloroethylene, hexachloroethane, carbon disulfide,
trichlorof1uoromethane, carbon tetrachloride, ethylene dibromide, dibromo-
chloropropane.
tAlthough discussed under the heading of Oral Administration: Gavage (Mouse),
this study in mice also includes subcutaneous injection, repeated skin application,
and initiation-promotion experiments with TCI and an initiation-promotion experi-
ment with TCI oxide.
8-35
-------
were done to estimate maximum tolerated doses (doses which did not affect body
weight gain or induce clinical signs of toxicity in a 6- to 8-week test which
included histopathologic examination of treatment site sections) for all test
groups in this study, TCI doses used in the main topical application experiments
were below those maximally tolerated. TCI doses used in the skin treatment
experiments were selected to permit a comparison with TCI oxide, with respect
to card nogenicity, on an equimolar basis. This study incorporated the following
experiments: 1) 30 females received topical applications of 1 mg TCI three times
weekly for approximately 581 days; 2) 30 females were treated topically with a
single application of 1.0 mg TCI followed 14 days later by applications of 2.5
ug phorbol myristate acetate (PMA) to the skin three times weekly until termination
of the study at 452 days. Thirty females similarly treated with 1.0 mg TCI
epoxide and 2.5 ug PMA were also tested; 3) 30 females were given subcutaneous
injections of 0.5 mg TCI once weekly for 622 days; and 4) 30 males and 30
females were administered 0.5 mg TCI by gavage once weekly for 622 days. A
vehicle-control group of 30 mice and an untreated control group of 100 mice
were included in each experiment. Dosing vehicles were acetone in the skin
treatment tests and trioctanoin in the other tests. In the initiation-promotion
experiment, 210 mice treated with PMA alone were also on test. The mice were 6
to 8 weeks old at the beginning of the study, and six animals were housed in each
cage. Test sites on the skin were shaved as necessary and were not covered,
and test samples were administered in ventilated hoods; however, it was the
authors' impression that TCI was immediately absorbed and that evaporation from
test sites was minimal. The animals were weighed monthly, and each animal was
examined by necropsy. Tumors and lesions were examined histopathologically.
Histopathologic evaluation was routinely done on skin, liver, stomach, and
kidney in the skin application experiments, injection site tissue and liver in
8-36
-------
the subcutaneous injection experiment, and stomach, liver, and kidney in the
gavage experiment.
Treatment with TCI and TCI oxide did not produce statistically significant
(P < 0.05) increases in tumor incidence in this study (Tables 8-12, 8-13, and 8-14),
In the initiation-promotion experiment, the numbers of mice with skin papillomas
(squamous cell carcinomas) were: 4 (1) treated with TCI, 15 (3) treated with
PMA alone, and none in the control groups. Repeated application to the skin
produced lung and stomach tumors in 9 and 1 TCI-treated mice, respectively, 11
and 2 vehicle-treated controls, respectively, and 30 and 5 untreated controls,
respectively. Lung tumors were diagnosed as benign papillomas, and stomach
tumors were described as papillomas of the forestomach. Tumors were not found
in TCI-treated and control animals in the experiment with subcutaneous doses.
In the experiment with gavage dosing, forestomach tumors were observed in one
female and two male mice given TCI, no vehicle-control animals, and five female
and eight male (one male had a squamous cell carcinoma in the forestomach)
untreated control mice. Three mice given repeated applications of TCI oxide
onto the skin had skin papillomas. Results in Tables 8-12, 8-13, and 8-14 show
that the mice were sensitive to positive control agents under the design of
this study. Median survival times approximated the number of days the animals
were on test, and body weights were comparable between treatment and control
groups in each experiment.
Use of TCI doses as high as those maximally tolerated could have more
strongly challenged the mice for carcinogenicity in the skin treatment experi-
ments, but Van Duuren et al. (1983) noted that low activity or lack of activity
of chloroolefins tested for carcinogenicity in skin treatment experiments in
their laboratory was expected because of evidence for the relatively low level
of epoxidizing enzymes in mouse skin relative to other organs such as liver.
8-37
-------
TABLE 8-12. MOUSE SKIN BIOASSAYS OF TRICHLOROETHYLENE AND TRICHLOROETHYLENE OXIDE
(30 FEMALE HA:ICR SWISS MICE PER GROUP)
(Van Duuren et al. 1979)
oo
i
VjJ
oo
Initiat ion-promotion3
Repeated applicationb
Compound
Dose, mg/ Days to Mice with Dose, mg/ Days to Mice with No. of mice
application/ first papillomas/ application/ first papi llomas/ with
mouse tumor total papillomas mouse tumor total papillomas distant tumors^
Trichl
oroethyl
ene
1.0
264
4/9
(1)C
1.
0
0
9
1
lung
stomach
Trichloroethylene
oxide 1.0
7,12-Dimethyl-
371
3/3
NT
benz[a]anthracenee 0.02
PMA controls
(120 mice) 0.0025
( 90 mice) 0.005
Acetone (0.1 ml )
No treatment
(100 mice)
54 29/317 (18) NT
P < 0.0005f
141 9/10 (1)
449 6/7 (2)
0.1 ml
-
-
0 11 lung
2 stomach
0 30 lung
5 stomach
aCompounds were applied to dorsal skin in 0.1 ml acetone once followed 14 days later with 2.5 mg PMA
in 0.1 ml acetone 3 times weekly.
Compounds applied in 0.1 ml acetone 3 times weekly. NT = Not tested.
cNumber of mice with squamous cell carcinomas are in parenthesis.
^Lung tumors were benign papillomas, and stomach tumors were papillomas of the forestomach.
ePositive control agent.
fStatistically significant by chi-square test compared to controls.
-------
GO
TABLE 8-13. SUBCUTANEOUS INJECTION OF TRICHLOROETHYLENE
(30 FEMALE HA:ICR SWISS MICE PER GROUP)
(Van Duuren et al. 1979)
Compound and
dose3
Trichloroethylene, 0.5
B-propiolactone,01 0.3
Trioctanoin, 0.05 ml
No treatment (100 mice)
Days on
test
622
378
631
649
No. of mice
with local
sarcomas'3 P value0
0 NS
24 <0.0005
0
0
aCompound subcutaneously injected in left flank once weekly in 0.05 ml trioctanoin. Dose is rng per
injection per mouse.
bHistopathologically characterized as fibrosarcomas.
CNS = Not significant. Statistical significance calculated hy chi-square test compared to controls.
^Positive control agent.
-------
oo
I
o
TABLE 8-14. CARCINOGENICITY OF TRICHLOROETHYLENE BY INTRAGASTRIC FEEDING IN
MALE AND FEMALE HA:ICR SWISS MICE (30 MICE PER GROUP)
(Van Duuren et al. 1979)
Compound and
dose3
Trichloroethylene, 0.5
B-Propiolactone, 1.0
Trioctanoin, 0. 1 ml
No treatment
(100 mice)
(60 mice)
Sex
M
F
M
F
M
F
M
F
Days on
test
622
622
539
582
631
635
649
636
No. of mice with
forestomach
tumorsb
1
2
24(23)
25(23)
0
0
5
8(1)
P valuec
NS
NS
<0.0005
<0.0005
••
-
—
-
aDose in mg per intubation per mouse. Compounds were given once weekly in 0.1 ml trictanoin.
^Number of mice with squamous cell carcinoma of the forestomach is given in parentheses.
CNS = Not significant. Statistical significance calculated by chi-square test compared to controls.
-------
Administration of TCI more than once each week in the experiments with oral and
subcutaneous treatment also might have more strongly challenged the animals for
carcinogenicity. Dr. Van Duuren noted (written communication) that once-weekly
subcutaneous injection would allow time for the injection site to heal before
subsequent injections; however, more than 1 injection per week might have more
effectively tested the carcinogenic potential of TCI at sites distant from the
injection site. Routine histopathologic examination of a greater range of
tissues and organs might have provided a broader evaluation for carcinogenicity.
8.1.3 Inhalation Exposure: Rats
8.1.3.1 Henschler et al. (1980)—Henschler et al. (198U) described a study on
the carcinogenic potential of TCI in Han:Wist rats. The initial age of the
animals was not given. Thirty male and 30 female rats were assigned to each of
three dose groups which were exposed to 0 (controls exposed to inhalation
chamber air only), 100 (0.55 mg/1 air), or 500 (2.8 mg/1 air) ppm TCI vapor 6
hours daily, 5 days/week, for 18 months. The animals were individually caged
in this study. Surviving rats were sacrificed at 36 months. All animals were
subjected to necropsy and histopathologic examination.
The TCI sample, stabilized with 0.0015% triethanolamine, contained the
following impurities by gas chromatography-mass spectrometry analysis: chloro-
form, carbon tetrachloride, 1,1,2-trichloroethane, 1,1,1-trichloroethane, and
1,2,2-tetrachloroethane, each present at less than 0.0000025% (w/w). Epoxides
were not detected in this test material. Exposure levels of TCI in the inhalation
chambers were monitored with direct ultraviolet spectrophotometry, and the flow
rate of the TCI air mixture was adjusted to yield a threefold turnover of the
total volume each hour. A description of the analytical method was provided by
D. Henschler, University of Wurzburg, as follows: Exposure levels were automa-
8-41
-------
tically controlled to 100 or 500 ppm, as appropriate, and were continuously
monitored in a gas-flow cell in an ultraviolet beam of 205 mm. The accuracy of
this method was determined by the noise of the electronic equipment, and the
registered band indicated _+ 7% of the median at 100 ppm.
No effect of TCI on body weight gain was observed. A statistically
significantly (P < 0.05) decrease in survival of treated rats was not found
(Table 8-15).
A carcinogenic effect of TCI was not evident in this study. Use of a
maximum tolerated dose is not indicated in the study report, and the com-
parability of survival and body weight between control and treatment groups
indicates that higher exposure levels of TCI possibly could have been used to
provide a broader evaluation of TCI carcinogenicity.
TABLE 8-15. ESTIMATION OF PROBABILITY OF SURVIVAL OF RATS (%)«
(Henschler et al. 1980)
Group
Control
Week
1
50
75
100
115
126
156
M
100.0
96.6
93.3
86.7
83.3
66.7
46.7
F
100.0
100.0
96.6
86.7
70.0
56.6
16.7
100
M
100.0
96.6
96.6
90.0
86.6
80.0
23.3
ppm
F
100.0
96.6
93.3
86.6
63.3
50.0
13.3
500
M
100.0
100.0
100.0
90.0
76.6
66.6
36.7
ppm
F
100.0
93.3
90.0
76.6
50.0
50.0
16.7
aBy the Kaplan and Meier (1958) method.
8-42
-------
8.1.3.2 BelT et al. (1978; audit of Industrial Bio-Test Laboratories, Inc. study)-
A carcinogenicity study of TCI vapor in Charles River rats was conducted at
Industrial Bio-Test Laboratories, Inc. (IBT) from 1975 to 1977, and audit
findings by the Manufacturing Chemists Association (MCA) on this study have
been reported (Bell et al. 1978). The TCI product used in this study contained,
on a weight percent basis, TCI, > 99%; diisobutylene, 0.023%; butylene oxide,
0.24%; ethyl acetate, 0.052%; N-methylpyrrole, 0.008%; and epichlorohydrin,
0.148%. Each of the three treatment groups plus an untreated control group
consisted of 120 rats/sex. Treated animals were exposed to nominal concentrations
of 100, 300, or 600 ppm (0.55, 0.165, or 0.330 mg/1 air) TCI vapor 6 hours/day,
5 days/week, for 24 months. Surviving animals were sacrificed on termination
of treatment. Thirty rats/sex scheduled for interim sacrifice to evaluate
hematology and clinical chemistry were not given pathologic examinations.
According to the audit report by the MCA, there were noticeable deficiencies
in the performance of this study. Examination of analytical data on the TCI
vapor concentrations in the inhalation chambers revealed wide deviations from
the nominal concentrations and high variability in the number of measurements
made daily which do not allow a precise determination of the actual exposure
levels. For example, histograms of measurements (Figures 8-9, 8-10, and 8-11)
made during the initial 120 days of exposure show a clustering of data around the
nominal concentrations; however, only 259/565, 393/623, and 389/598 readings
made during the first 6 months of the study fell within ± 30% of the 100, 300,
and 600 ppm nominal concentrations, respectively. Exposure data in the report
by Bell et al. (1978) cover the initial 6 months of this study, and additional
information from the Chemical Manufacturers Association has been requested
regarding exposure levels obtained during the final 18 months of this study.
8-43
-------
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Histopathologic reexamination of livers did not show a carcinogenic effect
from exposure to TCI. Hepatocellular carcinomas were found in 3/99 control
male and 3/99 low-dose male rats. Histopathologic reexamination of other
tissues was not evident.
8.1.4 Inhalation Exposure: Hamsters
8.1.4.1 Henschler et al. (1980)—Henschler et al. (1980) conducted a carcino-
genicity study on TCI in Syrian hamsters. The TCI sample and the experimental
conditions were those used in the carcinogenicity study of TCI in Han:Wist rats
by Henschler et al. (1980) discussed herein. Thirty male and 3U female hamsters
per exposure group were exposed to 0 (controls exposed to inhalation chamber
air only), 100, or 500 ppm TCI vapor 6 hours daily, 5 days per week, for IB
months. The initial age of the animals was not given. Terminal sacrifice was
at 30 months. All animals were subjected to necropsy and histopathologic
examination.
No effect of TCI on body weight gain was observed. A treatment-related
effect on survival of hamsters was not found (Table 8-16); however, a marked
increase in the death rate for all groups ensued after 75 weeks.
A carcinogenic effect of TCI was not evident in this study. Use of a
maximum tolerated dose is not indicated in the study report, and the similarity
of survival and body weight between control and treatment groups indicates that
higher exposure levels of TCI possibly could have been used to provide a broader
evaluation of TCI carcinogenicity.
8.1.5 Inhalation Exposure: Mice
8.1.5.1 Henschler et al. (1980)--Hensch1er et al. (1980) conducted a carcino-
genicity study on TCI in Han:NMRI mice. The TCI sample and the experimental
conditions were those used in the carcinogenicity study of TCI in Han:Wist rats
8-47
-------
by Henschler et al. (1980) discussed herein. Thirty male and 30 female mice
per exposure group were exposed to 0 (controls exposed to inhalation chamber air
only), 100, or 500 ppm TCI vapor 6 hours daily, 5 days per week, for 13 months.
The initial age of the animals was not given. Terminal sacrifice was at 30
months. All animals were subjected to necropsy and histopathologic examination.
TABLE 8-16. ESTIMATION OF PROBABILITY OF SURVIVAL OF HAMSTERS (%)«
(Henschler et al. 1980)
Week
1
50
75
100
115
126
156
Control
M F
100.0 100.0
100.0 100.0
100.0 83.6
56.7 23.3
40.0 6.7
13.3
-
Group
100 ppm
M F
100.0 100.0
100.0 100.0
100.0 89.7
63.3 20.7
26.7
6.7 -
-
500
M
100.0
100.0
100.0
70.0
40.0
26.7
-
ppm
F
100.0
90.0
76.6
30.0
-
-
-
aBy the Kaplan and Meier (1958) method.
No effect on body weight gain was observed. Survival patterns described in
Table 8-17 show a greater mortality in treated mice compared to controls, par-
ticularly after 50 weeks in males and 75 weeks in females. A statistically signifi
cant (P < 0.05) decrease in survival rates of males and females in both treatment
groups compared to controls was reported. The survival patterns indicate that
the TCI exposure levels used were toxic to male mice; increased mortality in
treated females was particularly evident during the period when lymphoma incidence
was increasing in these groups.
8-48
-------
TABLE 8-17. ESTIMATION OF PROBABILITY OF SURVIVAL OF MICE (%)a
(Henschler et al. 1980)
Group
Week
1
50
75
100
115
126
156
Control
M
T
100.0 100.0
93.3
83.3
66.7
33.3
23.3
-
93.3
83.3
46.7
33.3
26.7
-
100
M
100.0
86.7
63.3
36.6
16.6
10.0
-
ppm
F
100.0
96.6
73.3
30.0
13.3
3.3
-
500
M
100.0
83.3
56.6
26.7
13.3
3.3
-
ppm
F
100.0
100.0
32.8
37.9
10.3
3.4
-
aBy the Kaplan and Meier (1958) method.
A carcinogenic effect of TCI was not evident in male mice in this study;
however, the incidence (number affected/number examined) of malignant lymphomas
in female mice was as follows: 9/29 (control), 17/30 (100 ppm), and 18/28
(500 ppm). The response in the low (P = 0.042) and high (P = 0.012) dose
groups represents a statistically significant (P < 0.05) increase over the
control incidence by the Fisher Exact Test. The time-to-tumor occurrence was
shorter in treated females; however, the majority of lymphomas were apparent
after treatment was terminated at 78 weeks, as illustrated in Figure 8-12.
Induction of hepatocellular carcinomas by exposure to TCI was not observed in
this study. Hepatocellular adenoma was diagnosed in one control and two low-dose
male mice, and hepatocellular carcinoma was discovered in one control male mouse.
According to the authors, the increased incidence of malignant lymphoma in
female mice could have been due to an occurrence of immunosuppression capable
8-49
-------
20
15
to
5 10'
ce
UJ
ca
0 CONTROLS
A 100 ppm
O 500 ppm
A-
O
)
> O
/
o
o
o
X
a
36 40 50 60 70 80 90 100 110 120 130
WEEKS
Figure 8-12. Incidence of malignant lymphomas in female mice.
8-50
-------
of enhancing the susceptibility of tumor induction by specific, mostly inborn,
viruses. The 30% incidence of malignant lymphoma in matched control female
mice in the Henschler et al. (1980) study is higher than the 12 to 22% (average
16%) range of lymphoma incidence determined by Luz (1977) between 1968 and 1977
for 830 untreated female NMRI-Neuherberg mice used as control groups in long-
term experiments. It was the authors' impression that it was not clear whether
an immunosuppressl ve effect would have been exerted by TCI itself or by other
nonspecific influences, such as stress.
Under the conditions described in the report of their study, Sanders et al.
(1982) indicated an ability of orally administered TCI to reduce the immune
status in CD-I mice; therefore, enhancement of the lymphoma incidence in the
treated female mice possibly could have been related to an immunosuppressive
effect from TCI treatment. Kreuger (1972) presented an argument similar to
that of Henschler et al. (1980) in which a coexistence of persistent antigenic
stimulation and chronic immunosuppression is pathogenetically related to malignancy
in immunocompetent tissues. Furthermore, Kreuger (1972) indicated that a
significant increase in the incidence of lymphoreticular neoplasms corresponded
to a significantly shortened latent period of tumor formation in several experiments
cited in his report. The argument of Henschler et al. (1980), therefore, provides
a possible interpretation of the results of their study.
8.1.5.2 Bell et al. (1978; audit of Industrial Bio-Test Laboratories, Inc. study) —
A carcinogen!city study of TCI vapor in B6C3F1 mice was conducted at Industrial
Bio-Test Laboratories, Inc. (IBT) from 1975 to 1977, and audit findings by the
Manufacturing Chemists Association (MCA) have been reported (Bell et al. 1978).
The TCI test.sample and the experimental design were those used in the IBT
study in Charles River rats discussed elsewhere herein. Each of three treatment
8-51
-------
groups (exposed to nominal levels of 100, 300, or 600 ppm TCI vapor, 6 hours
daily, 5 days/week, for 24 months), as well as an untreated control group, con-
sisted of 140 mice of each sex. Except for 40 mice/sex/group assigned to
interim sacrifice for evaluation of clinical chemistry and hematology, a patho-
logic examination was scheduled for each animal. Surviving animals were sacri-
ficed on termination of treatment.
Serious flaws in the conduct of this study were described in the audit
report by the MCA. Wide deviations in actual exposure levels compared to
nominal concentrations and high variability in the number of vapor measurements
made daily, as found in laboratory records made early in the study, are des-
cribed in the discussion of the IBT carci nogenicity study in rats. Furthermore,
although all mice evidently were received from the same supplier, control mice
appear to have been selected from a shipment obtained approximately 3 weeks
earlier. Hence, the control group was not specifically matched with the treat-
ment groups. Histopathologic reexamination of livers revealed 39 discrepancies
between gross and microscopic observations, which were mostly the result of
improper sectioning of liver containing tumors found grossly. Twelve mice
(_< 3 per group) were identified as originally missexed during reexamination of
liver specimens. The sex of nine mice (_< 2 per group) could not be determined,
and for the tabulation of liver tumor data, the sex shown on laboratory records
was assumed.
Statistical analysis of liver tumor data from reexamination of liver patho-
logy was done by the Fisher Exact Test and the chi-square analysis (Table 8-18).
Significant (P < 0.05) increases in the incidences of hepatocel lular adenoma,
hepatocel lular carcinoma, and hepatocel lular adenoma/hepatocellular carcinoma
combined in treated males compared to untreated males and the incidence of
hepatocel lular adenoma/hepatocellular carcinoma combined in treated females
8-52
-------
TABLE 8-18. STATISTICAL ANALYSIS OF THE INCIDENCE OF PRIMARY HEPAfOCtLLULAR NEOPLASMS
IN CONTROL AND TRICMLOROETHYLENE TREATED B6C3F1 MICE
(Bell ct al. 1978)
Hepatocel lular adenoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocel lular carcinoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocel 1 ular adenoma/
hepatocel lular carcinoma
combi ned
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocel lular adenoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocel 1 ular carcinoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocellular adenoma/
hepatocel lular carcinoma
combi ned
Incidence
Percent
Chi square
Fisher Exact Test
*Significant at 95% level
a T-l: 100 ppm
b T-2: 300 ppm
c T-3: 600 ppm
Ontreated
control s
2/99
2.02%
i
18/99
18.18%
i
20/99
20.20%
Untreated
controls
2/99
2.02%
i
6/99
6.06%
i
8/99
8.08%
T-l«
7/96
7.37%
2.042
0.0752
28/95
29.47%
2.821
0.0463*
35/95
36.84%
5.815*
0.00778*
T-l
5/100
5.00%
0.5/2
0.2265
4/100
4.00%
0.116
0.8382
9/100
9.00%
U.OOO
0.5083
Males
T-2b
7/lu'O
7.00%
1.820
0.0873
31/100
31.00%
3.741
0.0262*
38/1UO
38.00%
6.794*
0.004407*
Females
T-2
1/94
1.064
0.002
0.8672
9/94
9.57J.
0.1418
0.2604
10/94
10.64%
0.132
0.3579
T-3C T-l vs. T-2 T-l vs. T-3 T-2 vs. T-3
10/97
10.31%
4.504* 0.032 0.214 0.329
O.OlbO* 0.6465 0.3223 0.2834
43/97
44.33%
14.431* 0.006 3.930* 3.184
0.000063* 0.4698 0.0235* 0.0371*
53/97
54.64%
23.408* 0.000 5.427* 4.836*
0.00000049* 0.4933 0.0098* 0.013U*
T-3 T-l vs. T-2 T-l vs. T-3 T-2 vs. 1-3
4/99
4.04%
0.172 1.364 0.000 0.719
0.3413 0.9824 0.7461 0.2007
13/99
13.131
2.D96 1.599 4.
-------
compared to untreated females were calculated as shown in Table 8-18. With
respect to the treatment groups which were on study concurrently, statistically
significant (P < 0.05) increases in both the number of hepatocellular carcinomas
alone and hepatocellular adenomas/hepatocel lular carcinomas combined in high-dose
vs. either low- or mid-dose males and in hepatocellular carcinomas in high-dose
vs. low-dose females were found. Increases in liver tumor incidence in high-
dose compared to low-dose mice may be related to clustering of exposure level
frequencies around nominal concentrations, as shown in Figures 8-9, 8-10, and 8-11
in the discussion of the IBT rat study, and increased hepatocellular carcinoma
incidence was evident in B6C3F1 mice treated with TCI by gavage in the NTP (1982)
and NCI (1976) bioassays discussed in this document. However, this study is
weakened by the deficiencies in its conduct described above. An ongoing carcino-
genicity study by Dr. C. Maltoni and associates can provide further evaluation
of the carcinogenic potential of TCI vapor in BC63F1 mice.
8.1.6 Other Carcinogenicity Evaluations of Trichloroethy1ene--A1though done
mainly to estimate general toxic effects of TCI, as discussed elsewhere in this
document, the studies summarized in Table 8-19, except for that by Laib et al.
(1978), were described by Waters et al . (1977) as studies in which carcinogenic
activity for TCI was not demonstrated. However, these studies involved small
groups of animals and durations of treatment and observation which were short
with respect to the lifetime of the animals or, in the study by Rudali (1967),
a duration which was unspecified.
Laib et al. (1979, 1978) histochemically evaluated livers from Wistar rats
exposed to either vinyl chloride (VC) or TCI, by the protocol shown in Table 8-19,
for focal deficiencies of nucleoside triphosphatase (ATPase) as evidence for
pre-malignant hepatocellular lesions. The authors stated that Friedrich-Freksa
8-54
-------
TABLE 8-19. SUMMARY OF NEGATIVE CARCINOGENICITY DATA FOR TRICHLOROETHYLENE
(Waters et al. 1977)
Species
Number
Exposure3
Results
Reference
oo
i
VJ1
Dogs
Rats
Guinea pigs
Monkeys
Rabbits
Cats
Mice
Rats
Guinea pigs
Monkeys
Rabbits
Dogs
16
12
11
2
4
28
15
15
3
3
2
Rats
37 newborn and 2
adults per group
Inhalation
150-750 ppm in air 20-48
hr/wk for 7-16 wk
Inhalation
3,000 ppm, 27 exposures, 36 days
100 ppm, 132 exposures, 85 days
200 ppm, 148 exposures, 212 days
200 ppm, 178 exposures, 248 days
Inhalation
200 ppm, 178 exposures, 248 days
Intragastric
0.1 ml in 40% oil solution
2/^k, unspecified treatment
period
Inhalation
3,825 mg/tn^, 8 hr/day, 5 days/wk,
6 months
189 mg/Ti3 continuous for 90 days
Inhalation
2,000 ppm, 8 hr/day, 5 days/wk,
10 weeks followed by 2 weeks
without treatment
No tumors; no deaths
Three rats died;
no tumors
No tumors; no deaths
No tumors ; no deaths
No tumors; no deaths
Se'ifter 1944
Adams et al.
1951
Mosinger and
Fiorentini 1955
Rudali 1967
Prendergast
et al. 1967
No tumorigenic potential Laib et al. 1979,
and Laib et al.
1978b
Surviving animals were sacrificed on approximately the final day of exposure or, in the studies by Laib et al.,
observation; however, the observation period is not specified in the report by Rudali (1967).
Addition to summary by Waters et al. (1977).
-------
had shown that focal deficiencies of distinct hepatocellular enzymes precede
formation of hepatocellular malignancies. There was no effect in adult female
rats exposed to either chemical, and ATPase-deficient foci were found in livers
from newborn rats exposed to VC, but not in livers from newborn rats exposed to
TCI. The authors concluded that this experiment did not indicate an oncogenic
potency for TCI in hepatocytes of newborn rats. However, it is not certain
whether a longer duration of exposure of newborn rats to TCI could ultimately
have resulted in enzyme-deficient foci in liver.
8.1.7 Trichloroethylene Oxide
8.1.7.1 Repeated Skin Application and Subcutaneous Administration: Mice
8.1.7.1.1 Van Duuren et al. (1983). Van Duuren et al. (1983) evaluated the
carcinogenicity of six epoxides of structurally related chloroalkenes by repeated
skin application and subcutaneous injection in chronic studies with female
ICR/Ha mice. The epoxides were cis-1-chloropropene oxide (ci_s_-CPO); trans-1-
chloropropene oxide (trans-CPO); cj_s_-l,3-dich1oropropene oxide (cis-DCPQ);
trans-l,3-dichloropropene oxide (trans-DCPO). trichloroethylene oxide (TCIO);
and tetrachloroethylene oxide (PCEO). The epoxides were prepared by a modifica-
tion of the method of Kline et al. (1978) to obtain pure samples. However,
cj_s_-DCPO and trans-DC PO contained 10 to 15% m-dichlorobenzene during the initial
14 months of the study, and TCIO contained 10% dichloroacetyl chloride early in
the study. Pure chemicals were stored at -70°C.
Mice were 6 to 8 weeks old when the study began. Six animals were housed
per cage. There were 30 mice in each experimental group, except for 100 mice
in each untreated control group. Maximum tolerated doses were estimated for the
chronic studies from doses not showing clinically apparent toxicity, including
histopathologic examination of treatment sites and liver, in preliminary toxicity
tests of 6 to 8 weeks duration.
8-56
-------
For the chronic skin application study, test sites were shaved initially
and about every 2 weeks thereafter. The epoxides were dissolved in 0.1 ml
acetone for application to the dorsal skin 3 times each week for the duration of
the study at the doses described in Table 8-20, except for PCEO, which, due to its
instability in acetone, was applied undiluted to skin followed immediately by
application of acetone. In the subcutaneous injection study, epoxides in 0.05 ml
pure tricaprylin were administered at the doses shown in Table 8-21 into the
left flank once weekly for the duration of the study.
Animals were observed daily and weighed bimonthly. Papillomas were
characterized as skin lesions of about 1 mm in diameter which persisted more
than 30 days. Each animal was necropsied, and each lesion and tumor was
histopathologically examined. Routine sections examined microscopically
included skin, liver, stomach, and kidney in the skin application study, and
injection site tissue and liver in the subcutaneous injection study.
As shown in Tables 8-20 and 8-21, vehicle and untreated control groups were
concurrently on study. Positive control groups were treated with dl-diepoxybutane.
The above-mentioned impurities rn-dichlorobenzene and dichloroacetylchloride were
tested at the levels in which they were present in the epoxides.
Results described in Table 8-20 show no induction of skin tumors from repeated
application with TCIO, and no significant (P < 0.05) increase in local tumors
resulted from subcutaneous injection of TCIO compared to controls (Table 8-21).
The positive control animals responded with development of highly significant
(P < 0.0005, chi-square test) tumor increases in both studies (Tables 8-20 and
8-21). The investigators stated that no significant (P < 0.05) incidences of
malignant distant tumors were observed.
As discussed by Van Duuren et al. (1983), chloroepoxides have been
considered as activated carcinogenic metabolites of chloroalkenes. The negative
8-57
-------
TABLE 8-20. CHRONIC SKIN APPLICATION IN MICE
(Van Duuren et al. 1983)
CO
I
CO
Thirty female ICR/Ha Swiss mice per group were painted on the dorsal skin 3 times weekly with halo-epoxides at the doses
indicated in 0.1 ml acetone by micropipet except where noted.
Compound
Time (days) to
first tumor
Median survival
time/test duration
in days
No. of mice with
papi1loma/total
papi1lomas
No. of mice with
squamous cell
carcinoma
P value3
cis-CPO, 10 mg
trans-CPO, 10 mg
cis-DCPO, 10 mg
trans-DCPO, 10 mg
TCIO, 2.5 mg
PCEO, 7.5 mgf
m-Dichlorobenzene,
3. 5 mg
Uichloroacetyl chloride
250 ug
j
dl-Diepoxybutane, 3.3 mg
(positive control )
Acetone, 0. 1 ml
No treatment (100
animals )
225
345
371
255
268
147
>432/432
513/574
522/574
511/573
526/575
>459/459
520/575
>576/576
462/573
>576/576
551/580
14/20b
15/20
16/19
20/27d
0
3/39
0
0
23/63
0
0
10
IOC
10
176
1
21"
<0.0005
<0.0005
<0.0005
<0.0005
0.014
<0.0005
aP values based on number of animals with benign and malignant tumors: X^ test with Yates' correction was used.
^Three were keratoacanthomas.
C0ne animal developed a fibrosarcoma and a squarnous cell carcinoma.
dOne was a histiocytoma, and one was a keratoacanthoma.
eOne was a fibrosarcoma in the treatment area.
fpCEO was applied at 5 ul (7.5 mg) by Eppendorf pipet followed immediately by 0.1 ml acetone by micropipet.
90ne was a keratoacanthoma.
"Two were fibrosarcomas in the treatment area.
-------
TABLE 8-21. CHRONIC S.C. INJECTION IN MICE
(Van Duuren et al. 1983)
Thirty female ICR/Ha Swiss mice per group were given injections once weekly of
halo-epoxides s.c. in the left flank at the doses indicated in U.05 ml tricaprylin
Median survival
time/test duration
Compound
cis-CPO, 1 mg
trans-CPO, 1 mg
cis-DCPO, 500 ug
trans-DCPO, 500 ug
TCIO, bOO ug
PCEU, 500 ug
m-Dichlorobenzene,
165 ug
Dichloroacetyl chloride,
50 ug
dl-Diepoxybutane, 330
ug (positive control)
Tri capryl in, 0. 05 ml
No treatment
(100 animals)
in days
482/551
418/556
500/557
498/557
547/558
491/558
525/560
>560/560
475/553
>524/561
551/580
with local tumors
1 fibrosarcoma
, . K
1 carcinoma"
1 leukemia
4 fi brosarcomas
1 carcinoma
3 fi brosarcomas
1 anaplastic sarcoma
1 carcinoma and
mammary tumor
5 fi brosarcomas0
1 fibrosarcoma
1 anaplastic sarcoma
1 leukemia
0
1 fi brosarcoma
9 fi brosarcomas6
2 carcinomas
1 mammary tumor
0
1 fibrosarcoma
P valued
0.052
0.003
0.003
0.003
_j
NSd
NS
NS
<0.0005
aAll mice with local tumors included in calculation of P values: X2 test with Yates'
correction was used. Each treated group was compared with the untreated control
group. C-CPO was marginally significant using the 0.050 level as cutoff point.
bSquamous cell carcinomas.
C0ne with a focus of angiosarcoma.
dNS = not significant, and P > 0.20.
eOne with a focus of anaplastic sarcoma.
8-59
-------
results for carcinogenicity with TCIO in the study by Van Duuren et al. (1983)
reflect the negative results for carcinogenicity with TCI reported by Van Duuren
et al. (1978) using the same experimental design. Presentation of data for each
epoxide tested by Van Duuren et al. (1983) is made herein to provide evidence
that the relative carcinogenic activity of the six epoxides was not specifically
dependent on stereochemistry (ci s or trans) or rate of hydrolysis (see Figure 8-14
herein). The authors further noted that vinyl chloride epoxide has a half-life
in aqueous solution similar to that of TCIO, but that the former compound has
demonstrated carcinogenicity by subcutaneous injection and initiating activity
in an initiationpromotion study. As indicated by the authors, the epoxides
tested would be direct-acting agents which would not have to rely on metabolic
activation at the sites of carcinogenic action in their study. Under the condi-
tions of the study by Van Duuren et al. (1983), TCIO was not carcinogenic in
female ICR/Ha mice. Routine histopathologic examination of additional tissues
and organs might have given a broader evaluation of pathology.
8.1.8 Cell Transformation Studies
8.1.8.1 Trichloroethylene
8.1.8.1.1 Price et al. (1978). Price et al. (1978) evaluated the potential of
TCI to transform Fischer rat embryo cells (F 1706, subculture 108) in vitro.
The TCI sample, obtained from Fisher Scientific Co., was >_ 99% pure and would
have been stabilized with 20 ppm triethylamine or 25 ppm diisopropylamine
(personal communication with Fisher Scientific Co.). Cells were grown in
Eagle's minimum essential medium in Earle's salts supplemented with 10% fetal
bovine serum, 2mM L-glutamine, 0.1 mM nonessential amino acids, 100 U penicillin,
and 100 ug streptomycin per ml. Liquid TCI was diluted 1:1000 in growth medium
before exposure of cells to desired TCI doses.
8-60
-------
Doses for the transformation assays were selected as those producing
minimal reduction in plating efficiency relative to medium-only control cells
in preliminary tests. In the preliminary tests, triplicate cultures were
exposed to 10-fold dilutions of TCI for 4 days before fixing and staining of
cells and counting of colonies. Minimal reduction in plating efficiency (3 to
16%) compared to controls was produced over the range of TCI doses tested;
hence, the two highest doses (1.1 x 104 and 1.1 x 103 uM) of TCI were selected
for the transformation assay.
In the transformation assay, quadruplicate cultures of F17U6 cells were
treated at 50% confluency with the two selected concentrations of TCI. Cells were
exposed to TCI for 48 hours and then washed and refed with growth medium alone.
Each culture was considered a separate series. Additional cultures were exposed
to the positive control agent 3-methylcholanthrene diluted in acetone (1:1000)
and growth medium to a dose of 0.37 uM. Negative control cultures were grown in
the presence of acetone (1:1000) in growth medium and in growth medium alone.
At each subculture, one set of flasks was retained for 2 weeks without sub-
division, and the other set of flasks was subdivided 1:2 each week to yield two
new sets of cultures, one for the holding series without subdivision, and one
for subdivision. Cell transformation was characterized by progressively
growing foci composed of cells lacking contact inhibition and orientation, and
by the ability of foci to grow in semi-solid agar four subcultures after treatment.
The ability of transformed cells to induce local fibrosarcomas after subcutaneous
injection of 1 x 10^ cells into newborn Fischer 344 rats was also evaluated.
Transformation of cells exposed to TCI was observed, as well as the
ability of transformed cells to grow in semi-solid agar and to induce local
fibrosarcomas after subcutaneous injection into rats (Table 8-22). Transforma-
tion of cells exposed to the positive control agent 3-methylcholanthrene was
8-61
-------
TABLE 8-22. IN VITRO TRANSFORMATION OF FISCHER RAT EMBRYO CELL CULTURES BY TRICHLOROETHYLENE
(adapted from Price et al. 1978)
Morphological Growth in
Compound Dose (uM) transformation3 agar at P + 4h
Trichloroethylene 1.1 x 104 +5 (44) 9
1.1 x 103 +5 (44) 8
3-Methylcholanthrene 3. 7 x 10~2 +4 (37) 124
oo
oj Acetone 1:1,000 (NA) 0
Medium (NA) 0
Tumor incidence0
No. of animals with tumors/
No. of animals inoculated
13/13 (48)
12/12 (55)
12/12 (27)
0/13 (82)
ND
aNumbers represent subcultures after treatment when transformed foci were first observed. The acetone and
medium control cultures were still negative at the termination of the experiment nine subcultures after treatment.
Cultures were held for 2 weeks at each subculture and stained prior to screening. Numbers in parentheses represent
number of days in vitro prior to observation of morphological transformation. NA = not applicable, as there was
no observed transformation by subculture 9 (81 days).
^Numbers represent the average number of microscopic foci per three dishes. Each dish was inoculated with 50,000
cells and held for 4 weeks at 37°C in a humidified CO? incubator prior to staining with 2-(p-iodophenyl}-3-(p-
nitrophenyl)-5-phenyl-2H-tetrazolium chloride.
cAnimals inoculated subcutaneously with cells that had been treated with chemical five subcultures earlier.
Numbers in parentheses represent days postinoculation to 100% tumor incidence. ND = not done.
-------
evident, as shown in Table 8-22. Spontaneous cell transformation was not found
in negative control cells (Table 8-22).
Although transformation of F1706 cells in the presence of TCI was observed
by Price et al. (1978), the potency of TCI was low relative to that of the
positive control chemical, 3-methylcholanthrene. The response to TCI may
reflect an ability of this cell line to convert TCI to active metabolites, and
further evaluation of the effect of TCI through active metabolites possibly
could have been made by additional testing with an exogenous metabolic activa-
tion system and by testing the ability of the F1706 cells to produce TCI
metabolites. The authors stated that the F1706 cell line, at the subculture
used (108), did not require addition of murine type "C" virus and was free of
any known infectious virus; however, these cells did contain genetic information
of the Rauscher leukemia virus. It is not clear whether cell transformation
was induced through action of the test chemicals on the host cells, their
integrated oncornavirus genome, or both. Additionally, cells were exposed to
liquid TCI diluted in growth medium; therefore, since TCI is a volatile sub-
stance, exposure of cells to TCI as a vapor could have indicated how different
exposure conditions possibly could have influenced its ability to transform
F17U6 cells.
8.1.8.1.2 Styles (1979). Styles (1979) reported an investigation on TCI in a
cell transformation system with BHK cells, using growth in semi sol id agar as an
endpoint, as part of a larger study (Purchase et al . 1978) done to screen
chemicals for carcinogenic potential. The BHK-agar transformation assay technique
used has been described by Styles (1977) and Purchase et al. (1978). In the
study reported by Styles (1979), baby Syrian hamster kidney (BHK-211C1 13) cells
were exposed to five different doses of test substance in vitro in serum-free
8-63
-------
liquid tissue culture medium in the presence of rat liver microsomal fraction
and cofactors (S-9 mix; Ames et al. 1975). The liver microsomal fraction was
obtained from Sprague-Dawley rats induced with Arochlor 1254.
Cells were grown and maintained in Dulbecco's modification of Eagle's
medium in an atmosphere of 20% C02 in air. Cells were maintained at 37°C until
confluent; subsequently, the cells were trypsinized and resuspended in fresh
growth medium. Resuspended cells were grown until 90% confluent for transfor-
mation assays or 100% confluent for stock. Only cells with normal morphology
were used for assays. To minimize spontaneous transformation frequency, cells
were obtained at low passage, grown to 90% confluency, and frozen in liquid
nitrogen. Cells were thawed at 37°C in growth medium for further use.
Test compounds were dissolved in DMSO or water as appropriate. Each dose
was tested in replicate assays. Cells incubated until 90% confluency were
trypsinized and resuspended in Medium 199 at a concentration of 10^ cells/ml.
Resuspended cells (10^) were incubated with test chemical and S-9 mix
at 37°C for 4 hours. To evaluate compound vapors, cells (2.5 x 10s5) were
plated and incubated until 90% confluent, at which time growth medium was
replaced with medium 199 and S-9 mix. Duplicate plates were exposed to test
chemical vapor at 37°C for 3 hours. After treatment, cells were centrifuged
and resuspended in growth medium containing 0.3% agar. Survival after treatment
was estimated by incubating 1,000 cells at 37°C for 6 to 8 days before counting
colonies. Transformation was evaluated by counting colonies after cells were
plated and incubated for 21 days at 37°C. The dose-response for transformation
was compared to that for survival. Styles (1977) accepted a fivefold increase
in transformation frequency above control values at the 1X59 as a positive
result. The spontaneous transformation frequency of BHK cells (72 experiments)
8-64
-------
in this study was 50 +_ 16 per 106 survivors. Suitability of the soft agar
medium for colony growth was checked by assays with polyomatransformed BHK-21/C1
13 cells or Hela cells.
Data reported by Styles (1979) on unstabilized TCI (source and purity not
reported) are compared with those found with vinyl chloride (VC) (ICI Mond
Division; purity not reported) by Styles (1977, 1979) in Figure 8-13. Results
were negative with exposure to TCI solution in DMSO added to culture medium in
a dose range including levels in which toxicity was observed. Vinyl chloride
was effective as a gas in producing transformation and toxicity; however,
VC solution in DMSO added to culture medium was ineffective to suggest that
cells were exposed to doses too low to produce an effect by this approach.
Although TCI doses high enough to produce toxicity did not induce transforma-
tion, exposure of cells to TCI as a vapor could have provided a comparison of
the transformation potential of TCI as a vapor and TCI in liquid solution, as
was done for VC.
The study by Purchase et al. (1978), which was done on 120 chemicals of
various classes, showed that the BHK-agar transformation assay system was about
90% accurate in discriminating between compounds with demonstrated carcinogenic
or noncarcinogenic activity, and was in approximately 83% agreement with results
from assays done by the authors with S. typhimurium (TA 1535, TA 1538, TA 98,
TA 100). Styles (1979) and Purchase et al. (1978) indicated, without presenting
numerical data, that results were obtained in the Salmonella assays on VC in
liquid solution, VC gas, and TCI in liquid solution that were similar to
results found in the transformation assays. Purchase et al. (1978) also observed
that metabolically activated agents transformed BHK cells more strongly in the
presence of S-9 mix, thus suggesting that BHK cells have limited intrinsic
metabolic capability.
8-65
-------
CO
cc
O
>
or
co
i
en
CTl
co
z
<
i «
£ >
z •- >
< oc o:
DC UJ D
h- CL CO
'00-
50-
0-
1.100-
900-
700-
ui
2
o
1
to
°
o
1
100-
CH2 = CHCI
._A --- A --- A --- A
cr.
O
a:
D
CO
39
10O-
50
0-
1.100-
900-
700-
t-
<
ir c 500-
0 O
LI. n> ir
co o >
z •- >
< DC °C
CC LU D
I- Q- co 300 •
100 —
0025 025 25 25
10 20 30 40
CONCENTRATION /ug/ml (SOLN)
CONCENTRATION % IN AIR
Survival and transformation of BHK
cells treated with vinyl chloride gas
( 0 0 ) and vinyl chloride solution
in DMSO (A— -—A)
250
50
\
CI2C = CHCI
J
1
025
25 25
CONCENTRATION p\/m\
250
2.500
Survival and transformation
of BHK cells treated with
unstabilized tnchloroethylene
solution in DMSO
Figure 8-13.
Cell transformation assays on vinyl chloride and trichloroethylene
in BHK-21/C1 13 jn_ vvtro. (Styles 1977, 1979)
-------
8.1.8.2 Trichloroethylene Oxide
8.1.8.2.1 DiPaolo and Donlger (1982). DiPaolo and Doniger (1982) assessed
the capacity of putative epoxide metabolites of six chloroalkenes, including TCI,
to induce morphologic transformation in cultured Syrian hamster cells. The
tested epoxides were cis-1-chloropropene oxide (c-CPO); trans-1-chloropropene
oxide (t-CPO); ci_s_-l,3-dichloropropene oxide (c-DCPO); trans-l,3-dichloropropene
oxide (t-CPO), trichloroethylene oxide (TCIO); and tetrachloroethylene oxide
(PCEO). TCIO and PCEO were prepared by auto-oxygenation of TCI and PCE with
removal of chloride by-products by extraction with 0.5N sodium hydroxide. The
other epoxides were synthesized by oxidation of the parent compounds in
m-chloroperbenzoic acid. Each epoxide was provided pure, as determined by
nuclear magnetic resonance analysis, by Drs. Kline and Van Duuren of New York
University.
Cells for culture were obtained from Syrian hamster embryos removed from
their mothers on the twelfth and thirteenth days of gestation. Primary cultures
were derived by seeding 1 x 107 cells/dish from trypsinized embryos. Secondary
cultures were made 2 to 3 days later by seeding 5 x 106 cells/10 ml complete
medium/plate. Mass cultures were grown in modified Dulbecco's minimum essential
medium supplemented with 10% fetal bovine serum (FBS) under an atmosphere of 11%
carbon dioxide.
For each plate, 300 cells from secondary cultures were plated in complete
medium with 20% FBS along with 6 x 104 feeder cells previously irradiated as
confluent monolayer cultures. After incubation for 2 days, the medium was
removed, the cells were washed with phosphate-buffered saline (PBS), and the
desired concentration of epoxide in PBS was added to the cells. After incuba-
tion of the cells in the presence of epoxide for 30 minutes at 37°C, the PBS was
removed, the cells were washed, and fresh complete medium was added. After
8-67
-------
further incubation for 5 days, the cells were washed with PBS, fixed, and
stained with Giemsa. Cell colonies _> 2 mm in diameter were counted for cloning
efficiency and were examined for morphologic transformation, exhibited as criss-
crossing and piled-up cells not seen in controls. Transformation frequency was
calculated relative to surviving colonies and initial number of cells plated.
Twelve dishes of cells were evaluated per dose.
Results of the cell transformation assay with TCIO show a weak transforma-
tion effect, with high concentrations (>1 mM) capable of producing cytotoxicity
in the form of a dose-related reduction in cell survival (Table 8-23). The
weak effectiveness of TCIO at high concentrations may relate to its short
stability half-life of 1.3 minutes in aqueous solution (Figure 8-14). However,
chemical stability in aqueous solution alone might not have been directly
correlated with the ability of the six epoxides to transform cells in this
study. The most stable of the six epoxides in aqueous solution, c-DCPO and
t-UCPO (Figure 8-14), were the most potent inducers of cell transformation
(Table 8-23), but the trans isomer was a stronger inducer than the cis.
Additionally, although PCEO and c-CPO had similar stability half-lives in
aqueous solutions (Figure 8-14), c-CPO was the stronger inducer of cell trans-
formation, which, as suggested by the authors, may reflect a more limited
ability of PCEO to interact with DNA. Although use of a positive control
agent was not indicated for this assay, the authors mentioned that transformed
colonies found in this study were similar to those induced by benzo[a]pyrene
in this cell line in their laboratory.
8.1.9 Summary of Animal and Cell Transformation Studies
Increased incidences of malignant tumors in animals treated with TCI were
found in carcinogenicity studies on male and female B6C3F1 mice with gavage or
inhalation treatment, female Han:MMRI mice with inhalation treatment, and male
8-68
-------
TABLE 8-23. TRANSFORMATION AND SURVIVAL OF SYRIAN HAMSTER EMBRYO
CELLS TREATED WITH DIVERSE CHLOROALKENE OXIDES3
(DiPaolo and Doniger 1982)
Compound
Acetone
c-CPO
t-CPO
c-DCPO
t-DCPO
TCEO
PCEO
Dose, mM
0.11
0.27
0.55
1.1
0.22
0.55
1.1
2.2
0.005
0.01
0.02
0.01
0.025
0.05
1.1
2.5
5.0
0.87
4.3
8.7
17.1
Percent
survival
100 (29)b
110
94
90
50
96
78
54
12
92
93
81
89
85
44
91
86
73
98
96
100
70
Total
transformed
colonies
0
4
5
6
12
0
2
6
1
1
3
1
8
8
4
1
1
4
0
3
2
5
Average
transformations /
cells seeded, x 103
1. 1
1.4
1.7
3.3
_
0.56
1.7
0.26
0.2b
0.83
0.26
2.2
2.2
1.1
0.26
0.26
1.1
_
0.83
0.56
1.4
aP1astic dishes (60 mm) were seeded with 300 cells from a secondary culture with
irradiated hamster cells (6 x 10^) for a feeder, grown for 2 days, and incubated with
the test chemical in PBS in the absence of growth medium for 30 min at 37°C. Cells
were washed, fed again, and incubated for 7 days after which colonies were stained,
counted for survival data, and scored for morphologic transformation. Twelve
dishes were used for each treatment.
''Number in parenthesis is the absolute cloning efficiency of solvent-treated
cultures.
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Compound
Structure
Half-Life (mm)
c/s-1-Chloropropene oxide
(c-CPO)
CH3
Cl
- v
H' V X
11
(l-CPO)
-Chloropropene oxide
o/V\
4.3
co
i
c/s-1,3-Dichloropropene oxide
(c-DCPO)
CICH2 Cl
290
trans 1,3-Dicliloropropene oxide
U-DCPO)
CICH
/
300
Trichloroelhylene oxide
(TCIO)
ci
ci
VCI
1 3
Tetrachloroelhylene oxide
(PCEO)
Cl
Cl'
O
.Cl
%CI
12
Ti gure 8- 14. Structures and stability of chloroalkene oxides. Half-life values are in minutes and were measured at
pH 7.4 and 37°C as determined by Kline et al. (1 978). (DiPaolo and Doniger 1982)
-------
Fischer 344 rats with gavage treatment. Incidences of malignant tumors were not
observed to be increased in animals treated with TCI in carcinogenicity studies
on female Fischer 344 rats, male and female Osborne-Mendel rats, male and female
Sprague-Dawley rats, and male and female ICR/Ha Swiss mice, all with gavage treat-
ment; male and female Charles River rats, male and female HanrWist rats, male
and female Syrian hamsters, and male Han:NMRI mice, all with inhalation treatment;
female ICR/Ha Swiss mice with subcutaneous injection; and female ICR/Ha Swiss
mice with topical application. Increased incidences of malignant tumors were
not found in carcinogenicity studies of TCI oxide in female ICR/Ha Swiss mice
with subcutaneous injection or topical application. Tumor-initiating activity
was not observed in studies where either TCI or TCI oxide was applied to the
skin of female ICR/Ha Swiss mice as the initiating agent. Cell transformation
activity by TCI in cultured Fischer rat embryo cells and by TCI oxide in Syrian
hamster embryo cells was reported. Cell transformation activity for TCI was
not found in cultured baby Syrian hamster kidney cells.
8.2 EPIDEMIOLOGIC STUDIES
There are currently nine epidemiologic studies which relate to the
issue of TCI exposure and cancer. In only four of these studies, however, were
individuals actually identified as having trichloroethylene exposure. Because
trichloroethylene has been used to some extent in the dry-cleaning business in
the United States, three proportionate mortality studies of decendents who had
worked in the dry-cleaning business as well as two case control studies in which
the cases and controls were asked about history of employment, including employment
in the dry-cleaning business, are also discussed here. In one of the case-control
studies, the subjects were also asked about consumption of decaffeinated coffee
(TCI has been used as a decaffeinating agent.)
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8.2.1 Axel son et al. (1978)
Axel son et al. (1978) found no excess mortality due to cancer in a cohort
study of 518 Swedish male TCI production workers. Workers with >^ 10 years of
observation were identified as a subcohort. The authors did not indicate the
number of workers in this subcohort, but did indicate that the subcohort included
3,643 of the 7,688 total person-years of observation in the study. Since the
1950s the TCI manufacturer had provided, free of charge, two urinary analyses
per year per worker of the TCI metabolite trichloroacetic acid (TCA). Additional
analyses were reported to have been provided at low cost. Using this information,
the subcohort with >_ 10 years of observation was divided into low (average
reading of TCA in the urine of less than 100 mg/1) and high (average reading of
TCA in the urine of more than 100 mg/1). A level of 100 mg/1 of TCA in urine
was reported to correspond roughly to an 8-hour time-weighted average exposure
of 30 ppm TCI in the air, which is the Swedish occupational standard for TCI.
The expected number of deaths was calculated by multiplying the person-years of
observation by cause- and age-specific national death rates from the respective
calendar years and summing the fractional age and calendar-year contributions
over the entire cohort. Table 8-24 compares the expected and observed numbers
of deaths for the cohort and subcohort.
There were no significant differences between the observed and the expected
numbers of cancer deaths in the total cohort or in any of the subcohorts. The
high exposure group, with _> 10 years of observation, had a risk ratio of 1.7 for
cancer deaths, but this was not statistically significant. Two deaths from stomach
cancer and two from leukemia occurred in the cohort; the authors did not indicate
what the expected number of deaths for these sites would be, however.
Although the data presented in this study do not demonstrate that TCI is a
human carcinogen, there are several limitations in the study design, or at least
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TABLE 8-24. A COHORT STUDY OF MORTALITY AMONG MEN EXPOSED TO TRICHLOROETHYLENE
IN THE 1950s AND 1960s AND FOLLOWED THROUGH DECEMBER 1975<*
(adapted from Axelson et al. 1978)
Cohort
Total cohort
Subcohort
(_> 10
years of
latency
time)
- high exp.
- low exp.
Cause Person-yrs.
of at
death observation
All deaths 7688
Tumor deaths
All deaths 3643
Tumor deaths
All deaths 548
Tumor deaths
All deaths 3095
Tumor deaths
Observed
49
11
37
9
8
3
29
6
Expected
62.0
14.5
39.8
9.5
7.6
1.8
32.2
7.7
Risk
ratio
0.8
0.8
0.9
0.9
1.1
1.7
0.9
0.8
Confidence
95% limits
risk ratio
0.6 -
0.4 -
0.7 -
0.4 -
0.5 -
0.3 -
0.6 -
0.3 -
1.0
1.4
1.3
1.8
2.1
4.9
1.3
1.7
aExposure categorization is based on trichloroacetic acid (TCA) in the urine,
with low exposure referring to those not exceeding a concentration of 100 mg/1
TCA in urine on average.
in the manner in which the data are reported, that would restrict any conclusions
regarding this study. For example, little information is available concerning
the duration of exposure of the workers. No definition of minimum exposure
period seems to have been made by the authors, so that workers who were exposed
for a very short period of time could have been included in the study cohort.
Secondly, the definition of high and low exposure for the subcohort was based
on the average of TCA levels in the urine with no regard to how long the
individual may have worked in the high or low exposure area. Thus, an
individual may have had very low TCA levels over a short period of time, despite
the fact that most of his working time was in a high-exposure area where few
samples had been taken. Thirdly, the number of workers in the study (518) was
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relatively small for the detection of a significant risk of cancer from TCI
exposure. Furthermore, it should be noted in this regard that only half of
the total person-years of observation were for the group with > 10 years of
observation, indicating that most of the persons in the cohort had less than
10 years of observation. (It takes more people, when there are fewer person-
years per individual, to equal the number of person-years generated by people
with more person-years per individual.) Thus, less than 259 people had been
followed for as long as 10 years, which itself may not be a long enough
observation period in which to observe an increase in cancer mortality, con-
sidering that a latency period for cancer may be as long as 20 years. Finally,
no analysis was made with regard to specific tumor sites. If TCI is a
carcinogen, it is more likely to act on one particular organ or tissue site
than on all organ or tissue sites uniformly. It is interesting that 2 of the
11 deaths from cancer in this cohort were from leukemia (18%), whereas
approximately 4% of cancer deaths among males in Sweden are leukemia deaths.
In summary, this study does not indicate that TCI is a human carcinogen; how-
ever, there are severe limitations in interpreting the data. It should also be
noted that the primary author has reported in personal communications (Axelson 1980
and 1983), that the study is being updated through 1980 and that the cohort will be
expanded to include 1100 workers who were exposed to TCI between 1970 and 1975.
8' 2-2 loJJLJLt-AL'-JLli8-0!
An ongoing cohort study on workers in Finland exposed to TCI was reported
by Tola et al. (1980). According to the authors, the cohort is to be followed
and analyzed every fifth year. The cohort consisted of 1,148 men and 969 women
known to have been occupationally exposed to TCI at some time between 1963 and
1976. Names of workers were obtained from the biochemical laboratory of the
Institute of Occupational Health in Finland, where most of the analyses for
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urinary trichloroacetic acid (TCA), a TCI metabolite, were done as part of the
routine medical examinations for workers. Urinalyses were done by the Fujiwara
reaction, which was reported to be sensitive to levels as low as 1 ing TCA/1 urine.
The list of names of persons whose urinary TCA levels had been measured was supple-
mented with the names of persons who had been reported to have TCI poisoning,
according to the Occupational Disease Register of Finland, and with the names
of persons who had been reported by employees to have been exposed to TCI.
After confirming worker identities, the vital status and the causes of death of
those who died as of November 30, 1976 were obtained from the Population Data
Register of the Central Statistical Office in Finland, and cancer death informa-
tion was made available by the Finnish Cancer Registry.
Urinary TCA levels for 2,004 workers were reported. The majority (91%) of
the workers had TCA levels below 100 ug/1 urine, which, according to Axelson et
al. (1978), corresponds to an ambient exposure level of 30 ppm TCI. Furthermore,
78% of the workers had urinary TCA levels below 50 mg/1 urine. An additional 80
workers registered with clinical poisoning from exposure to TCI were also part of
the cohort; however, urinary TCA measurements were not available for these workers.
The observed numbers of all deaths and of cancer deaths were compared with
those expected based on national mortality statistics for 1971. A similar
comparison was done for a subcohort of workers exposed before 1970.
Results on the total cohort and subcohort are summarized in Table 8-25. No
statistically significant (P < 0.05) differences between the observed and the
expected numbers of deaths from cancer were found for either the total cohort
or the subcohort. Tumor sites for those dying from cancer included the gall
bladder, lung, breast, uterus, testis, and multiple myeloma. Two workers with
more than 100 ug TCA/1 urine and one worker with clinical poisoning had cancer;
however, the difference of three cases from the expected number was not statis-
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TABLE 8-25. COMPARISON OF WORKERS EXPOSED TO TRICHLOROETHYLENE WITH THE TOTAL
FINNISH POPULATION FOR MORTALITY FROM ALL CAUSES AND FOR CANCER MORTALITY
(adapted from Tola et al. 1980)
Total cohort
All deaths Neoplasms
Person-years Expected Observed Expected Observed
Males
7,041 52.1 40.0 6.6 5.0
Females
6,892 32.2 18.0 7.7 6.0
Subcohort (workers exposed before 1970)
Males
4,269 36.1 33.0 6.3 5.0
Females
4,822 25.8 13.0 6.0 4.0
tically significant (P < 0.05).
An excess cancer incidence among workers exposed to TCI was not found in this
study. The study, however, had a number of limitations. The duration of
follow-up was short; the age structure of the cohort was such that 60% of the
males and 40% of the females were less than 40 years old at the end of follow-up;
the possibility exists, as indicated by the authors, that some workers with low
urinary TCA levels actually could have been exposed to another degreasing
solvent but were included due to the use of an improper exposure test by the
attending physician; and the actual durations of exposure were unknown.
8.2.3 Malek et al. (1979)
Malek et al. (1979) studied the incidence of cancer among a group of 57
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dry-cleaners in Prague, Czechoslovakia. This group was reported to represent
86% of all men in Prague who had spent at least one year working in this type
of service since the 1950s. The follow-up was from 5 to 50 years with 50% of
the workers reported to have been followed for more than 20 years. Six cases
of cancer were reported for the group, which was reported to he not significantly
(P < 0.05) different from what would be expected. The expected number of cancer
cases was not included in the authors' report, however. The primary weakness of
this study is, of course, the small sample size, with the resulting weakness in
the power of the study to detect a significant increase in cancer incidence
above that expected.
8.2.4 Paddle (1983)
Paddle (1983) obtained from the Mersey Regional Cancer Registry in England
a list of all the liver cancer cases for the period 1951-77. Information on the
cases included name, address at registration, and dates of birth and registration.
This list was then reduced to those who had both primary liver cancer and an
address near Runcorn, which is the location of an Imperial Chemical Industries
(ICI) plant that has manufactured trichloroethylene since 1909. This resulting
list of 95 was then compared with a list of the "tens of thousands" of employees
of the Runcorn plant who had worked during the period 1934-76. None of the 95
primary liver cancer cases who had lived near Runcorn were found to have worked
in the ICI plant. As a further check, the files in the company's computerized
medical records system were searched for records of past employees dying with
liver cancer as the underlying cause. One liver cancer death was found for the
Runcorn plant and one liver cancer was found for a neighboring plant. The
cancer registry records, however, showed that the first of these had suffered a
primary cancer of the esophagus with a secondary liver tumor; the other patient
had had multiple secondary deposits from an unknown primary tumor.
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There are several deficiencies in this study. In addition, there are
limitations in the author's description which make the study difficult to
evaluate. One problem is that the author chose only those individuals from the
cancer registry who had addresses near Runcorn. Obviously, had a worker lived
farther away or moved from Runcorn following employment at the ICI plant, he
would not have been included in the study. Another problem is that the author
provides no indication as to the likelihood of finding an ICI worker with TCI
exposure among the 95 primary liver cancer cases. Although the author reported
that the list of 9b liver cancer cases was compared with a list of "tens of
thousands" of employees at the ICI plant at Runcorn, he also indicated in his
report that only about "1,000 or so" had TCI exposure. No controls were used
in the study; thus there is no basis for a statistical comparison. Furthermore,
it is not known whether the personnel lists used for comparison included the
names of all workers who had worked in the ICI plant during the period 1934-76
or only the names of those who had retired from the plant. If the lists included
only the names of retirees, then workers who may have developed liver cancer
but left employment before retiring would not have been included in the study.
Lastly, for those workers with TCI exposure, it is not known how long they were
exposed, or how recent their exposures might have been.
8.2.5 Blair et al. (1979)
Blair et al. (1979) reported, as the preliminary results of a cohort study
of 10,000 laundry and dry-cleaning workers, a proportionate mortality analysis
of 330 of the workers, who had died during the period 1957-77. Deaths were
identified from the mortality records of two union locals in St. Louis, Missouri.
The distribution by cause of death among the 330 was compared to that expected
based on the proportionate mortality experience of the U.S. Only 279 of these
330 had worked exclusively in dry-cleaning shops; however, the authors did not
8-78
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report the proportionate mortality analyses of these 279. Furthermore, it is
unknown what dry-cleaning agent the dry-cleaners in this study may have used.
Among the 330 deaths, deaths from cancer of the lung, cervix, and skin contributed
to the significant (P < 0.05) excess proportion of deaths from cancer at all
sites that was found. For lung cancer, there were 17 observed deaths versus 10
expected (P < 0.05), for cervical cancer there were 10 observed deaths versus
4.8 expected (P < 0.05), and for skin cancer there were 3 observed deaths versus
0.7 expected (P < 0.05). On the other hand, a significant deficit of deaths
occurred in the cause category identified as "all circulating diseases," with
100 observed versus 125.9 expected (P < 0.005), which could account for the excess
proportion of cancers.
Conclusions from this study regarding the potential carci nogenicity of TCI
are obviously limited. The distribution of deaths for dry-cleaners alone was
not reported. Furthermore, it is unknown what dry-cleaning agent these workers
may have used. Lastly, confounding variables, such as smoking with regard to
the lung cancer excess, socioeconomic status with regard to the cervical cancer
excess, and sunlight exposure with regard to the skin cancer excess, were not
considered in the analysis of the results.
8.2.6 Katz and Jowett (1981)
Katz and Jowett (1981) analyzed the death certificate records of 671 white
female laundry and dry-cleaning workers for the period 1963-1977. Dry-cleaning
workers, who are more likely to experience exposure to TCI than laundry workers,
could not be separated from laundry workers because they shared the same occupa-
tional code. Furthermore, the authors did not state what dry-cleaning agent
the dry-cleaning workers used.
Cause-specific proportionate mortality for the deceased laundry and dry-
cleaning workers was compared to that for the deaths of all other working females
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in Wisconsin during the same time period, and to deaths of females in "lower-wage
occupations" in Wisconsin during the same time period. Significantly elevated
proportionate mortality ratios were found for deaths from cancer of the genitals
(unspecified) (P < 0.01) and for deaths from cancer of the kidney (P < 0.05)
when both deaths among women of all occupations and deaths among women of
"lower-wage occupations" were used as the comparison groups.
In summary, this study, although suggesting an association of employment in
the dry-cleaning industry with an excess risk of certain types of cancer, cannot
be said to demonstrate an association between TCI exposure and an excess risk of
cancer because of the possible confounding influence of other dry-cleaning agents
and because of the inability, from the data available for study, to distinguish
between laundry and dry-cleaning workers.
8-2-7 Lin and Kessler (1981)
Lin and Kessler (1981) did a case-control study of 109 pancreatic cancer
cases diagnosed during the time period 1972-75 from 115 hospitals in five
metropolitan areas. Controls were subjects free of cancer but similar in age
(+_ 3 years), sex, race, and marital status, and were selected at random from
among contemporary admissions to the same hospital. Cases and controls were
asked about demographic characteristics, residental history, toxic exposures,
animal contacts, smoking habits, diet, medical history, medications, and family
history. Males were asked about sexual practices and urogenital conditions.
Females were questioned on these topics and also on their marital, obstetric, and
gynecologic practices. Among other statistically significant associations, an
association was found between pancreatic cancer and the drinking of decaffeinated
coffee. TCI has been used as a decaffeinating agent, but it is not known
whether the coffee that was drunk by the pancreatic cancer cases was decaffeinated
with TCI, or if it was, whether the decaffeinated coffee was contaminated with
8-80
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TCI. An association was also found between pancreatic cancer and employment
either as a dry-cleaner or in a job involving exposure to gasoline. It is not
known, however, how many pancreatic cancer cases were employed as dry-cleaners
and how many were employed in occupations involving exposure to gasoline. Further-
more, the dry-cleaners may have used a variety of cleaning agents. Thus this
study, although suggesting an association between employment as a dry-cleaner
and an excess risk of pancreatic cancer, cannot be said to demonstrate an
association between pancreatic cancer and TCI exposure.
8.2.8 Asa! (personal communication 1983)
Two cancer epidemiology studies conducted by Dr. Nabih Asa! of the University
of Oklahoma found an excess of cancer among dry-cleaners (personal communication
1983). Published reports of the studies are not yet available, however. One
of the studies was a kidney cancer case-control study in which Asal found an
association between kidney cancer and employment as a dry-cleaner. The second
study was a proportionate mortality study of dry-cleaning workers in Oklahoma.
Asal reported that results from that study suggested an increased mortality
from kidney and lung cancer among the workers. Again, as in the Blair et al.,
Katz and Jowett, and Lin and Kessler studies, exposure in the studies by Asal was
not specific for TCI. Moreover, the conclusions of the study cannot objectively
be evaluated until published descriptions of the studies are available.
8.3 QUANTITATIVE ESTIMATION
This quantitative section deals with the unit risk for TCI in air and water
and the potency of TCI relative to other carcinogens 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 throughout their lifetimes to a concentration of
1 ug/m3 of the agent in the air they breathe, or to 1 ug/1 in the water they
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drink. This calculation is done to estimate in quantitative terms the impact of
the agent as a carcinogen. Unit risk estimates are used for two purposes: 1)
to compare the carcinogenic potency of several agents with each other, and 2) to
give a crude indication of the population risk which might be associated with
air or water exposure to these agents, if the actual exposures are known.
These evaluations are made independently of the overall weight of evidence
that TCI is carcinogenic in animals. Therefore, the attempt here to do a
quantitative estimate should not be interpreted as implying that the evidence
for carcinogenicity is strong. For TCI that evidence is limited.
8.3.1 Pjjcedu_res__fp_T_jtetermjnati_on__of_ IInLit_ Risk
The data used for quantitative estimation are one or both of two types:
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 the extrapolation model.
There is no solid scientific basis for any mathematical extrapolation model
that relates carcinogen exposure to cancer risks at the extremely low concentra-
tions that must be dealt with in evaluating environmental hazards. 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 carcino-
genic process involves the concept that most agents that cause cancer also cause
irreversible damage to DMA. This position is reflected by the fact that a very
large proportion of agents that cause cancer are also mutagenic. There is reason
to expect that the quanta! type of biological response, which is characteristic
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of mutagenesis, is associated with a linear non-threshold dose-response relation-
ship. 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). There is also
some evidence from animal experiments that is consistent with the linear non-
threshold model (e.g., liver tumors induced in mice by 2-acetylaminofluorene in
the large scale EDgi study at the National Center for Toxicological 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 to low levels 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
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for estimating the largest possible linear slope (in the 95% confidence limit
sense) at low extrapolated doses that is consistent with the data at all dose
levels of the experiment.
8.3.1.1 Annas
8. 3. 1 . 1. 1 jfejj:jJjpJ;Jjw_jrf_J:Jie^ model . Let P (d )
represent the lifetime risk (probability) of cancer at dose d. The multistage
model has the form
P(d) = 1 - exp C-(qQ + qjd + q2d2 + ... + qkdk)]
where
qi 2. 0, i = 0, 1, 2 ..... k
Equi valently,
Pt(d) = 1 - exp [-(qjd + q2d2 + ... + qkdk)]
where
is the extra risk over background rate at dose d or the effect of treatment.
The point estimate of the coefficents qi, i =0, 1, 2, ..., k, and
consequently the extra risk function Pt(d) at any given dose d, is calculated
by maximizing the likelihood function of the data.
The point estimate and the 95% upper confidence limit of the extra risk,
Pt(d), are calculated by using the computer program GLOBAL79, developed by Crump
and Watson (1979). At low doses, upper 95% confidence limits on the extra risk
and lower 95% confidence limits on the dose producing a given risk are determined
from a 95% upper confidence limit, q*, on parameter qj. Whenever qi > 0, at low
doses the extra risk P^(d) has approximately the form ?t(d) = qj x d. Therefore,
q* x d is a 95% upper confidence limit on the extra risk and R/q* is a 95% lower
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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 m to a value q* such that when the log-likelihood is remaximized
subject to this fixed value, q*, for the linear coefficient, the resulting
maximum value of the log-likelihood LI satisfies the equation
2 (LQ - Lj) = 2.70554
where 2.70554 is the cumulative 90% point of the chi-square distribution with
one degree of freedom, which corresponds to a 95% upper-limit (one-sided). This
approach of computing the upper confidence limit for the extra risk Pt(d) is
an improvement on the Crump et al. (1977) model. The upper confidence limit for
the extra risk calculated at low doses is always linear. This is 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^(d) will be
abbreviated as P.)
In fitting the dose-response model, the number of terms in the polynomial is
chosen equal to (h-1), where h is the number of dose groups in the experiment
including the control group.
Whenever the multistage model does not fit the data sufficiently well, data
at the highest dose is deleted, and the model is refit to the rest of the data.
This is continued until an acceptable fit to the data is obtained. To determine
whether or not a fit is acceptable, the chi-square statistic
h 2
X 2 = £ i JL i_ i
8-85
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is calculated where N^ is the number of animals in the itn dose group, X^ is
the number of animals in the itn dose group with a tumor response, P^ is the
probability of a response in the itn dose group estimated by fitting the multi-
stage model to the data, and h is the number of remaining groups. The fit is
determined to be unacceptable whenever X^ is larger than the cumulative 99%
point of the chi-square distribution with f degrees of freedom, where f equals
the number of dose groups minus the number of non-zero multistage coefficients.
8.3.1.1.2 Selection of data. For some chemicals, several studies in different
animals species, strains, and sexes, each 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. It may also be appropriate to correct
for metabolism differences between species and absorption factors via different
routes of administration. The procedures used in evaluating these data are
consistent with the approach of making a maximum-likely risk estimate. They
are li sted 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 statistically significantly higher than the
control for at least one test dose level and/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 that has the larger sample size is
selected for calculating the carcinogenic potency.
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2. If there are two or more data sets of comparable size that are identical
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 A]_, A2, ..., Am is defined as
(A Y A y x A }^/m
\r\ 1 A "*Q " • • • A m /
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.
8.3.1.1.3 Calculation of human equivalent dosages from animal data. Following
the suggestion of Mantel and Schneiderman (1975), we assume that mg/surface area/
day is an equivalent dose between species. Since, to a close approximation, the
surface area is proportional to the 2/3rds power of the weight as would be the
case for a perfect sphere, the exposure in mg/day per 2/3rds 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 i n 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=]eXm
Le x W2/3
8.3.1.1.3.1 Oral. Often exposures are not given in units of mg/day and it
becomes necessary to convert the given exposures into mg/day. For example, in
8-87
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most feeding studies exposure is in terms of ppm in the diet. Similarly in
drinking water studies exposure is in ppm in the water. In these cases the
exposure in mg/day is
m = ppm x F x r
where ppm is parts per million of the carcinogenic agent in the diet or water, F
is the weight of the food or water consumed per day in kg, and r is the absorption
fraction. In the absence of any data to the contrary, r is assumed to be equal
to one. For a uniform diet, the weight of the food consumed is proportional to
the calories required, which in turn is proportional to the surface area or 2/3rds
power of the weight. Water demands are also assumed proportional to the surface
area, so that
m « ppm x W*-/3 x r
or
m « ppm
rW2/3
As a result, ppm in the diet or in water is often assumed to be an
equivalent exposure between species. However, we feel that this is not
justified since the caloriesAg of food are very different in the diet of man
compared to laboratory animals primarily due to moisture content differences.
Consequently, the amount of drinking water required by each species differs
also because of the amount of moisture in the food. Therefore, we use an
empirically-derived factor, f = F/VJ, which is the fraction of a species body
weight that is consumed per day as food or water. We use the following rates:
Fraction of Body
Weight Consumed as
Species W ffood *water
"Man700.028 0.029
Rats 0.035 0.05 0.078
Mice 0.03 0.13 0.17
8-88
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Thus, when the exposure is given as a certain dietary or water concentration in
ppm, the exposure in mg/W^/3 is
m
= ppm x F = ppm x f x W = ppm x f x W1/3
rW2/3 w2/3 w2/3
When exposure is given in terms of mg/kg/day = m/Wr = s, the conversion is
simply
m = s x W1/3
rW2/3
8.3.1.1.4 Calculation of the unit risk from animal studies. The 95% 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 simply the risk corresponding to an exposure of X = 1.
To estimate this value we simply find the number of mg/kg2/3/day corresponding to
one unit of X and substitute this value into the above relationship. Thus, for
example, if X is in units of ug/m3 in the air, we have that for case 1, d = 0.29 x
7L)1/3 x 10"3 mg/kg2/3/day, and for case 2, d = 1, when ug/m3 is the unit used to
compute parameters in animal experiments.
If exposures are given in terms of ppm in air, we may simply use the fact
that
o
1 ppm = 1.2 x rcoTecuTar weight (gas) mg/nr
molecular weight (air)
Note, an equivalent method of calculating unit risk would be to use mg/kg/day
for the animal exposures and then increase the jth polynomial coefficient by an
amount
(Wh/Wa)J/3 j = 1, 2 k
and use mg/kg equivalents for the unit risk values.
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8.3.1.1.4.1 Adjustment for less than lifespan duration of experiment. If
the duration of experiment, Le, is less than the natural lifespan of the test
animal, L, the slope, q*, or more generally the exponent, g(d), is increased by
multiplying a factor (L/L_e)3. We assume that if the average dose, d, is continued,
the age-specific rate of cancer will continue to increase as a constant function
of the background rate. The age-specific rates for humans increase at least by
the 2nd power of the age and often by a considerably higher power as demonstrated
by Doll (1971). Thus, we would expect the cumulative tumor rate to increase by
at least the 3rd power of age. Using this fact, we assume that the slope, q*, or
more generally the exponent, g(d), would also increase by at least the 3rd power
of age. As a result, if the slope q* [or g(d)j is calculated at age Le, we would
expect that if the experiment had been continued for the full lifespan, L, at the
given average exposure, the slope q* [or g(d)] would have been increased by at
least (L/Le)3.
This adjustment is conceptually consistent with the proportional hazard
model proposed by Cox (1972) and the time-to-tumor model considered by Crump
(1979) where the probability of cancer by age t and at dose d is given by
P(d,t) = 1 - exp C-f(t) x g(d)]
8.3.1.1.5 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
8-90
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on a mg/surface area basis is virtually without experimental verification
regarding carcinogenic response. Finally, human populations are variable with
respect to genetic constitution and diet, living environment, activity patterns,
and other cultural factors.
The unit risk estimate can give a rough indication of the relative potency
of a given agent compared with other carcinogens. The comparative potency of
different agents is more reliable when the comparison is based on studies in
the same test species, strain, and sex, and by the same route of exposure,
preferably by inhalation.
The quantitative aspect of the 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, it should be recognized that the estimation of cancer risks to humans
at low levels of exposure is uncertain. At best, the linear extrapolation model
used here provides a rough but plausible estimate of the upper-limit of risk;
i.e., it is not likely that the true risk would be much more than the estimated
risk, but it could very well be considerably lower. The risk estimates presented
in subsequent sections should not be regarded as an accurate representation of
the true cancer risks even when the exposures are accurately defined; however,
the estimates presented may be factored into regulatory decisions to the extent
that the concept of upper risk limits is found to be useful.
8.3.1.1.6 Alternative methodological approaches. The methods used by the
Carcinogen Assessment Group (CAG) for quantitative assessment are consistently
conservative, i.e., tending 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
8-91
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alternative models have not been used by the CAG in the following analysis
because it feels that the limited evidence of carcinogenicity for TCI does not
warrant that complete an analysis. Furthermore, the CAG feels that with 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. The position is taken by
the CAG that the risk estimates obtained by use of the linear non-threshold
model are plausible upper limits, and that the true risk could be lower.
In terms of the choice of animal bioassay as the basis for extrapolation,
the general approach is to use the most sensitive responder on the assumption
that humans are as sensitive as the most sensitive animal species tested. For
TCI, the average response of all of the adequately tested bioassay animals
was used; this is because three well-conducted valid drinking water studies
using different strains of rats showed similar target organs and about the
same level of response.
Extrapolations from animals to humans could also be done on the basis of
relative weights rather than relative surface areas. The latter approach, used
here, has more basis in human pharmacological responses; it is not clear which
of the two approaches is more appropriate for carcinogens. In the absence of
information on this point, it seems appropriate to use the most generally employed
method, which also is more conservative. In the case of the TCI gavage study,
the use of extrapolation based on surface area rather than weight increases the
unit risk estimates by a factor of 12 to 13.
8.3.1.2 Humans--Mode1 for Estimation of Unit Risk Based on Human Data—If human
epidemiologic studies and sufficiently valid exposure information are available
for the compound, they are always used in some way. If they show a carcinogenic
effect, the data are analyzed to give an estimate of the linear dependence of
8-92
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cancer rates on lifetime average dose, which is equivalent to the facter B^. If
they show no carcinogenic effect when positive animal evidence is available,
then it is assumed that a risk does exist, but it is smaller than could have been
observed in the epidemiologic study, and an upper limit to the cancer incidence is
calculated assuming hypothetically that the true incidence is below the level
of detection in the cohort studied, which is determined largely by the cohort
size. Whenever possible, human data are used in preference to animal bioassay
data.
Very little information exists that can be utilized to extrapolate from
high exposure occupational studies to low environmental levels. However, if a
number of simplifying assumptions are made, it is possible to construct a crude
dose-response model whose parameters can be estimated using vital statistics,
epidemiologic studies, and estimates of worker exposures.
In human studies, the response is measured in terms of the relative risk of
the exposed cohort of individuals compared to the control group. The mathematical
model employed assumes that for low exposures the lifetime probability of death
from lung cancer (or any cancer), PQ, may be represented by the linear equation
P0 = A + BHx
where A is the lifetime probability in the absence of the agent, and x is the
average lifetime exposure to environmental levels in some units, say ppm. The
factor, BH, is the increased probability of cancer associated with each unit
increase of the agent in air.
If we make the assumption that R, the relative risk of lung cancer for
exposed workers compared to the general population, is independent of the length
or age of exposure but depends only upon the average lifetime exposure, it
follows that
8-93
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R = JL = A + BH (X1 + x?)
PU A + BH xi
or
RPQ - A + BH (X1 + x2)
where xi = lifetime average daily exposure to the agent for the general
population, xg = lifetime average daily exposure to the agent in the
occupational setting, and PQ = lifetime probability of dying of cancer with
no or negligible TCI exposure.
Substituting PQ = A + B^ x xi and rearranging gives
BH = PO (K - i)Az
To use this model, estimates of R and X2 must be obtained from the epidemiologic
studies. The value PQ is derived from the age-cause-specific death rates for
combined males found in the 1976 U.S. Vital Statistics tables using the life
table methodology. For lung cancer the estimate of Pg is 0.036. This
methodology is used in the section on unit risk based on human studies.
8.3.2 Unit Risk from Animal Studies
8.3.2.1 Water—None of the six available epidemiologic studies provided positive
results from which to derive a quantitative risk estimate for TCI exposure. For
the animal data only the 1982 NTP gavage study and the 1976 NCI gavage study
provided sufficient evidence of treatment-related carcinogenicity; both of
these studies produced treatment-related hepatocellular carcinomas in male and
female B6C3F1 mice. The results of all the carcinogenicity bioassays have been
previously presented (Table 8-1). The NTP tumor data for male and female mice
have also been presented (Table 8-6 and 8-7). The NCI tumor data for male and
female mice have been presented in Table 8-11. These hepatocellular carcinoma
data are also shown in Table 8-26, together with the human equivalent dosage
8-94
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TABLE 8-26. INCIDENCE RATES OF HEPATUCELLULAR CARCINOMAS IN MALE AND FEMALE MICE IN
THE NTP (1982) AND NCI (1976) GAVAGE STUDIES. CONTINUOUS HUMAN EQUIVALENT DOSAGES,
AND ESTIMATES OF q*. 95% UPPER-LIMIT SLOPE, FROM THE LINEARIZED MULTISTAGE MODEL
Continuous human
equivalent (animal
Incidence rates
95% upper-)imit
slope estimate
nominal ) doses
Study mg/kg/day
Male NTP 0
59. 3d
NCI 0
56.3
112.6
Female NTP 0
56.7
NCI 0
38.7
77.3
(0)
(1,000)
(°) K
(l,169)b
(2,339)b
(0)
(1,000)
(0)
(869)b
(l,739)b
No. with tumor/total (%)
8/48
30/50
1/20
26/50
31/48
2/48
13/49
0/20
4/50
11/47
(17%)
(60%)
(5%)
(52%)
(65%)
(4%)
(27%)
(0%)
(3%)
(23%)
"I
(mg Ag/dav)-1
1.8 x 10-2
2.0 x
1.1 x
6.5 x
,„-*
10'3
10-3c
aAll 95% upper-limit slopes q* calculated using continuous human equivalent doses.
Equivalent human dosage = animal dose x 5 days x e x (Wa/70) '
where Wa = weight of the mice. The average weight of males is taken as 40 grams
for dosed males and 35 grams for dosed females for the NTP study; for the NCI
study the average weights are 33 grams for males and 26 grams for females. Le
= 2 years and le, the duration of exposure, is 2 years for the NTP study and
1.5 years for the NCI study.
bTime-weighted average gavage dose over 78-week treatment period; see Table 8-8.
cSlope is increased by (104/90)3 = 1.54 due to the duration of the experiment's
being less than the natural lifespan of the test animal.
8-95
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conversions and the 95% upper-limit slope factor, q*. As seen in the table,
the largest estimates are those from hepatocellular carcinomas in the males.
These values are q* = 1.8 x 10-2 (mg Ag/day)"l from the NTP gavage study and
2.0 x 10"2 (mg/kg/day )-* from the NCI gavage study. Since these are essentially
the same study, we take the geometric mean of
q* = 1.9 x 10-2 (mgAg/day)'1
as the unit risk value.
In order to estimate a unit risk for a concentration of 1 ug/1 drinking
water, the following conversion is used.
1 mg/kg/day x 70 kg x 103 ug_ x l_day = 3. 5 x 104 ug/1
mg 2 1
Therefore, the unit risk corresponding to 1 ug TCI per liter of water is
q* = 1.9 x 10-2 (mg/kg/day)-1 x _J_jng^£/d_ay___ - 5.4 x 10-7 (Ug/l
3.5 x 10 ug/1
8.3.2.2 IjiJiaJj|tJ_on--Two animal inhalation studies showed positive results in
mice. These were the Henschler et al. (1980) study showing increased malignant
lymphomas in females and the Bell et al. (1978) study showing increased hepato-
cellular carcinomas in males. As discussed in the qualitative section, however,
both studies showed serious defects and are considered inadequate for calculating
unit risk estimates. As an alternative the unit risk estimate based on both the
NTP (1982) and NCI (1976) gavage studies can be used. That slope estimate must
first be converted to units of ug/m3 by the following conversion.
1 mg/kg/day = 1 mg/kg/day x _7_OJ
-------
or
1 ug/m3 =2.86 x 10~4 mg/kg/day.
Data presented in the metabolism section show pulmonary absorption levels of TCI
in humans as high as 75% of the inhaled amount. Assuming that 75% of inhaled TCI
can be absorbed into the body, 1 ug/m3 in the air would result in an absorbed
effective dose of 2.15 x 10~3 mgAg/day. The unit risk can then be estimated as
q* = 1.9 x ID'2 (mg/kg/day)-1 x 2.15 x 10~4 nLg_Ag_/d_ay_ = 4.1 x 10"6 (ug/m3)-1
ug/m3
8. 3. 2.3 UjpJgJjjjjnjj^jJ^
H_umans--Although the epidemiologic studies on TCI provide no evidence for
its carcinogenicity, they can still be used to provide information on an upper
limit of risk to be expected. The CAG adopts this procedure of using negative
epidemiological studies for providing upper limits when animal studies are
positive. Such use of negative human studies can modify estimates of potency
from the positive animal studies.
The following 95% upper-limit estimates on relative risk are derived from
data in Table 8-24 obtained by Axelson et al. (1978). These upper-limit
estimates are calculated according to the following approach.
The 95% upper limit = u/expected number of cancer deaths (Table 8-27),
where u is the solution to the sum of Poisson probabilities
*n l^iUlU 0.025
i=0 i!
One might also interpret u as the upper limit of the number of workers with
cancer deaths given the observed frequency, and X = number of observed workers
with cancer deaths. Hence, the 95% upper-limit estimates for the cohort and
each subcohort are as follows (Table 8-27).
8-97
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TABLE 8-27. NUMBER OF WORKERS WITH CANCER DEATHS
Cohort
Total cohort
Subcohort with
10 yr. latency
(high and low
exposure)
High exposure
Low exposure
Observed
X
11
9
3
6
Upper limit Expected
u if no effect
19.7 14.5
17.2 9.5
8.7 1.8
13.3 7.7
Relative risk
95/i upper 1 imi t
1.4
1.8
4.8
1.7
These upper-limit relative risks can be used to provide estimates of the
upper-limit probability of death from cancer due to a lifetime exposure to 1 ug/m
of TCI. This upper-limit probability estimate is 1.7 x 1U5 per ug/m3 of TCI in
air, and it is four times greater than the risk estimate based on the NCI (1976)
and NTP (1982) carcinogenicity gavage bioassays. In showing these risk calcula-
tions below, the CAG emphasizes that:
1. This upper-limit value is derived from a negative epidemiologic study.
Not only was there no specific target organ for cancer, but the total
number of cancer deaths was less than expected.
2. Since there was no specific target organ, the upper-limit lifetime
probability estimate is based on all cancer deaths. Since the
lifetime probability of death from cancer to humans is 0.19 (based on
1977 U.S. death rates, race and sex combined), any even moderately
conservative confidence limit will provide a large upper-limit risk.
The calculations are based on the subcohort data from the Axelson et al.
(1978) study, specifically the low exposure group with at least a 10-year latency
8-98
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period. Table 8-27 projects an upper-limit relative risk of 1.7. Since the low
exposure referred to those not exceeding a concentration of 100 nig trichloroaceti c
acid per ml in urine on the average, we equate this exposure to be not more than
30 ppm TCI in air. We use the 30 ppm as the 8-hour time-weighted average dose.
This corresponds to 30 ppm x 103 x 1.2 x MTCI/Mair = 30 x 103 x 1.2 x 131.4/28.3 =
1.64 x 10^ ug/m3. A lower dose estimate would provide an even higher upper-limit
probability of death.
For years exposed, in the absence of any other information, we chose 10
years as a point assumed to be somewhere in the middle of the range.
With the above information we estimate the total lifetime dose of TCI
to the low-dose exposure workers as:
(0 ppm + 1.64 x 105) x 240 x _10 = 7. 7 x 103 ug/m3 continuous
2 365 70
where the 1.64 x 105 ug/fa3 TCI of the working day must be averaged with the
0 ug/m3 of the nonworking day. We assume that one-half the air breathed during
a day will be during working hours. Thus, the upper-limit estimate of the
probability of death from 1 ug^i3 TCI in air (continuous exposure) is estimated
as:
P = « (R - l)dl = 0.19 (1.7 - 1)1 = 1.7 x 1Q-5 per ug/m3
d2 7.7 x 103
where R = the relative risk, dj = 1 ppm, ^2 - dose of workers, and a = the
background lifetime probability of death from all cancers.
Comparing this upper-limit estimate with that from the animal studies, the
Axelson et al. study provides no evidence that human potency is less than that
of animals.
8.3.3 Relative Potency
One of the uses of the concept of unit risk is to compare the relative
8-99
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potencies of carcinogens. To estimate relative potency on a per mole basis,
the unit risk slope factor is multiplied by the molecular weight, and the
resulting number is expressed in terms of (mg/Mol Ag/day)~*. This is called
the relative potency index.
Figure 8-15 is a histogram representing the frequency distribution of
potency indices of 54 chemicals evaluated by the GAG as suspect carcinogens.
The actual data summarized by the histogram are presented in Table 8-28.
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
animal oral studies; this allows potency comparisons by route.
The potency index for TCI based on mouse hepatocellular carcinomas in the
NTP and NCI gavage studies is 5.8 x ICT1. This is derived as follows: the mean
slope estimate from both studies, 1.9 x 10~2 (mgAg/day)"1, is multiplied by the
molecular weight of 131.4 to give a potency index of 2.5 x 10U. Kounding off to
the nearest order of magnitude gives a value of 10°, which is the scale presented
on the horizontal axis of Figure 8-15. The index of 2.5 is among the least
potent of the 54 suspect carcinogens, ranking in the lowest quartile. 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
for 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. <
8-100
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4th 3rd 2nd 1st
quartile quartile quartile quartile
1 1 1
1x10*1 4x10*2 2x10*3
CN
1
-2
eo
j
0
I
1
i
2
CO
1
CN
I
4
I
8
Log of Potency Index
Figure 8-15. Histogram representing the frequency distribution of the potency indices of 5^ susoect
carcinogens evaluated by the Carcinogen Assessment Group.
8-101
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TABLE 8-28. RELATIVE CARCINOGENIC POTENCIES AMONG 54 CHEMICALS EVALUATED BY
THE CARCINOGEN ASSESSMENT GROUP AS SUSPECT HUMAN CARCINOGENS1.2.3
Slope Molecular
Compounds (mg/kg/day)_i weight
Acrylonitrile
Aflatoxin BI
Aldrin
Ally! Chloride
Arsenic
B[a]P
Benzene
Benzidene
Beryl lium
Cadmium
Carbon Tetrachloride
Chlordane
Chlorinated Ethanes
1,2-dichloroethane
hexachloroethane
1 ,1,2,2-tetrachl oroethane
1,1, 1-trichloroethane
1,1,2-trichl oroethane
Chloroform
Chromium
DDT
Dichlorobenzidine
1,1-dichloroethylene
Dieldrin
0.24(W)
2924
11.4
1.19 x 10-2
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 ID'3
5.73 x 10-2
7 x ID'2
41
8.42
1.69
1.47 x 10-1(1)
30.4
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
Potency
i ndex
1 x 10+1
9 x 10+b
4 xlO+3
9 x 1U-1
2 x 1U+3
3 x 10+3
4 x ll)U
4 x 10+4
4 x 1U+1
7 x 1U+2
2 x 10+1
7 x 10+2
7 x ll)U
3 x 11)0
3 x 10+1
2 x 10-1
8 x 11)0
8 x 10U
4 x 10+3
3 x 10+3
4 x 10+2
1 x 10+1
1 x 10+4
Order of
magni tude
(logio
i ndex)
+1
+6
+4
0
+3
+3
+1
+5
+2
+3
+1
+3
+1
0
+1
-1
+1
+1
+4
+3
+3
+1
+4
(continued on the following page)
8-102
-------
TABLE 8-28. (continued)
Compounds
Dinitrotol uene
Diphenylnydrazine
Epichlorohydrin
Bi s(2-chloroethyl )ether
Bi s(chloromethl )ether
Ethylene Dibromide (EDB)
Ethylene Oxide
Formaldehyde
Heptachlor
Hexachlorobenzene
Hexachlorobutadiene
Hexachlorocyclohexane
technical grade
alpha isomer
beta isomer
gamma i somer
Methylene Chlori de
Nickel
Ni trosamines
Dimethylnitrosamine
Diethylnitrosamine
Dibutylnitrosamine
N-ni trosopyrroli dine
N-ni troso-N-ethyl urea
N-ni troso-N-methyl urea
N-nitroso-diphenylamine
PCBs
Slope Molecular
(mg/kg/day)_i weight
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*)
1
43.5(not by q*)
1
5.43
2.13
32.9
302.6
4.92 x 10-3
4.34
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
324
Potency
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
1 x 10+3
3 x 10+3
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
2 x 10+2
4 x 10+3
3 x 10+4
1 x 11)0
1 x 10+3
Order of
magni tude
OOQIO
i ndex )
+2
+2
0
+2
-t-6
+3
+1
0
+3
+3
+1
+3
+3
+3
+3
-1
+2
+3
+4
+3
+2
+4
+4
0
+3
8-103
(continued on the following page)
-------
TABLE 8-28. (continued)
Compounds
Phenols
2,4,6-trichlorophenol
Tetrachlorodioxin
Tetrachloroethylene
Toxaphene
Trichloroethy lene
Vinyl Chloride
Slope Molecular
(mgAg/day)_i weight
1.99 x
4.25 x
3.5 x
1.13
1.9 x
1.75 x
10-2
10*
10-2
10-2
10-2(1)
197.4
322
165.8
414
131.4
62.5
Potency
index
4 x 10°
1 x 10+8
6 x 10°
5 x 10+2
2.5 x 10U
1 x 10°
Order of
magni tude
(logio
i ndex)
+ 1
+8
+ 1
+3
0
0
Remarks:
1. Animals slopes are 95% upper-limit slopes based on the linearized 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). Hunan slopes are
point estimates based on the linear non-threshold model.
2. The potency index is a rounded-off slope in (rrMol /kg/day)"•*• and is cal-
culated by multiplying the slopes in (mg/kg/day)"^- 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.
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8.4 SUMMARY AND CONCLUSIONS
8.4.1 Qualitative Asssessment
8.4.1.1 Animal Studies—In a 90-week carci nogenicity bioassay with male and
female B6C3F1 mice given estimated maximally (2,339 mg/kg in males and 1,739
mg/kg in females) and one-half maximally tolerated doses of technical grade,
epoxide-stabilized TCI in corn oil daily, 5 days/week, for 78 weeks, statistically
significant increases in the incidence of hepatocellular carcinomas in male and
female mice compared to corn oil controls were found [National Cancer Institute
(NCI) 1976]. Statistically significant increases in hepatocellular carcinoma
incidence in treated male and female mice compared to vehicle-control mice were
also observed in a repeat of the above carcinogenicity bioassay in which male
and female B6C3F1 mice were treated with purified TCI, containing no detectable
epoxides, in corn oil by gavage at a dose of 1,000 mg/kg/day, 5 days/week, for
103 weeks [National Toxicology Program (NTP) 1982J. In effect, this repeat study
indicated that epoxide stabilizers were not necessary factors in the TCI-induced
increases in hepatocellular carcinoma incidence in male and female B6C3F1 mice
under the conditions of these bioassays. It is noted that spontaneous hepato-
cellular tumor formation is a characteristic of the B6C3F1 mouse strain.
A 110-week carcinogenicity study on male and female Osborne-Mendel rats
given technical grade, epoxide-stabilized TCI in corn oil by gavage at estimated
maximally (1,097 mg/kg) and one-half maximally tolerated doses daily, 5 days/
week, for 78 weeks, was negative for carcinogenicity; however, this study may
be inconclusive because of high mortality in the treatment groups (NCI 1976).
In a carcinogenicity study on male and female Fischer 344 rats treated with
purified TCI, containing no detectable epoxides, in corn oil by gavage at
estimated maximally (1,000 mg/kg) and one-half maximally tolerated doses for
103 weeks, a small incidence of renal adenocarcinomas in high-dose males at
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terminal sacrifice (3/16) was calculated to be a statistically significant
increase over that in matched vehicle treated controls (0/33) by life table
and incidental tumor tests, which adjust for mortality, but not by the Fisher
Exact Test (NTP 1982). However, the NTP, sponsor of the study, has concluded
that this study is inadequate to evaluate the presence or absence of a carcino-
genic response to TCI. Under the conditions of this study in Fischer 344
rats, there was a statistically significant increase in mortality in treated
males and 2, 6, and 20% of the matched vehicle-control, low-dose, and high-dose
males, respectively, were accidentally killed during the study. Toxic nephrosis
characterized as cytomegaly was commonly found in the treated male and female
Fischer 344 rats, with a stronger effect evident in males. A carcinogenicity
study of purified TCI administered by gavage in olive oil to male and female
Sprague-Dawley rats daily, 4 or 5 days/Aveek for 52 weeks, at doses of 250 or 50
mg/kg followed by lifetime observation was negative, but the 52-week exposure
period was below potential lifetime exposures for these animals (Maltoni 1979).
Inhalation exposure of male Han:NMRI mice, male and female Han:Wist rats,
and male and female Syrian hamsters to purified TCI vapor at levels of 100 ppm
and 500 ppm 6 hours/day, 5 days/week, for 78 weeks followed by lifetime obser-
vation did not induce a carcinogenic effect (Henschler et al. 1980). Overt
toxicity of TCI was not evident in rats and hamsters, which suggests that
testing at higher levels of TCI was possible in this study, but mortality was
greater in male mice compared to controls. A statistically significant increase
in malignant lymphoma incidence was found in low-dose and high-dose female
Han:NMRI mice compared to controls in this study; however, the spontaneous
incidence of malignant lymphomas in controls was high (30%), and it was postu-
lated in the study report that the increased incidence of malignant lymphomas
in female mice could have been a result of an immunosuppressive response capable
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of enhancing susceptibility to tumor induction by specific, mostly inborn,
viruses (Henschler et al. 1980).
A chronic inhalation study of technical grade, epoxide-stabilized TCI as a
vapor was done in which male and female Charles River rats and male and female
B6C3F1 mice were exposed to nominal levels of TCI at 100, 300, and 600 ppm 6
hours/day, 5 days/week for 24 months (Bell et al. 1978). An audit of liver
pathology revealed no carcinogenic effect in rats and a dose-related increase
in the incidence of hepatocellular tumors in male and female B6C3F1 mice;
however, this study is weakened by wide variability in the actual TCI exposure
levels and lack of a concurrent matched control group of mice.
A series of studies done to evaluate the carcinogenic potential of purified
TCI in lifetime treatment studies with female ICR/Ha Swiss mice included an
initiation-promotion study in which a single topical application of 1 mg TCI in
acetone was followed by lifetime repeated topical applications with the promoter
phorbol myristate acetate; a study of complete carcinogenicity with repeated
topical applications of 1 mg TCI in acetone; a study with once weekly sub-
cutaneous injections of 0.5 mg TCI in trioctanoin; and a study in males as
well as females with once-weekly gavage administrations of 0.5 mg TCI in
trioctanoin (Van Duuren et al. 1979). Complete carcinogenic and initiating
activities for TCI were not found in these studies. The dose used for topical
application was below that estimated as maximally tolerated, but the authors
concluded that no activity or low activity of chloroolefins tested for carcino-
genicity on mouse skin would be expected because of a relatively low level of
epoxidizing enzymes in mouse skin. More than once-weekly dosing by gavage and
subcutaneous administration could have permitted a broader evaluation of TCI
card nogenicity.
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Carcinogenic activity for TCI was not observed in several animal studies
designed for an evaluation of general toxicity; however, these studies involved
small groups and durations of treatment and observation that were less than
the lifetime of the animals or, in one study, a duration that was not specified.
Exposure of newborn Wistar rats for ID weeks to 2,000 ppm TCI or vinyl chloride,
8 hours/day, 5 days/week induced ATPase-deficient foci, reported as premalignant
lesions, in liver only in rats exposed to the latter agent (Laib et al. 1978, 1979);
however, while only vinyl chloride was effective with this exposure duration,
it is not certain whether longer exposure to TCI could ultimately have induced
similar foci .
The carcinogenic potential of TCI oxide, a putative direct-acting metabolite
of TCI, has also been evaluated in lifetime treatment studies with female
ICR/Ha Swiss mice (Van Duuren et al. 1983). These studies included an initiation-
promotion study in which a single topical application of 1 mg TCI oxide in
acetone was followed by lifetime repeated topical applications with phorbol
myristate acetate; a study of complete carcinogenicity with repeated topical
applications of 2.5 mg TCI oxide in acetone; and a study with once-weekly
subcutaneous injections of 0.5 mg TCI oxide in trioctanoin. These studies were
negative for initiating activity and complete carcinogenic activity at estimated
maximum tolerated doses.
8.4.1.2 Cell Transformation $tudies--Ce11 transformation activity by TCI, at
minimally toxic doses, and 3-methylcholanthrene, a positive control agent, was
found in an in vitro study with Fischer rat embryo cells (F1706, subculture
108) containing genetic information of the Rauscher leukemia virus (Price et
al. 1978). Cell transformation has been developed as an endpoint for predicting
carcinogenic activity; however, it is not certain whether these agents elicited
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transformation in this Fischer rat cell line through an action on the host
cells, their integrated oncornavirus, or both.
Exposure of baby Syrian hamster kidney (BHK-211C1 13) cells in the presence
of rat liver microsomes in vitro to TCI solution in DMSO at doses including
those producing toxicity did not induce cell transformation, whereas vinyl
chloride induced cell transformation when administered as a gas at levels
including those producing toxicity, but not as a solution in DMSO tested at
levels where toxicity was not clearly evident (Styles 1979). A broader evalua-
tion could have been made with an additional test where cells were exposed to
TCI as a vapor.
Cell transformation was induced by high concentrations (>1 mM) of TCI oxide
capable of producing dose-related cytotoxicity in cultures of Syrian hamster
embryo cells. The transformed colonies in this system were similar to those
induced by benzo[a]pyrene and other chloroalkene epoxides (UiPaolo and Doniger
1982).
8.4.1.3 Human Studies—Three cohort studies of workers exposed to TCI did not
find that these workers experienced an excess risk of cancer (Axel son et al. 1978,
Tola et al. 1980, and Malek et al. 1979). Each of these studies, however,
suffered from one or more of the following deficiencies: small sample size,
lack of analysis by tumor site, problems with exposure definition, and problems
with length of exposure.
Paddle (1983) found that none of the primary liver cancer cases residing
in an area near an industrial plant that produced TCI had been employed by the
plant. This study had several deficiencies, and there are also limitations in
the author's description of the study which make it difficult to evaluate. No
controls were used in the study, and the author provided no indication of the
probability of finding a liver cancer case who had been an employee of the
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plant and had been exposed to TCI. The author provided no information on the
dates or lengths of exposure of TCI workers or on whether the employee personnel
lists included retirees only or all workers who had ever worked at the plant.
Furthermore, persons with liver cancer who did not live in the area of the
plant or who had moved from the area were excluded from the study by
study definition.
Three proportionate mortality studies of dry-cleaning workers found some
excesses for certain types of cancer deaths (Blair et al. 1979, Katz and Jowett
1981, and (Asal 1983, personal communication), and one of the studies (Blair et al,
1979) found an excess for deaths from all malignant neoplasms. None of these
studies, however, identified what dry-cleaning agent or agents were used by the
workers. Thus, any conclusions from these studies regarding an association of
TCI with death from cancer are obviously limited.
A case-control study of pancreatic cancer cases found a statistically
significant association between pancreatic cancer and the drinking of caffeine-
free coffee (Lin and Kessler 1981). However, it is not known whether the
decaffeinating agent for the coffee was TCI, or, if it was, whether any TCI was
actually consumed by those who drank the decaffeinated coffee. An association
was also found in this study between pancreatic cancer and occupational exposure
in the dry-cleaning industry or occupational exposure to gasoline (service
stations and garages). It is not known, however, how many of the pancreatic
cancer cases involved dry-cleaning exposure and how many involved gasoline
exposure. Secondly, the dry-cleaning agent used by the dry-cleaning workers was
not identified. A kidney cancer case-control study found an .association between
kidney cancer and employment as a dry-cleaner (Asal 1983, personal communication).
Again, however, the dry-cleaning agent used by the dry-cleaning workers was not
identified.
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In summary, there is no human evidence that TCI is a carcinogen. Although
some studies have found an association between cancer and employment in the
dry-cleaning industry, none of these studies indicated that TCI was the dry-
cleaning agent that was used.
8.4.1.4 Concl usions--Evidence for the carci nogenicity of TCI in experimental
animals includes increases in the incidences of hepatocellular carcinoma in
male and female B6C3F1 mice, malignant lymphoma in female Han:NMRI mice, and
renal adenocarinoma in male Fischer 344 rats. However, as discussed in this
document, the inhalation study in B6C3F1 mice was weakened by deficiencies in
its conduct; there was a 30% incidence of spontaneous lymphoma in control mice
and the possibility of an indirect effect in treated mice in the inhalation
study in Han:NMRI mice; and the gavage treatment study in Fischer 344 rats was
concluded to be inadequate for a judgment of TCI carcinogenicity by the study's
sponsor (NTP) because of experimental deficiencies. The gavage treatment
study of purified TCI in male and female B6C3F1 mice was done with basically
the same experimental design as the gavage treatment study of technical grade
TCI in male and female B6C3F1 mice to evaluate the necessity of epoxide stabi-
lizers in TCI in the induction of hepatocellular carcinomas.
Cell transformation activity by TCI in Fischer rat embryo cells containing
rat oncornavirus was found, but it is not certain whether the effect could have
occurred in the host genetic material, the oncornavirus, or both. As concluded
in this document, currently available data provide suggestive evidence that
commercial grade TCI is weakly active as an indirect mutagen, and data on pure
TCI do not allow a conclusion to be drawn about its mutagenic potential; although,
if it is mutagenic, the available data suggest that pure TCI would be a very
weak indirect mutagen. As discussed in the metabolism chapter herein, there is
evidence that TCI metabolite(s) can react with cellular protein macromolecules
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In vivo and in vitro and with exogenous DNA and RNA in vitro, whereas a study
of purified radiolabeled TCI binding to DNA in liver in vivo in male BC63F1
mice given a gavage dose of 1,200 mgAg of TCI-14C (>500 uCi/animal) suggested a
low order of binding [maximum of 0.62 (j^O.42) alkylated bases/lO6 nucleotides].
Cell transformation with high doses of TCI oxide, a putative direct-acting
carcinogenic metabolite of TCI, has been observed in vitro; however, animal studies
of initiating and complete carcinogenic potential of TCI oxide were negative.
TCI is positive for the induction of malignant tumors of the liver in both
male and female B6C3F1 mice. This constitutes a signal that TCI might be a
carcinogen for humans. The positive lymphoma response in females of the HanrNMRI
mouse strain does qualify as a separate strain, but that result is only suggestive
of a carcinogenic response because it occurs only in females, has a high
spontaneous rate, and has been found to involve an indirect mechanism of action.
It should be recognized that there is a substantial body of opinion in the
scientific community to the effect that the mouse liver overreacts to chlorinated
organic compounds in contrast to that of the rat, and that the induction of
liver cancer in the mouse represents only a promoting action for spontaneous
liver tumors, which normally occur with substantial incidence. Furthermore,
this promoting action might be related to liver damage associated only with
high-level exposures to chlorinated agents such as TCI. However, the evidence
is inconclusive either for this restrictive position on the mouse liver car-
cinogenic response to chlorinated organics, or to the position that the mouse
liver is as good as any other mammalian indicator of carcinogenicity for these
compounds. The kidney tumor results in the NTP rat study are not strong indica-
tions of a response in a second species because of the small number of animals
responding (3 of 49 animals) and because of the high mortality, although the
statistical significance after mortality corrections suggests that a carcinogenic
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effect may be taking place. The classification of the TCI carcinogenicity
results under the criteria of the International Agency for Research on
Cancer (IARC) could be either "sufficient" or "limited" depending on the differing
current scientific views about the induction of liver tumors in mice by chlorinated
organic compounds. Since there are no adequate epidemiological data in humans,
the overall ranking of TCI under the criteria of the IARC could be either Group
2B or Group 3, i.e., the more conservative scientific view would regard TCI as
a probable human carcinogen, but there is considerable scientific sentiment for
regarding TCI as an agent that cannot be classified as to its carcinogenicity
for humans.
Additional ongoing carcinogenicity studies on purified TCI (no detectable
epoxides) include those sponsored by the NTP with lifetime gavage treatment in
four strains of rats (Osborne-Mendel, Marshall 540, August 288U7, and ACI);
those performed in the laboratories of Dr. C. Maltoni with lifetime inhalation
exposure in BC63F1 mice, Swiss mice, and Sprague-Dawley rats; and those performed
in the laboratories of Dr. D. Henschler with gavage treatment in ICR/Ha Swiss
mi ce.
8.4.2 Quantitative Assessment
None of the epidemiologic studies cited above provide positive evidence
from which to estimate a unit risk for exposure to TCI. As an alternative, an
upper-limit risk has been estimated from one negative study for comparison with
the two positive animal gavage studies. This upper-limit risk for air of 1.7 x
10~5 per ug/m^ based on the human study is approximately four times greater than
the estimate based on the mean of the two gavage studies, q* = 4.1 x 1U~6 (ug/m^)-!
both of which showed hepatocellular carcinomas in mice. Hepatocellular carcinomas
were also present in mice in one inhalation study, but this study displayed suf-
ficient defects to exclude its use for a unit risk estimate. The results from
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the gavage studies were also used to estimate the unit risk from 1 ug/1 of drink-
ing water; this value was q* = 5.4 x 10"? (ug/l)~l. The relative potency of
TCI is 2.5, ranking it in the lowest quartile of 54 chemicals which the GAG has
evaluated as suspect carcinogens. Furthermore, these estimates are valid upper
limits only under the assumption that TCI is a potential human carcinogen. As
discussed in the qualitative section, there is only limited evidence to support
that assumption.
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